Bispecific antibodies and uses thereof

Abstract

The invention provides a bispecific antibody targeting CD3 and tumor targets and application thereof. The bispecific antibody comprises a first antigen binding region, a second antigen binding region and an Fc part, the Fc part comprises a first Fc peptide 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 connected with the N end of the scFv fragment, and the C end of the scFv fragment is connected with the N end 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 connected with the N end of the second Fc peptide fragment; the first molecule and the second molecule are the same, and the scFv fragment has CD3 binding activity. The bispecific antibody is weak in combination with T cells, strong in combination with tumor cells, good in safety, high in anti-cancer activity and high in clinical application and drug development value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to a bispecific antibody and its application. More specifically, the present invention relates to an antibody, a nucleic acid molecule, a vector or a 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, there are about 19 million new cancer cases and 10 million cancer deaths each year worldwide, and the incidence and mortality rates are on the rise. In addition to surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have large side effects and are prone to recurrence. In recent years, immunotherapy, including tumor-targeted antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hot spot and new hope in anti-cancer. Immunotherapy represented by PD-1 / L1 has shown great potential, but even the PD-1 / L1 therapy, which has the widest approved indications, has an overall response rate of only 30%, and more patients cannot benefit from it. One of the main reasons is that immune checkpoint therapy is ineffective for "cold tumors". T cells recognize neo-antigens (neo-antigens), that is, antigens with tumor gene mutations, through the T cell receptor (TCR) on the surface. Some tumors have low gene mutation frequencies and few types of neo-antigens, which are called "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 therapies for "cold tumors".

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

[0004] One way to solve the safety problem of CD3 bispecific antibodies is to increase the affinity of CD3 bispecific antibodies for binding to tumor targets, while reducing their affinity for CD3 and weakening their binding to T cells, so that CD3 bispecific antibody drugs are more distributed to the tumor, increasing local tumor drug concentration, reducing peripheral drug concentration, and reducing off-target toxicity. Therefore, clinically, there is an urgent need to develop CD3 bispecific antibodies with better safety and higher clinical application value. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, one object of the present invention is to provide a bispecific antibody targeting CD3 and a tumor target, which can bind to CD3 and a tumor target, and has low CD3 binding activity, weak binding to T cells, and weak ability to bridge T cells and tumor cells, and can effectively promote the activity of PBMC in killing tumor cells, has improved T cell activation performance, and can promote the distribution of drugs to the local tumor rather than the peripheral or lymphoid tissues. Therefore, the bispecific antibody of the present invention has good safety, high anticancer activity, and high clinical application and drug development value.

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

[0007] In order to improve the safety of CD3 and MICA bispecific antibodies, the inventors obtained a new configuration of CD3 and MICA bispecific antibodies through a large number of screening and experimental verifications. Further experimental results show that: the CD3 and MICA bispecific antibody has weak binding to T cells, strong binding to tumors, and high promoting killing activity, effectively promoting PBMC to kill tumor cells, and has good anti-cancer activity; and the bispecific antibody has reduced T cell binding activity, high T cell activation performance, higher safety, and good clinical application and drug development value.

[0008] Therefore, in the first aspect of the present invention, the present invention provides a bispecific antibody. According to an embodiment of the present invention, the bispecific antibody comprises: a first antigen binding region, a second antigen binding region and an Fc portion;

[0009] The Fc portion comprises a first Fc peptide segment and a second Fc peptide segment;

[0010] 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;

[0011] 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;

[0012] The first molecule and the second molecule are the same, and the scFv fragment has CD3 binding activity.

[0013] The bispecific antibody according to the embodiment of the present invention can bind to CD3 and tumor targets. The bispecific antibody can bind to CD3 and tumor targets, and has low CD3 binding activity, weak binding to T cells, and weak ability to bridge T cells and tumor cells. At the same time, it can effectively promote the activity of PBMC in killing tumor cells, has improved T cell activation performance, and can promote the distribution of drugs to local tumors rather than peripheral or lymphoid tissues. Therefore, the bispecific antibody of the present invention has good safety, high anti-cancer activity, and high clinical application and drug development value.

[0014] In a second aspect of the present invention, 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 tumor targets.

[0015] In the third aspect of the present invention, the present invention provides a vector or a transformant. According to an embodiment of the present invention, the vector or the transformant contains the aforementioned nucleic acid. Thus, the constructed vector or the transformant can effectively express the aforementioned bispecific antibody.

[0016] In a fourth aspect of the present invention, the present invention provides a cell. According to an embodiment of the present invention, 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 vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0017] In the fifth aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned cell. According to an embodiment of the present invention, the bispecific antibody can effectively promote PBMC to kill tumor cells and has high anti-cancer activity; the bispecific antibody has low CD3 binding activity and good safety. The drug thus obtained can be further used to prevent and / or treat related diseases mediated by CD3 and / or tumor targets.

[0018] In a sixth aspect of the present invention, the present invention provides a kit. According to an embodiment 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 an embodiment of the present invention, the kit can be combined with CD3 protein and / or tumor target, and can effectively identify CD3 protein and / or tumor target.

[0019] 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 herein.

[0020] 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 the preparation of a kit for detecting CD3 and / or tumor targets.

[0021] 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.

[0022] 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, wherein the drug is used to prevent and / or treat related diseases mediated by CD3 and / or tumor targets.

[0023] 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 described in detail here.

[0024] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0026] Figure 1 A schematic diagram of a CD3×MICA bispecific antibody according to a specific embodiment of the present invention;

[0027] Figure 2 This 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;

[0028] Figure 3 This 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 protein according to a specific embodiment of the present invention;

[0029] Figure 4 This 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;

[0030] Figure 5 This 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;

[0031] Figure 6 This 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;

[0032] Figure 7 This is a result diagram showing that Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A10022:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill HCT-15 colorectal cancer cells;

[0033] Figure 8 This is a result diagram showing that Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill NCI-H1299 lung cancer cells;

[0034] Fig. 9This is a result diagram showing that Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill PANC-1 pancreatic cancer cells;

[0035] Fig.10 This is a result diagram showing that Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill A498 renal cancer cells;

[0036] Fig.11 This is a result diagram showing that Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill PC-3 prostate cancer cells;

[0037] Fig.12 This is a result diagram showing that Cross3×h5A1002 1:1 and Cross3×h5A1002 2:1 antibodies according to a specific embodiment of the present invention promote the secretion of immune-activating cytokines IL-2 and IFN-γ after co-incubation of PBMC with HCT-15 colorectal cancer cells;

[0038] Fig.13 This is a result diagram showing that Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1, and SP34×h5A1002 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill HCT-15 colorectal cancer cells;

[0039] Fig.14 This is a result diagram showing that Cross3×h5A1002 2:1, Cross3×1D5 1:1, and Cross3×1D5 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill HCT-15 colorectal cancer cells;

[0040] Fig.15 This is a result diagram showing that Cross3×h5A1002 2:1, Cross3×13A9 1:1, and Cross3×13A9 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill HCT-15 colorectal cancer cells;

[0041] Fig.16This is a result diagram showing that Cross3×h5A1002 2:1, Cross3×619 1:1, and Cross3×619 2:1 antibodies according to a specific embodiment of the present invention promote PBMC to kill HCT-15 colorectal cancer cells;

[0042] Fig.17 This 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;

[0043] Fig.18 This 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 reconstructed mouse NCI-H1299 human lung cancer model;

[0044] Fig.19 This is a graph showing the in vivo efficacy results of Cross3×h5A1002 2:1 antibody promoting immune reconstitution in PC-3 human prostate cancer models in mice according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0045] 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.

[0046] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0047] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the 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 as specifically disclosed in this article.

[0048] Terms and Definitions

[0049] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0050] 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 (Miller et al. (2003) Jour. of Immunology 170: 4854 4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy chains, full-length light chains, complete immunoglobulin molecules; or immunologically active portions of any of these polypeptides, i.e., molecules or portions thereof that contain an antigen binding site that immunospecifically binds to a target antigen of interest, such targets include but are not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases.

[0051] Herein, certain regions in the variable region have a higher degree of variation in amino acid composition and arrangement order, which are called "hypervariable regions (HVR)". The hypervariable region is the location where the antigen and antibody bind, so it is also called the complementarity-determining region (CDR). There are three CDR regions in both the heavy chain variable region and the light chain variable region. 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 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or from a "hypervariable loop" (e.g., amino acid residues around 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable region, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0052] 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".

[0053] Herein, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment", which may include a portion of an intact antibody, generally an antigen-binding region or a variable region. Including but not limited to: Fv, scFv, Fab, Fab', Fab'-SH, scFab (single chain Fab fragment, scFab), F(ab')2, scFv-Fc fragments or bispecific antibodies (Bispecific antibodies, BsAbs), linear antibodies or any fragments that should be able to increase the half-life by chemical modification or by incorporation into liposomes, the chemical modification is, for example, the addition of poly (alkylene) glycols, such as polyethylene glycol ("PEGylation, PEGylation") (called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG PEGylated fragments) ("PEG" is polyethylene glycol).

[0054] 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).

[0055] Herein, the term "humanized antibody" refers to a recombinant antibody obtained by replacing the amino acid sequence of non-CDR (Fv framework region (FR)) amino acids in the constant region and variable region of a monoclonal antibody from one species (such as mouse) with the amino acid sequence of non-CDR amino acids in the constant region and variable region of an antibody from another species (such as 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 non-CDR amino acids in 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, which will not be repeated here.

[0056] With respect to nucleotides, the terms "homology", "identity" or "similarity" are used to describe or compare the degree of nucleotide similarity of 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 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 to be 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 arrangements for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0057] For 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 their designated sequences when optimally aligned and compared (where nucleotides are appropriately inserted or deleted). The % homology between two sequences varies with the number of identical positions shared by these sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions × 100), wherein the 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 determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0058] As used herein, the term "conservatively modified forms of an amino acid sequence such as" 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 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 by an amino acid residue with 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 non-polar 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).

[0059] 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 the expression of 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 that contains the vector.

[0060] 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 combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active bispecific antibody with a liquid carrier, a finely divided solid carrier, or both.

[0061] 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.

[0062] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the bispecific antibodies of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0063] In this article, the term "administration" refers to the introduction of a predetermined amount of material into a patient by a suitable method. Antibodies or antigen binding fragments, recombinant proteins, bispecific antibodies, conjugates or pharmaceutical compositions of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are expected, including peritoneal, intravenous, intramuscular, subcutaneous injections, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, compositions of the present invention are administered by intravenous or subcutaneous injection.

[0064] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking 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 an individual's disease, including but not limited to administering a drug containing the bispecific antibody described herein to an individual in need.

[0065] 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.

[0066] The present invention provides a bispecific antibody, nucleic acid molecule, vector or transformant, cell, pharmaceutical composition, kit and application thereof targeting CD3 and tumor target, which will be described in detail below.

[0067] Bispecific Antibodies

[0068] The present invention provides a bispecific antibody. The bispecific antibody comprises: a first antigen binding region, a second antigen binding region and an Fc part;

[0069] The Fc portion comprises a first Fc peptide segment and a second Fc peptide segment;

[0070] 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;

[0071] 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;

[0072] The first molecule and the second molecule are the same, and the scFv fragment has CD3 binding activity.

[0073] The bispecific antibody according to the embodiment of the present invention can bind to CD3 and tumor targets. The bispecific antibody can bind to CD3 and tumor targets, and has low CD3 binding activity and weak ability to bridge T cells and tumor cells. At the same time, it can effectively promote the activity of PBMC in killing tumor cells, has improved T cell activation performance, and can promote the distribution of drugs to local tumors rather than peripheral or lymphoid tissues. Therefore, the bispecific antibody of the present invention has good safety, high anti-cancer activity, and high clinical application and drug development value.

[0074] 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.

[0075] According to an embodiment of the present invention, the first molecule and the second molecule are both 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. Thus, the bispecific antibody has low binding activity with T cells and high binding activity with tumors, can effectively promote PBMC to kill tumor cells, and can promote drug distribution to the local tumor rather than peripheral or lymphoid tissues. Thus, the bispecific antibody has good safety and high anticancer activity.

[0076] 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.

[0077] 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.

[0078] It should be noted that the amino acid sequences involved in the present invention are all shown from N-terminus to C-terminus.

[0079] 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.

[0080] 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.

[0081] 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

[0082] The C-terminus of the CH1 fragment of the second scFab fragment is connected to the N-terminus of the second Fc peptide segment.

[0083] According to the bispecific antibody of the embodiment of the present invention, the scFv fragment is successfully shielded by the first Fab fragment or the first scFab fragment in configuration. As a result, the CD3 binding activity of the bispecific antibody is reduced, the ability to bridge T cells and tumor cells is weakened, and the safety is good. Contrary to the expectations of the inventors, the tumor cell binding activity of the bispecific antibody of this configuration is significantly improved.

[0084] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment have MICA binding activity. According to the bispecific antibody of the embodiment of the present invention, its CD3 antigen binding portion has little effect on the affinity of the MICA antigen binding portion. Therefore, in order to obtain a bispecific antibody with good safety and high anti-cancer activity, it is preferred that the MICA antigen binding portion selects 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.

[0085] In some embodiments of the invention, the first Fab fragment and the second Fab fragment are not identical.

[0086] In some embodiments of the invention, the first Fab fragment and the second Fab fragment are identical.

[0087] 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,

[0088] 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,

[0089] 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,

[0090] 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,

[0091] 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,

[0092] 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,

[0093] The second light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 12 or a conservatively modified version thereof.

[0094] 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.

[0095] It should be noted that one or more amino acid residues in the heavy chain variable region CDR sequence or the light chain variable region CDR sequence of the present invention 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 by the functional assay method described herein. Preferably, the conservative modification does not exceed 1 or 2 in number.

[0096] 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.

[0097] 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 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.

[0098] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment each independently comprise: a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:29 or a 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.

[0099] 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;

[0100] 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.

[0101] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment each independently comprise:

[0102] 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.

[0103] 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,

[0104] The first heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1,

[0105] The first heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2,

[0106] The first heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3,

[0107] The first light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 4,

[0108] The first light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 5,

[0109] The first light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO:6.

[0110] 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.

[0111] 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.

[0112] 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 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.

[0113] It should be noted that the immunoglobulin described herein can be any type (e.g., IgG, IgE, IgM, IgD and IgA), category (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 immunoglobulin can be derived from any species.

[0114] According to an embodiment of the present invention, the scFv fragment comprises:

[0115] 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.

[0116] 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 by the functional assay method described herein. Preferably, the conservative modification does not exceed 1 or 2 in number.

[0117] According to an embodiment of the present invention, the scFv fragment further comprises: a connecting peptide 1;

[0118] 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

[0119] 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.

[0120] 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.

[0121] In some embodiments of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:11.

[0122] 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.

[0123] According to an embodiment of the present invention, the first antigen binding region further comprises a connecting peptide 2;

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] According to an embodiment of the present invention, the human Fc peptide segment is a human IgG1 Fc peptide segment.

[0130] 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.

[0131] According to an embodiment of the present invention, the bispecific antibody comprises:

[0132] 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;

[0133] 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;

[0134] 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;

[0135] 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.

[0136] It should be noted that, without substantially affecting the binding activity of the multispecific antibody CD3 and MICA (retaining at least 95% of the activity), a person skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the amino acid sequence of the polypeptide chain contained in the multispecific antibody of the present invention to obtain a variant of the amino acid sequence of the polypeptide chain of the bispecific antibody. They are all deemed 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 variant of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% consistency (or homology) with the reference amino acid sequence.

[0137] According to an embodiment of the present invention, the bispecific antibody comprises:

[0138] 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

[0139] 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

[0140] 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

[0141] 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

[0142] 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

[0143] 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.

[0144] Nucleic Acids

[0145] The present invention provides a nucleic acid. The nucleic acid encodes the above-mentioned antibody or antigen-binding fragment thereof. The nucleic acid molecule encodes the aforementioned bispecific antibody. The nucleic acid molecule according to the embodiment of the present invention can encode a bispecific antibody that can simultaneously target CD3 and tumor targets.

[0146] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, if the amino acid sequence of the molecule in the present invention includes a DNA form or an RNA form, disclosing one of them means that the other is also disclosed.

[0147] Vector or transformant

[0148] The present invention provides a vector or transformant. The vector or transformant contains the above-mentioned nucleic acid. The vector or transformant according to an embodiment of the present invention may include an optional control sequence, such as, the control sequence is operably connected to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The vector or transformant thus constructed can effectively express the aforementioned bispecific antibody.

[0149] When the nucleic acid molecule is connected to the vector or transformant, such as an expression vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. The nucleic acid molecule can be operably connected to the control element.

[0150] 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 connecting the nucleic acid to a commercially available vector (such as a plasmid or a viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0151] In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as the amino acid sequence of transcription control such as and the amino acid sequence of translation control such as, can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, phages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the nucleic acid molecules described above can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of protein encoded by the nucleic acid molecules.

[0152] According to an embodiment of the present invention, the vector is a eukaryotic vector or a prokaryotic vector.

[0153] According to an embodiment of the present invention, the vector includes at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

[0154] cell

[0155] 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 vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0156] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

[0157] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0158] According to an embodiment of the present invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0159] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0160] 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.

[0161] According to an embodiment of the present invention, the cell is a mammalian cell.

[0162] According to an embodiment of the present invention, the cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0163] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the antibody or its antigen-binding fragment described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the antibody or its antigen-binding fragment include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, 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 antibody according to the specific environment of the laboratory.

[0164] Pharmaceutical composition

[0165] 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 embodiment of the present invention can effectively promote PBMC to kill tumor cells, and has high anti-cancer activity; and has low CD3 binding activity and good safety. The drug obtained thereby can be further used to prevent and / or treat related diseases mediated by CD3 and / or tumor targets.

[0166] According to an embodiment of the present invention, it further includes a pharmaceutically acceptable excipient.

[0167] According to an embodiment of the present invention, the auxiliary material includes: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0168] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0169] 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, each component can be administered to the subject simultaneously or sequentially.

[0170] The drug of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable adjuvant. Such adjuvants include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the drug preparation should match the mode of administration. The dosage form of the drug of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The drug is preferably manufactured under sterile conditions.

[0171] 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 selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

[0172] 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 match the mode of administration, which are well known to those of ordinary skill in the art.

[0173] Reagent test kit

[0174] 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 the embodiment of the present invention can be combined with CD3 protein and / or tumor target, and can effectively identify CD3 protein and / or tumor target.

[0175] 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 herein.

[0176] In some embodiments of the present invention, the tumor target is MICA. As mentioned above, the bispecific antibody according to the embodiment of the present invention can specifically bind to CD3 and MICA. The CD3 protein and / or MICA protein-related kit developed using this property can be used for CD3 protein and / or MICA protein-related research, such as for detecting and / or enriching and / or separating and purifying CD3 protein and / or MICA protein of humans or other mammals. The kit can effectively detect, enrich or separate and purify CD3 protein and / or MICA protein in biological samples, and is further used in scientific research, such as qualitatively or quantitatively detecting CD3 protein and / or MICA protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, etc., which involve using CD3 protein and / or MICA protein and antibody specific binding properties for detection. These kits may contain any one or more of the following: antagonists, bispecific antibodies of the present invention or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents. The bispecific antibodies of the present invention can be used for different types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins can be detected 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.

[0177] Use in the preparation of kits

[0178] 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.

[0179] In some embodiments of the invention, the tumor target is MICA.

[0180] 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.

[0181] Use in the preparation of medicines

[0182] The present invention provides the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, the aforementioned cell or the aforementioned pharmaceutical composition in preparing a drug, wherein the drug is used to prevent and / or treat CD3 and / or tumor target-mediated related diseases.

[0183] 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 described in detail here.

[0184] According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0185] 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.

[0186] According to an embodiment of the present invention, the tumor target is MICA. The bispecific antibody and nucleic acid, vector or transformant or pharmaceutical composition according to an embodiment of the present invention can be further prepared into a drug, which can be used clinically to prevent or treat diseases related to CD3 and / or MICA.

[0187] 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.

[0188] 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.

[0189] Disease treatment methods

[0190] 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 a pharmaceutically acceptable amount of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, the aforementioned cell or the aforementioned pharmaceutical composition to a subject.

[0191] It should be noted that the terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal that is evaluated for treatment and / or 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, etc. 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, etc.

[0192] 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 with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0193] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0194] 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.

[0195] In some specific embodiments, the MICA-mediated disease is cancer, disease caused by transplant rejection, autoimmune disease, infectious disease.

[0196] In some specific 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, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0197] The amino acid sequences involved in the present invention are described in detail in Table 1.

[0198] Table 1 Amino acid sequence description

[0199]

[0200]

[0201]

[0202]

[0203] The present invention will be described in detail below through examples. In the examples or test examples, experimental methods without specific conditions are all carried out under conventional conditions.

[0204] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the reagents or instruments used are not indicated by the manufacturer, they are all conventional products that can be obtained by commercial purchase.

[0205] Example 1: Production of Antibodies

[0206] The specific experimental operation of antibody production is as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution. (2) When the dual antibody is of A configuration ( Figure 1 ), add the pcDNA3.4 vector containing CD3 antibody, MICA antibody heavy chain, and MICA antibody light chain (commissioned by Nanjing GenScript for synthesis) into 2 mL OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a; or, when the dual antibody is in the B 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) was added to 2 mL OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a. (3) 160 μL Express Fectamine CHO transfection reagent (purchased from Thermo Fisher) was added to 2 mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Then, solution a and solution b were mixed to obtain a transfection mixture, and the transfection mixture was added to 50 mL ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C and 5% CO2 for 1 day, 8 mL Feed and 300 μL Enhancer (purchased from Thermo Fisher) were added, and the culture was transferred to 32°C and 5% CO2 for 9 days. The culture supernatant was harvested, and 8 mL Feed was added on the 5th day. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nano-Micro).

[0207] In this example, 11 CD3 and MICA bispecific antibodies were prepared to investigate the various properties of the bispecific antibodies of the present invention. Figure 1 , Table 1 and Table 2.

[0208] Table 2 Bispecific antibody structure and corresponding amino acid sequence in Example 1

[0209]

[0210] Note: For the configuration of class A and B antibodies, see Figure 1 .

[0211] Example 2: ELISA binding experiment of the antibody of the present invention

[0212] ELISA experiments are used to detect the binding properties of bispecific antibodies. The antigen protein is coated into 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.

[0213] (1) MICA-His protein (purchased from Acro) was diluted to 2 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and then 200 μl / well PBS / 10% BSA was added and incubated at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μl / well of the bispecific antibodies Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), UCHT1×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) were added. NO:18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), control hIgG1LALA (purchased from Baiying Bio), and incubated at 37°C for 1h. Remove the reaction system, wash the plate with PBST 6 times, dilute the HRP (horseradish peroxidase) labeled anti-human IgG antibody secondary antibody (purchased from Jacksonlab) with PBST / 0.05% BSA at 100μl / well, and incubate at 37°C for 1h. After washing the plate with PBST 6 times, add 80μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3min, and add 80μl / well 4M sulfuric acid to terminate the reaction. Read the absorbance value at 450mm with an enzyme reader.

[0214] The results are as follows Figure 2 As shown in the figure, Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies can all bind to MICA protein, and their binding abilities are comparable.

[0215] (2) CD3E&D protein (purchased from Acro) was diluted to 2 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween20) buffer, and then 200 μl / well PBS / 10% BSA was added and incubated at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μl / well of the bispecific antibodies Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), UCHT1×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) were added. NO:18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), control hIgG1LALA (purchased from Baiying Bio), and incubated at 37°C for 1h. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled anti-human IgG antibody secondary antibody (purchased from Southern biotech) with PBST / 0.05% BSA at 100μl / well, and incubate at 37°C for 1h. After washing the plate with PBST 6 times, add 80μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3min, and add 80μl / well 4M sulfuric acid to terminate the reaction. Read the absorbance value at 450mm with an enzyme reader.

[0216] The results are as follows Figure 3 As shown. Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A10022:1 antibodies can all bind to CD3E&D protein; Cross3×h5A1002 2:1 has a weaker ability to bind to CD3E&D than Cross3×h5A1002 1:1; and Cross3×h5A1002 2:1 has the weakest activity in binding to CD3.

[0217] The above test results show that after adjusting the structure of the bispecific antibody ( Figure 1 , configuration B), the antigen-binding fragment of the anti-CD3 antibody is successfully shielded in the configuration, and its ability to bind to CD3 is weakened.

[0218] Example 3: Antibody ELISA bridging experiment

[0219] ELISA experiments were used to detect the bridging binding properties of bispecific antibodies. CD3E&D antigen proteins were coated into 96-well plates. After the addition of antibodies, biotin-labeled MICA proteins were 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.

[0220] MICA-His protein (purchased from Acro) was diluted to 2 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween20) buffer, and then 200 μl / well PBS / 10% BSA was added and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μl / well of the bispecific antibodies Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), UCHT1×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), and UCHT1×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) were added. NO:18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), control hIgG1LALA (purchased from Baiying Bio), and incubated at 37°C for 1h. Remove the reaction system, wash the plate with PBST 6 times, add MICA*002α3-Fc-Biotin diluted to an appropriate concentration, and incubate at 37°C for 1h. Remove the reaction system, wash the plate with PBST 6 times, dilute HRP (horseradish peroxidase) labeled Streptavidin secondary antibody (purchased from Southern biotech) with PBST / 0.05% BSA at 100μl / well, and incubate at 37°C for 1h. After washing the plate with PBST 6 times, add 80μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3min, and add 80μl / well 4M sulfuric acid to terminate the reaction. Read the absorbance value at 450mm with an enzyme reader.

[0221] The results are as follows Figure 4As shown in the figure, Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A10022:1 antibodies can bridge CD3E&D and MICA proteins; the bridging ability of Cross3×h5A1002 2:1 antibody is weaker than that of Cross3×h5A1002 1:1; and Cross3×h5A1002 2:1 antibody has the weakest bridging ability.

[0222] according to Figure 2 , 3 The results showed that the weak bridging ability of Cross3×h5A1002 2:1 antibody was due to the low affinity of Cross3×h5A1002 2:1 antibody to CD3.

[0223] Example 4: Antibody Flow Cytometry Binding Experiment

[0224] 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.

[0225] (1) Dilute PBMC to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added thereto, and blocked at 4℃ for 30min. Serial dilutions of the bispecific antibodies Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), UCHT1×h5A1002 1:1 (the amino acid sequence of the 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), and UCHT1×h5A1002 were added. 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), control hIgG1LALA (purchased from Baiying Bio), and incubated at 4°C for 30min. Add 1ml PBS to the EP tube, centrifuge at 4°C 3500rpm×5min, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μl / tube PBS, add 1μl / tube Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson lab) and 0.5μl / tube PerCP-Cy5.5-labeled anti-human CD8 antibody, and incubate at 4°C in the dark for 30min. Wash twice with PBS, discard the supernatant after centrifugation. Resuspend the cells with 200μl / tube PBS and detect them using a flow cytometer.

[0226] The results are as follows Figure 5 As shown. Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A10021:1, UCHT1×h5A1002 2:1 antibodies can bind to CD8 T cells, and the 2:1 configuration (configuration B) bispecific antibody has a weaker ability to bind to T cells than the 1:1 configuration (configuration A) bispecific antibody. Among them, Cross3×h5A10022:1 bispecific antibody has the weakest binding activity to T cells.

[0227] (2) HCT-15 tumor cells were diluted to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added thereto, and blocked at 4℃ for 30min. Serial dilutions of the bispecific antibodies Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), and UCHT1×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15) were added. NO: 15), control hIgG1LALA (purchased from Baiying Bio), incubate at 4°C for 30 min. Add 1 ml PBS to the EP tube, centrifuge at 4°C 3500 rpm × 5 min, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100 μl / tube PBS, add 1 μl / tube Alexa-647 labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson lab), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200 μl / tube PBS and detect using a flow cytometer.

[0228] The results are as follows Figure 6 As shown. (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 Cross3×h5A1002 2:1 and UCHT1×h5A1002 2:1 antibodies have comparable tumor binding activity.

[0229] Example 5: Experiment on Bispecific Antibodies Promoting PBMCs to Kill Tumor Cells

[0230] 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.

[0231] (a) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate on the machine;

[0232] (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;

[0233] (c) The serially diluted bispecific antibodies Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO:13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), UCHT1×h5A1002 1:1 (the amino acid sequence of the 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), UCHT1×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), control hIgG1LALA (purchased from Bio-Bio) (the highest concentration is 0.2 μg / ml, 8 gradients, 5-fold dilution), added to the RTCA plate obtained in step (2), the added volume is 20 μl / well;

[0234] (4) PBMC (purchased from Sai Li Biotechnology) were diluted to 1.25×10 6pcs / ml, added to the RTCA plate obtained in step (3), with the added volume being 80 μl / well;

[0235] (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 instrument.

[0236] The results are as follows Figure 7 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, UCHT1×h5A1002 2:1 antibodies promote PBMC to kill HCT-15 colorectal cancer cells. (2) Contrary to the inventors' expectations, the killing ability of the 2:1 configuration (configuration B) bispecific antibody is stronger than that of the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A10022:1 has the highest killing activity.

[0237] The results are as follows Figure 8 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, UCHT1×h5A1002 2:1 antibodies promote PBMC to kill NCI-H1299 lung cancer cells. (2) Contrary to the inventors' expectations, the killing ability of the 2:1 configuration (configuration B) bispecific antibody is stronger than that of the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A1002 2:1 has the highest killing activity.

[0238] The results are as follows Fig. 9 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, UCHT1×h5A1002 2:1 antibodies promote PBMC to kill PANC-1 pancreatic cancer cells. (2) Contrary to the inventors' expectations, the killing ability of the 2:1 configuration (configuration B) bispecific antibody is stronger than that of the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A1002 2:1 has the highest killing activity.

[0239] The results are as follows Fig.10 (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoted PBMC to kill 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.

[0240] The results are as follows Fig.11As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promote PBMC to kill PC-3 prostate cancer cells. (2) Contrary to the inventors' expectations, the killing ability of the 2:1 configuration (configuration B) bispecific antibody is stronger than that of the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A1002 2:1 has the highest killing activity.

[0241] The above results show that the 2:1 configuration (configuration B) bispecific antibody of the present invention significantly enhances the ability of PBMC 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. Among them, when the CD3 antigen binding region is the antigen binding fragment of the Cross3 antibody, the 2:1 configuration (configuration B) bispecific antibody of the present invention has the strongest activity in promoting PBMC to kill tumor cells.

[0242] Example 6: Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0243] The bispecific antibody was added to the PBMC and tumor cell co-incubation system, and the culture supernatant was collected after 48 hours of culture. The cytokine content in the supernatant was detected to determine the characteristics of the bispecific antibody in inducing cytokine release.

[0244] (1) Dilute the tumor to 1×10 using complete RPMI 1640 medium. 5 / ml, added to 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0245] (2) Using complete RPMI 1640 medium, the bispecific antibody Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15), and the control hIgG1LALA (purchased from Bio-Innovative) were serially diluted and added to a 96-well plate at 20 μl / well;

[0246] (3) PBMC (purchased from Sai Li Biotechnology) were diluted to 1.25×10 6 / ml, added into 96-well plate, 80 μl / well;

[0247] (4) The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 48 h;

[0248] (5) Centrifuge at 300 g for 10 min at room temperature and collect the cell culture supernatant;

[0249] (6) Use CBA kit (purchased from BD) to detect the cytokine content in the supernatant.

[0250] The results are as follows Fig.12 As shown. (1) Cross3×h5A1002 1:1 and Cross3×h5A1002 2:1 antibodies promote PBMC to secrete immune-activating cytokines IL-2 and IFN-γ. (2) Cross3×h5A1002 2:1 antibody has a stronger ability to promote PBMC to secrete immune-activating cytokines.

[0251] The above results show that the 2:1 configuration (configuration B) bispecific antibody of the present invention has an enhanced ability to promote PBMC to secrete immune-activating cytokines. Considering that the 2:1 configuration (configuration B) bispecific antibody has weak binding to T cells and strong binding to tumor cells, after administration, more drugs can be distributed to the tumor site, enhancing the anti-cancer effectiveness.

[0252] Example 7: Experiment on promoting PBMC to kill tumor cells with bispecific antibodies at different effector-target ratios

[0253] The ability of the bispecific antibody of the present invention to promote PBMC to kill HCT-15 colorectal cancer cells was detected under different effector-target ratios (PBMC: tumor).

[0254] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0255] (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;

[0256] (3) The serially diluted bispecific antibodies Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), UCHT1×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), SP34×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 19, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15) were added to complete RPMI-1640 medium. NO: 15), control hIgG1LALA (purchased from Bio-Bio) (maximum concentration of 0.2 μg / ml, 8 gradients, 5-fold dilution), added to the RTCA plate obtained in step (2), with an added volume of 20 μl / well;

[0257] (4) PBMC (purchased from Sai Li Biotechnology) were diluted to 1.25×10 6 / ml (effectiveness-target ratio 10:1), 0.625×10 6 / ml (efficacy-target ratio 5:1), 0.3125×10 6 / ml (efficacy-target ratio 2.5:1), added to the RTCA plate obtained in step (3), with an added volume of 80 μl / well;

[0258] (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 instrument.

[0259] The results are as follows Fig.13 As shown in the figure. Under different effector-target ratios, Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1, and SP34×h5A1002 2:1 antibodies can promote PBMC to kill HCT-15 colorectal cancer cells, and Cross3×h5A1002 2:1 has the highest killing activity.

[0260] Example 8: Experiments on promoting PBMC to kill tumor cells with other MICA dual antibodies

[0261] The ability of the bi-antibody of the present invention with a 2:1 configuration (configuration B) formed based on other MICA antibodies to promote PBMC to kill HCT-15 colorectal cancer cells was detected to evaluate whether the ability of the bi-antibody of the present invention with a 2:1 configuration (configuration B) to promote PBMC to kill tumor cells is generally better than that of the 1:1 configuration (configuration A). Other MICA antibodies are selected from: 1D5 (Genentech monoclonal antibody, US20200055939A1), 13A9 (Genentech monoclonal antibody, US20200055939A1), 619 (Cullinan monoclonal antibody, US20210253711A1).

[0262] 1. Expression of 2:1 configuration (configuration B) antibody: the same method as in Example 1.

[0263] 2. Investigation of the ability of PBMC to kill tumor cells: Referring to Example 7, proceed as follows.

[0264] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0265] (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;

[0266] (3) The serially diluted bispecific antibodies Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), Cross3×1D5 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 21), Cross3×1D5 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody 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), and Cross3×13A9 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:23, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:24), Cross3×13A9 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO:25, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:23, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:24), Cross3×619 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:26, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:27), Cross3×619 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO:28, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: NO:26, and the amino acid sequence of the light chain of the MICA antibody is shown in SEQ ID NO:27), control hIgG1LALA (purchased from Bio-Bio) (the highest concentration is 0.2 μg / ml, 8 gradients, 5-fold dilution), added to the RTCA plate obtained in step (2), the added volume is 20 μl / well;

[0267] (4) PBMC (purchased from Sai Li Biotechnology) were diluted to 1.25×10 6pcs / ml, added to the RTCA plate obtained in step (3), with the added volume being 80 μl / well;

[0268] (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 instrument.

[0269] The results are as follows Fig.14 As shown. (1) The killing ability of the 2:1 bispecific antibody based on 1D5 is weaker than that of the 1:1 bispecific antibody. (2) In addition, Cross3×h5A1002 2:1 has a higher killing activity than the bispecific antibody based on 1D5.

[0270] The results are as follows Fig.15 As shown. (1) The killing ability of the 2:1 dual antibody based on 13A9 is weaker than that of the 1:1 dual antibody. (2) In addition, Cross3×h5A1002 2:1 has higher killing activity than the dual antibody based on 13A9.

[0271] The results are as follows Fig.16 As shown. (1) The killing ability of the 2:1 dual antibody based on 619 is weaker than that of the 1:1 dual antibody. (2) In addition, Cross3×h5A1002 2:1 has higher killing activity than the dual antibody based on 619.

[0272] 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.

[0273] Example 9: Effect of CD3×MICA antibody on anti-cancer in mice

[0274] The in vivo efficacy experiment was used to detect the function of the Cross3×h5A1002 2:1 bispecific antibody of the present invention in promoting the anti-cancer effect in immune reconstructed mice.

[0275] (1) On day -14, human PBMC (purchased from Sai Li Biotechnology) was transfused into NSG mice (purchased from Southern Model) via the tail vein at an injection volume of 5×10 6 / Only;

[0276] (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;

[0277] (3) On day 0, the mice were weighed and the tumor volume was measured. The mice were divided into 6 groups according to the weighing results and tumor volume;

[0278] (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 solvent control PBS, 250 μl per mouse, through the tail vein;

[0279] (4) After injection of the above antibodies, the tumor volume was measured and the mice were weighed twice a week.

[0280] The results are as follows Fig.17 , 19 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, and the tumor can be completely regressed.

[0281] The above experimental results show that the bispecific antibody of the present invention can bind to CD3 and MICA, thereby promoting T cell activation, secreting cytokines, 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 cells to fight cancer, has good anti-cancer activity, and higher safety, and has high clinical application value and drug development value.

[0282] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

[0283] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0284] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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, Betacatenin, 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; Optionally, the first molecule and the second molecule are both MICA; Optionally, 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; Optionally, 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; Optionally, 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; Optionally, 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.

3. The bispecific antibody according to claim 2, characterized in that The first Fab fragment and the second Fab fragment have MICA binding activity; Optionally, 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 form thereof; Optionally, 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; Optionally, at least a portion of the second heavy chain framework region and / or the second light chain framework region is 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; Optionally, 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 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; Optionally, 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; Optionally, 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.

4. The bispecific antibody according to any one of claims 1 to 3, 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; Optionally, the scFv fragment comprises a first heavy chain framework region and / or a first light chain framework region; Optionally, at least a portion of the first heavy chain framework region and / or the first light chain framework region is 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; Optionally, 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; Optionally, the scFv fragment further comprises: 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; Optionally, 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; Optionally, the scFv fragment has the amino acid sequence shown in SEQ ID NO: 35; Optionally, the first antigen binding region further comprises 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; Optionally, 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; Optionally, the connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO: 36 or 37.

5. The bispecific antibody according to claim 1 or 2, characterized in that: The first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure; Optionally, the first Fc peptide segment and the second Fc peptide segment are both human Fc peptide segments; Optionally, the human Fc peptide is a human IgG1 Fc peptide; Optionally, 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.

6. The bispecific antibody according to claim 2, 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; Optionally, 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.

7. A nucleic acid, characterized in that Encoding the multispecific antibody according to any one of claims 1 to 6.

8. A vector or transformant, characterized in that: Containing the nucleic acid according to claim 7; Optionally, the vector is a eukaryotic vector or a prokaryotic vector; Optionally, the vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

9. A cell, characterized in that Carrying the nucleic acid of claim 7, the vector or transformant of claim 8; or Expressing the multispecific antibody according to any one of claims 1 to 6; Optionally, the cell is a prokaryotic cell, a eukaryotic cell, or a bacteriophage.

10. A pharmaceutical composition, characterized in that Comprising: the multispecific antibody according to any one of claims 1 to 6, the nucleic acid according to claim 7, or the vector or transformant according to claim 8; Optionally, further comprising a pharmaceutically acceptable excipient; Optionally, the auxiliary materials include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Optionally, the pharmaceutical composition is an injection.

11. A kit, characterized in that: The method comprises: the multispecific antibody according to any one of claims 1 to 6, the nucleic acid according to claim 7, the vector or transformant according to claim 8, or the cell according to claim 9.

12. Use of the multispecific antibody according to any one of claims 1 to 6, the nucleic acid according to claim 7, the vector or transformant according to claim 8, or the cell according to claim 9 in the preparation of a kit for detecting CD3 and / or tumor targets.

13. Use of the multispecific antibody according to any one of claims 1 to 6, the nucleic acid according to claim 7, the vector or transformant according to claim 8, the cell according to claim 9 or the pharmaceutical composition according to claim 10 in the preparation of a drug for preventing and / or treating CD3 and / or tumor target-mediated related diseases; Optionally, the CD3-mediated related diseases include autoimmune diseases; Optionally, the autoimmune disease comprises 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; Optionally, the tumor target is MICA; Optionally, the MICA-mediated disease comprises cancer or transplant rejection, autoimmune disease and infectious disease; Optionally, the cancer comprises 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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