Antibodies against cd3 and uses thereof

Abstract

The application provides an antibody against CD3 and application thereof, the antibody comprises CDR sequences selected from at least one of the following or amino acid sequences with at least 95% identity: heavy chain variable region CDR sequences: SEQ ID NO: 1-3, light chain variable region CDR sequences: SEQ IN NO: 4-6. The antibody of the embodiment of the application can be combined with human and monkey CD3 proteins, and the bispecific antibody prepared by using the antibody has higher human and monkey CD3 binding activity, and can effectively treat and / or prevent CD3-mediated related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an anti-CD3 antibody and its application. Background Technology

[0002] T lymphocytes play a crucial role in adaptive immune responses and the precise regulation of these responses. T cell receptors (TCRs) on the surface of T cells exist as heterodimers of α and β or γ and δ. After receiving antigenic peptide signals from antigen-presenting cells, TCRs do not directly mediate intracellular signaling; they need to form a complex with CD3 molecules. Not only do α and β TCRs form complexes with CD3, but γ and δ TCRs also do. Deletion of either the TCR or CD3 gene can lead to the disappearance of the TCR-CD3 complex from the T cell surface. The CD3 molecule is composed of three pairs of peptide chains, each consisting of five polypeptide chains. 90% of CD3 molecules are composed of γε, δε, and ζη chains, while the remaining 10% are composed of γε, δε, and ζζ chains. The tyrosine activation motifs of the immune receptors in the intracellular regions of each CD3 polypeptide chain are phosphorylated by the intracellular tyrosine protein kinase p56, recruiting and binding tyrosine kinases such as ZAP-70, and then participating in mediating TCR activation signals.

[0003] CD3 is a widely chosen target in T cell retargeting bispecific antibodies. Unlike immune checkpoint blocking antibodies, CD3-associated bispecific antibodies can bypass the TCR and peptide-major histocompatibility complex (pMHC) to mediate T cell activation, but the molecular mechanism at the synapse is similar to the classical TCR-pMHC interaction. The activity of bispecific antibodies is influenced by CD3 affinity; high-affinity CD3 bispecific antibodies show better cytotoxic effects in vitro but pose a higher risk of releasing factor syndrome in vivo. Meanwhile, studies have found that very low-affinity CD3 antibody sequences can also effectively stimulate T cell activation after being used to construct bispecific antibodies. When the CD3 antibody affinity is within an appropriate range and the tumor-targeting antibody has high affinity, the bispecific antibody can induce T cells to selectively localize to the tumor site rather than circulating in the periphery, avoiding systemic activation.

[0004] Many known CD3 antibodies are species-specific; for example, clones like UCHT-1, OKT3, and L2K only react with chimpanzee CD3 and not with CD3 in other primates such as cynomolgus monkeys and rhesus monkeys, or even mouse CD3. Since new drug development requires rigorous non-clinical trials before clinical studies, identifying relevant species is crucial for preclinical safety evaluation. However, using chimpanzees for drug safety testing is highly restricted. Among known CD3 antibodies with human-monkey cross-linking, the SP34 antibody exhibits an order-of-magnitude decrease in affinity after forming a single-chain antibody.

[0005] Therefore, developing CD3 antibodies that have specific cross-reactivity with other non-human primates has significant practical application value. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art:

[0007] The inventors of this application have successfully screened a murine anti-CD3 monoclonal antibody that exhibits high binding activity to human or monkey CD3 protein. Furthermore, the inventors partially humanized the constant region of the obtained murine antibody while retaining the CDR of the murine anti-CD3 monoclonal antibody to obtain a chimeric antibody. Further, the inventors humanized the framework region of the light chain variable region or heavy chain variable region of the chimeric antibody to obtain a fully humanized anti-CD3 antibody. The chimeric antibody and the humanized antibody not only specifically target and bind to human and monkey CD3 proteins but also exhibit low immunogenicity, effectively treating and / or preventing CD3-mediated diseases such as autoimmune diseases.

[0008] Bispecific antibodies (biantibodies) or multispecific antibodies (polyantibodies) prepared using the anti-CD3 antibodies can also specifically target and bind to human CD3 protein and monkey CD3 protein. Generally, based on the specificity of the bispecific or polyantibodies, the bispecific or polyantibodies can target T cells to other antigens and eliminate the cells that produce such antigens through T cell-mediated cell killing, thereby treating a variety of diseases, such as tumors.

[0009] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment. According to embodiments of the invention, it comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity with it: heavy chain variable region CDR sequence: SEQ ID NO: 1-3, light chain variable region CDR sequence: SEQ ID NO: 4-6. The antibody or antigen-binding fragment according to embodiments of the present invention can bind to human or monkey CD3 protein, effectively treating or preventing CD3-mediated diseases.

[0010] According to embodiments of the present invention, the above-described antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0011] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 2, and 3, or amino acid sequences having at least 95% identity with SEQ ID NO:1, 2, and 3, respectively; and / or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:4, 5, and 6, or amino acid sequences having at least 95% identity with 4, 5, and 6, respectively.

[0012] According to embodiments of the present invention, the antibody or antigen-binding fragment includes: the heavy chain variable region CDR1 sequence as shown in SEQ ID NO:1, the heavy chain variable region CDR2 as shown in SEQ ID NO:2, the heavy chain variable region CDR3 as shown in SEQ ID NO:3, the light chain variable region CDR1 as shown in SEQ ID NO:4, the light chain variable region CDR2 as shown in SEQ ID NO:5, and the light chain variable region CDR3 as shown in SEQ ID NO:6.

[0013] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes at least one of a heavy chain FR region and a light chain FR region.

[0014] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of human antibodies, primate antibodies, and mouse antibodies or mutants thereof.

[0015] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO:7-10; or at least one of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO:15-18.

[0016] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO:11-14 respectively; or at least one of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO:19-22 respectively.

[0017] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: at least one of the heavy chain frame region sequences HFR1, HFR2, HFR3 and HFR4 as shown in SEQ ID NO:7-10; at least one of the light chain frame region sequences LFR1, LFR2, LFR3 and LFR4 as shown in SEQ ID NO:11-14; or at least one of the heavy chain frame region sequences HFR1, HFR2, HFR3 and HFR4 as shown in SEQ ID NO:15-18; or at least one of the light chain frame region sequences LFR1, LFR2, LFR3 and LFR4 as shown in SEQ ID NO:19-22.

[0018] According to embodiments of the present invention, the antibody or antigen-binding fragment includes: a heavy chain variable region as shown in SEQ ID NO:23 or SEQ ID NO:27; and / or a light chain variable region as shown in SEQ ID NO:24 or SEQ ID NO:28.

[0019] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes: 1) a heavy chain variable region as shown in SEQ ID NO:23 and a light chain variable region as shown in SEQ ID NO:24; or 2) a heavy chain variable region as shown in SEQ ID NO:27 and a light chain variable region as shown in SEQ ID NO:28.

[0020] According to embodiments of the present invention, the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody, a mouse antibody, or a mutant thereof.

[0021] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse IgG antibodies or their mutants, or human IgG antibodies or their mutants.

[0022] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse IgG1 antibody or its mutant or human IgG1 antibody or its mutant.

[0023] According to embodiments of the present invention, the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO:29 or 33 and / or a light chain constant region of the amino acid sequence shown in SEQ ID NO:30 or 34.

[0024] According to embodiments of the present invention, the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence shown in any one of SEQ ID NO:35, 37 and 39 and a light chain having an amino acid sequence shown in any one of SEQ ID NO:36, 38 and 40.

[0025] According to embodiments of the present invention, the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:35 and a light chain of the amino acid sequence shown in SEQ ID NO:36; the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:37 and a light chain of the amino acid sequence shown in SEQ ID NO:38; or the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:39 and a light chain of the amino acid sequence shown in SEQ ID NO:40.

[0026] According to embodiments of the present invention, the antibody or antigen-binding fragment includes monoclonal antibodies or polyclonal antibodies.

[0027] According to embodiments of the present invention, the monoclonal antibody includes at least one of full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody, and minimum recognition unit.

[0028] In a second aspect, the present invention provides a bispecific antibody. According to embodiments of the invention, it includes a first binding region comprising the antibody or antigen-binding fragment described in the first aspect; and a second binding region having BCMA or B7H6 binding activity. The bispecific antibody according to embodiments of the invention can bind to human or monkey CD3 and BCMA proteins, or to human or monkey CD3 and B7H6 proteins, and can be used in scientific research, or to effectively treat or prevent CD3 and BCMA, or CD3 and B7H6-mediated diseases.

[0029] Those skilled in the art will understand that the binding activity of the second binding region is not particularly limited and may also have other binding activities, as long as the bispecific antibody has the antibody or antigen-binding fragment described in the first aspect, and both the antibody or antigen-binding fragment and the second binding region can effectively function. Furthermore, the antibody or antigen-binding fragment described in this application can be used to prepare more specific antibodies, such as trispecific, tetraspecific, or pentaspecific antibodies. Based on the multispecificity of the antibodies, the antibody or antigen-binding fragment of this invention can target T cells to other antigens, eliminating cells producing such antigens through T cell-mediated cell killing.

[0030] According to embodiments of the present invention, the above-mentioned dual antibodies may further include at least one of the following additional technical features:

[0031] According to an embodiment of the present invention, the dual antibody includes symmetric dual antibodies or asymmetric dual antibodies.

[0032] According to an embodiment of the present invention, the dual antibody is a symmetrical dual antibody.

[0033] According to an embodiment of the present invention, the antibody or antigen-binding fragment is an anti-CD3 single-chain antibody.

[0034] According to embodiments of the present invention, the second binding region includes at least one of the following: a full-length antibody, an Fv, a single-chain antibody, a Fab, a single-domain antibody, and a minimum recognition unit, all possessing BCMA or B7H6 binding activity. Those skilled in the art will understand that the specific composition of the second binding region is not particularly limited, as long as it possesses BCMA or B7H6 binding activity; it can be a complete full-length antibody or an antibody fragment (antigen-binding fragment).

[0035] According to an embodiment of the present invention, the second binding region includes an anti-BCMA single-chain antibody or an anti-B7H6 single-chain antibody.

[0036] According to an embodiment of the present invention, the anti-CD3 single-chain antibody includes an anti-CD3 antibody light chain variable region and an anti-CD3 antibody heavy chain variable region, wherein the anti-CD3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:23 or 27, and the anti-CD3 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:24 or 28.

[0037] According to an embodiment of the present invention, the anti-CD3 single-chain antibody further includes a linker peptide 1, wherein the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the heavy chain of the anti-CD3 antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the light chain of the anti-CD3 antibody; or the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the light chain of the anti-CD3 antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the heavy chain of the anti-CD3 antibody.

[0038] According to an embodiment of the present invention, the anti-BCMA single-chain antibody includes an anti-BCMA antibody light chain variable region and an anti-BCMA antibody heavy chain variable region, wherein the anti-BCMA antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:42, and the anti-BCMA antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:61.

[0039] According to an embodiment of the present invention, the anti-BCMA single-chain antibody further includes a linker peptide 2, wherein the N-terminus of the linker peptide 2 is connected to the C-terminus of the variable region of the heavy chain of the anti-BCMA antibody, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the variable region of the light chain of the anti-BCMA antibody; or the N-terminus of the linker peptide 2 is connected to the C-terminus of the variable region of the light chain of the anti-BCMA antibody, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the variable region of the heavy chain of the anti-BCMA antibody.

[0040] According to an embodiment of the present invention, the anti-B7H6 single-chain antibody includes an anti-B7H6 antibody light chain variable region and an anti-B7H6 antibody heavy chain variable region, wherein the anti-B7H6 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:63, and the anti-B7H6 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:62.

[0041] According to an embodiment of the present invention, the anti-B7H6 single-chain antibody further includes a linker peptide 3, wherein the N-terminus of the linker peptide 3 is connected to the C-terminus of the variable region of the heavy chain of the anti-B7H6 antibody, and the C-terminus of the linker peptide 3 is connected to the N-terminus of the variable region of the light chain of the anti-B7H6 antibody; or the N-terminus of the linker peptide 3 is connected to the C-terminus of the variable region of the light chain of the anti-B7H6 antibody, and the C-terminus of the linker peptide 3 is connected to the N-terminus of the variable region of the heavy chain of the anti-B7H6 antibody.

[0042] According to an embodiment of the present invention, the linker peptide 1 has an amino acid sequence (GGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0043] According to an embodiment of the present invention, at least one of the linker peptide 2 and linker peptide 3 has an amino acid sequence (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.

[0044] Those skilled in the art will understand that the linker peptides 1, 2, and 3 described herein are not particularly limited, and any conventional linker peptides in the art, such as conventional flexible or rigid amino acid fragments, may be used.

[0045] According to an embodiment of the present invention, the linker peptide 1 has the amino acid sequence shown in SEQ ID NO:48.

[0046] According to an embodiment of the present invention, the linker peptides 2 and 3 have the amino acid sequence shown in SEQ ID NO:41.

[0047] According to an embodiment of the present invention, the anti-CD3 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:45.

[0048] According to an embodiment of the present invention, the anti-BCMA single-chain antibody has an amino acid sequence as shown in SEQ ID NO:46.

[0049] According to an embodiment of the present invention, the anti-B7H6 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:47.

[0050] According to an embodiment of the present invention, the first antigen-binding region further includes a first Fc peptide segment, wherein the N-terminus of the first Fc peptide segment is connected to the C-terminus of the antibody or antigen-binding fragment.

[0051] According to an embodiment of the present invention, the second antigen-binding region further includes a second Fc peptide, the N-terminus of which is linked to the C-terminus of the anti-BCMA single-chain antibody or the anti-B7H6 single-chain antibody.

[0052] According to an embodiment of the present invention, the first Fc peptide has the amino acid sequence shown in SEQ ID NO:49.

[0053] According to an embodiment of the present invention, the second Fc peptide has the amino acid sequence shown in SEQ ID NO:50.

[0054] According to embodiments of the present invention, the first Fc peptide and the second Fc peptide are linked by a knob-into-hole structure. Those skilled in the art will understand that the amino acid sequences of the first Fc peptide and the second Fc peptide may be the same or different. For example, when they have the same amino acid sequence, the first Fc peptide and the second Fc peptide can be linked by disulfide bonds.

[0055] According to an embodiment of the present invention, the first antigen-binding region has the amino acid sequence shown in SEQ ID NO:51, and the second antigen-binding region has the amino acid sequence shown in SEQ ID NO:52 or 53.

[0056] The nucleic acid encoding the antibody or its antigen-binding fragment of the present invention or the bispecific antibody is within the scope of the present invention. Based on its amino acid sequence, those skilled in the art can easily obtain the corresponding nucleic acid sequence.

[0057] Therefore, in a third aspect of the invention, the invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment or the bispecific antibody described in the first aspect. According to some specific embodiments of the invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule can bind to human or monkey CD3 protein, effectively treating or preventing CD3-mediated diseases. The bispecific antibody encoded by the nucleic acid molecule can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, effectively treating or preventing CD3 and BCMA, or CD3 and B7H6-mediated diseases.

[0058] According to embodiments of the present invention, the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features:

[0059] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0060] It should be noted that those skilled in the art should understand that the nucleic acids mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one means that the other is also disclosed.

[0061] In a fourth aspect, the present invention provides an expression vector carrying the aforementioned nucleic acid molecule. The expression vector may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence may be one or more control sequences that direct the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiments of the present invention can efficiently and massively express the antibody or antigen-binding fragment in suitable host cells.

[0062] In this article, "operably ligated" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. When ligating the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Those skilled in the art will understand that nucleic acid molecules used to encode antibody or antigen-binding fragments can be independently inserted into different vectors, but commonly they are inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, etc., such as the Plasmid-X plasmid.

[0063] In a fifth aspect, the present invention provides a method for preparing the aforementioned antibody or antigen-binding fragment or bispecific antibody, comprising: introducing the aforementioned expression vector into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment or bispecific antibody. The method proposed according to some specific embodiments of the present invention can effectively obtain the antibody or antigen-binding fragment or bispecific antibody in large quantities in vitro.

[0064] According to some specific embodiments of the present invention, the above-described methods for preparing the antibodies or antigen-binding fragments or bispecific antibodies may further include at least one of the following additional technical features:

[0065] According to some specific embodiments of the present invention, the cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used.

[0066] According to some specific embodiments of the present invention, the cells are eukaryotic cells.

[0067] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells. According to some specific embodiments of the present invention, when the cells are eukaryotic cells, such as mammalian cells, the expression efficiency of the recombinant antibody is higher.

[0068] In a sixth aspect, the present invention provides a recombinant cell, wherein the recombinant cell expresses the aforementioned nucleic acid, or expression vector, or is capable of expressing the aforementioned antibody or antigen-binding fragment, or the bispecific antibody. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently and extensively express the aforementioned antibody or antigen-binding fragment, or the bispecific antibody, under suitable conditions.

[0069] It should be noted that the recombinant cells described in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0070] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody, antigen-binding fragment, or bispecific antibody described in this application. Those skilled in the art will readily understand that suitable conditions for antibody, antigen-binding fragment, or bispecific antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the antibody, antigen-binding fragment, or bispecific antibody based on the specific environment of their laboratory.

[0071] In a seventh aspect, the present invention provides an immunoconjugate comprising the aforementioned antibody or antigen-binding fragment or the bispecific antibody, and a therapeutic agent. As previously described, the antibody or antigen-binding fragment of the embodiments of the present invention can effectively bind to CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, effectively treating or preventing CD3 and BCMA, or CD3 and B7H6-mediated diseases. Therefore, the immunoconjugate containing the antibody or antigen-binding fragment can also bind to human or monkey CD3 protein, and the immunoconjugate containing the bispecific antibody can also bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein. The immunoconjugate has good preventive and / or therapeutic effects on CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated diseases.

[0072] In an eighth aspect, the present invention provides a composition comprising the aforementioned antibody or antigen-binding fragment, bispecific antibody, nucleic acid molecule, expression vector, or recombinant cell. As previously described, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and effectively inhibit the proliferation of tumor cells. Therefore, the composition containing the above substances can also effectively bind to human or monkey CD3 protein, or to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and has good preventive and / or therapeutic effects on CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases. The type of composition is not particularly limited and can be a food composition or a pharmaceutical composition.

[0073] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the compositions may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention can also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit CD3 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with upstream or downstream CD3 signal transduction.

[0074] It should be noted that the compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0075] In a ninth aspect, the present invention provides a medicament comprising the aforementioned antibody or antigen-binding fragment, bispecific antibody, nucleic acid molecule, expression vector, recombinant cell, or composition. As previously described, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein. Therefore, a medicament containing an effective amount of the antibody or antigen-binding fragment or the active ingredient of the bispecific antibody, or a series thereof, can also effectively bind to human or monkey CD3 protein, or to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, exhibiting good preventive and / or therapeutic effects on CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases.

[0076] According to embodiments of the present invention, the above-mentioned drug may further include at least one of the following additional technical features:

[0077] According to embodiments of the present invention, the drug may further include a pharmaceutically acceptable carrier.

[0078] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0079] The effective amount of the antibody or antigen-binding fragment or the bispecific antibody described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0080] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0081] The medicament of this invention contains a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched with the route of administration, which may be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal administration. The dosage forms of the medicament of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The medicament is preferably manufactured under sterile conditions. The antibody or antigen-binding fragment or bispecific antibody can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0082] Of course, the anti-CD3 monoclonal or bispecific antibodies mentioned in this article can also be made into kits or other diagnostic reagents as needed.

[0083] In a tenth aspect, the present invention provides a kit containing the aforementioned antibody or its antigen-binding fragment, a bispecific antibody, a nucleic acid molecule, an expression vector, or recombinant cells. As previously described, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein. Therefore, a kit containing the antibody or antigen-binding fragment can effectively perform qualitative or quantitative detection of human or monkey CD3 protein, and a kit containing the bispecific antibody can effectively perform qualitative or quantitative detection of human or monkey CD3 protein and BCMA protein, or human or monkey CD3 protein and B7H6 protein. The kit provided by the present invention can be used, for example, in immunoblotting, immunoprecipitation, and other kits involving the detection of human or monkey CD3 protein using the specific binding properties of antibodies. These kits may contain any one or more of the following: antagonist, anti-CD3 antibody, or pharmaceutical reference material; protein purification column; immunoglobulin affinity purification buffer; cell assay diluent; instructions or literature, etc. Anti-CD3 antibodies can be used in various diagnostic tests, such as detecting the presence of a wide range of diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be tested on the serum or blood of a subject to detect related diseases. These related diseases may include CD3-related diseases, such as cancers, including at least one of multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma. The antibodies or antigen-binding fragments provided herein can also be used for radioimmunoassay and radioimmunotherapy of the aforementioned diseases. The bispecific antibodies described above are also applicable to these applications, and will not be elaborated further here.

[0084] The kit may also include conventional reagents for detecting CD3, or CD3 and BCMA, or CD3 and B7H6, such as coating solutions.

[0085] In an eleventh aspect, the present invention proposes the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant cells, or compositions in the preparation of pharmaceuticals for the prevention and / or treatment of CD3-mediated diseases. As previously stated, the antibodies or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to human or monkey CD3 proteins. Therefore, pharmaceuticals containing an effective amount of the aforementioned antibodies or antigen-binding fragments or a series of substances thereof can also effectively bind to human or monkey CD3 proteins, exhibiting good preventive and / or therapeutic effects against CD3-mediated diseases.

[0086] According to embodiments of the present invention, the use of the above-mentioned prepared drug may further include at least one of the following additional technical features:

[0087] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0088] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0089] In a twelfth aspect, the present invention provides for the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, or compositions in the preparation of a medicament for the prevention and / or treatment of CD3 and BCMA, or CD3 and B7H6-mediated diseases. As previously described, the bispecific antibodies of some specific embodiments of the present invention can bind to human or monkey CD3 and BCMA proteins, or to human or monkey CD3 and B7H6 proteins. Therefore, a medicament containing an effective amount of the active ingredient of the bispecific antibody or a series thereof can also effectively bind to human or monkey CD3 and BCMA proteins, or to human or monkey CD3 and B7H6 proteins, and has good preventive and / or therapeutic effects on CD3 and BCMA, or CD3 and B7H6-mediated diseases.

[0090] According to embodiments of the present invention, the use of the above-mentioned prepared drug may further include at least one of the following additional technical features:

[0091] According to embodiments of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.

[0092] According to embodiments of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma.

[0093] In a thirteenth aspect of the invention, the present invention provides for the use of the aforementioned antibody or antigen-binding fragments, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of a kit for detecting CD3. As previously described, the antibody or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein and block the binding of said CD3 protein to its receptor. Therefore, the antibody or antigen-binding fragments can be used to prepare a kit for detecting CD3 protein, which can effectively perform qualitative or quantitative detection of human or monkey CD3 protein.

[0094] In a fourteenth aspect, the present invention provides for the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of kits for the detection of CD3 and / or BCMA, or CD3 and / or B7H6. As previously described, the bispecific antibodies of some specific embodiments of the present invention can effectively bind to human or monkey CD3 and BCMA proteins, or to human or monkey CD3 and B7H6 proteins. Therefore, the bispecific antibodies can be used to prepare kits for the detection of CD3 and / or BCMA, or CD3 and / or B7H6, which can effectively perform qualitative or quantitative detection of human or monkey CD3 and / or BCMA, or CD3 and / or B7H6.

[0095] In a fifteenth aspect, the present invention provides a method for treating or preventing CD3, or CD3 and BCMA, or CD3 and B7H6-mediated diseases. According to embodiments of the invention, the method comprises administering to a subject at least one of the following: 1) the aforementioned antibody or antigen-binding fragment; 2) the aforementioned bispecific antibody; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; 5) the aforementioned recombinant cells; 6) the aforementioned composition; and 7) the aforementioned drug. As previously stated, the bispecific antibody is capable of effectively binding to human or monkey CD3 and BCMA proteins, or to human or monkey CD3 and B7H6 proteins, and the antibody or antigen-binding fragment is capable of binding to human or monkey CD3 protein, thus effectively treating or preventing CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases, such as autoimmune diseases or cancer. Therefore, the method according to embodiments of the invention is capable of effectively treating or preventing CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases, preferably autoimmune diseases or cancer.

[0096] According to embodiments of the present invention, the above-described method for treating or preventing diseases may further include at least one of the following additional technical features:

[0097] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0098] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0099] According to embodiments of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.

[0100] According to embodiments of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma.

[0101] In a sixteenth aspect of the invention, a method for diagnosing CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases is provided. According to embodiments of the invention, the method includes detecting CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in a test sample using at least one of the following: 1) the aforementioned antibody or antigen-binding fragment; 2) the aforementioned bispecific antibody; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; and 5) the aforementioned recombinant cells, determining the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample based on the detection results of the aforementioned CD3, or CD3 and / or BCMA, or CD3 and / or B7H6. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, and recombinant cell-expressed antibodies or antigen-binding fragments proposed in this application can all effectively bind to human or monkey CD3 protein. The bispecific antibodies, or nucleic acid molecules, expression vectors, and recombinant cell-expressed bispecific antibodies can all effectively bind to CD3, or CD3 and / or BCMA, or CD3 and / or B7H6. Therefore, the method described in this application can effectively detect the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample derived from the test individual, and can effectively diagnose related diseases caused by CD3, or CD3 and / or BCMA, or CD3 and / or B7H6.

[0102] According to embodiments of the present invention, the above-described method for diagnosing diseases may further include at least one of the following additional technical features:

[0103] According to an embodiment of the present invention, the requirement that the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample is not lower than the minimum standard for disease is indicated by the fact that the test sample originates from a patient suffering from a disease related to CD3, or CD3 and / or BCMA, or CD3 and / or B7H6. The minimum standard value can be determined through comparative analysis and verification of the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in test samples from a large number of individuals suffering from diseases related to CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 and a large number of healthy individuals.

[0104] According to embodiments of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0105] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0106] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0107] According to embodiments of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.

[0108] According to embodiments of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma.

[0109] In a seventeenth aspect of the invention, a method for staging CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases is provided. According to embodiments of the invention, the method includes detecting CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in a test sample using at least one of the following: 1) the aforementioned antibody or antigen-binding fragment; 2) the aforementioned bispecific antibody; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; and 5) the aforementioned recombinant cells, and determining the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample based on the detection results of the aforementioned CD3, or CD3 and / or BCMA, or CD3 and / or B7H6. The bispecific antibodies, or nucleic acid molecules, expression vectors, and recombinant cell-expressed bispecific antibodies proposed in this application can effectively bind to human or monkey CD3 and / or BCMA, or CD3 and / or B7H6. The antibody or antigen-binding fragment, or nucleic acid molecules, expression vectors, and recombinant cell-expressed antibody or antigen-binding fragment, can effectively bind to human or monkey CD3 protein. Therefore, the method described in this application can effectively detect the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample derived from the test individual, and assess the stage of the related disease caused by CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 based on the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6.

[0110] According to embodiments of the present invention, the above-described method for staging diseases may further include at least one of the following additional technical features:

[0111] According to embodiments of the present invention, the presence of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample at levels not lower than the standard level for stage IV tumor lesions indicates that the test sample originated from a patient with stage IV tumor lesions; the presence of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample between the standard levels for stage IV and stage III tumor lesions indicates that the test sample originated from a patient with stage III tumor lesions; the presence of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample between the standard levels for stage III and stage II tumor lesions indicates that the test sample originated from a patient with stage II tumor lesions; and the presence of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample between the standard levels for stage I and stage II tumor lesions indicates that the test sample originated from a patient with stage I tumor lesions. Those skilled in the art will understand that the levels of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in tumors at stages I, II, III, and IV vary depending on the type of tumor. Determining the tumor stage can be achieved by comparing the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the sample to be tested with the standard levels of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 for the corresponding tumor stage, or by comparing the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the sample to be tested with the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in samples from individuals or groups with known disease stages. The standard levels for tumor stages I, II, III, and IV can be determined by comparing and verifying the differences in CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 content in test samples from a large number of individuals with angiogenesis-related diseases and a large number of healthy individuals.

[0112] According to embodiments of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0113] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0114] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0115] According to embodiments of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.

[0116] According to embodiments of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma.

[0117] In an eighteenth aspect of the invention, the invention provides a method for assessing the prognosis of diseases mediated by CD3, or CD3 and / or BCMA, or CD3 and / or B7H6. According to embodiments of the invention, the method includes detecting CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in a test sample using at least one of the following: 2) the aforementioned bispecific antibody; 2) the aforementioned antibody or antigen-binding fragment; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; and 5) the aforementioned recombinant cells, determining the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample based on the detection results of the aforementioned CD3, or CD3 and / or BCMA, or CD3 and / or B7H6. As mentioned earlier, the levels of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6, have a significant impact on cancer. After treatment, monitoring the levels of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in tissues or excreta, such as peripheral blood and urine, can effectively assess the prognosis of this type of disease. For example, comparing the levels of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in subjects before and after treatment, or comparing the levels of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in subjects after treatment with the levels in normal or diseased individuals, etc. In this application, the bispecific antibody, or the bispecific antibody expressed by a nucleic acid molecule, expression vector, or recombinant cell can effectively bind to CD3 and / or BCMA, or CD3 and / or B7H6. The antibody or antigen-binding fragment, or the antibody or antigen-binding fragment expressed by a nucleic acid molecule, expression vector, or recombinant cell can effectively bind to human or monkey CD3. Therefore, the method described in this application can effectively detect the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample derived from the test individual, and assess the prognosis of diseases related to CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 based on the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6.

[0118] According to embodiments of the present invention, the above-described method for assessing disease prognosis may further include at least one of the following additional technical features:

[0119] According to an embodiment of the present invention, the test sample is derived from a patient with CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated related diseases before or after treatment.

[0120] According to embodiments of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0121] According to embodiments of the present invention, the prognostic effect of CD3, CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases is determined based on the content of CD3, CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases in test samples from patients who have such diseases before or after treatment.

[0122] According to embodiments of the present invention, a decrease in the levels of CD3, CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample of a patient with CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated related diseases after treatment is an indicator of a good prognosis for the patient.

[0123] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0124] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0125] According to embodiments of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.

[0126] According to embodiments of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma.

[0127] In a nineteenth aspect, the present invention proposes the use of the aforementioned bispecific antibodies, antibody or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions, or drugs in the treatment or prevention of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases. As previously stated, the bispecific antibodies exhibit high binding activity to both CD3 and / or BCMA, or CD3 and / or B7H6, and the antibody or antigen-binding fragment can effectively bind to human or monkey CD3, thereby effectively treating or preventing CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases.

[0128] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0129] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0130] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0131] According to embodiments of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.

[0132] According to embodiments of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma.

[0133] In a twentieth aspect of the invention, the invention proposes the use of the aforementioned bispecific antibodies, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors or recombinant cells in the diagnosis of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases, in staging of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases, or in assessing the prognosis of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6-mediated diseases. As previously stated, the bispecific antibodies, or nucleic acid molecules, expression vectors, or recombinant cell-expressed bispecific antibodies proposed in this application can effectively bind to CD3 and / or BCMA, or CD3 and / or B7H6. The antibody or antigen-binding fragment, or nucleic acid molecules, expression vectors, or recombinant cell-expressed antibody or antigen-binding fragment, can effectively bind to human or monkey CD3. Therefore, the method described in this application can effectively detect the content of CD3, or CD3 and / or BCMA, or CD3 and / or B7H6 in the test sample derived from the test individual, and can effectively diagnose, stage, and assess the prognosis of diseases mediated by CD3, or CD3 and / or BCMA, or CD3 and / or B7H6.

[0134] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0135] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0136] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0137] According to embodiments of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.

[0138] According to embodiments of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma, and glioma.

[0139] The term "subject" or "individual" in this invention generally refers to mammals, such as primates and / or rodents, particularly humans, monkeys, or rats.

[0140] The beneficial effects of this invention are:

[0141] 1) The mouse-derived anti-CD3 monoclonal antibody obtained in this invention has higher CD3 protein binding activity than existing CD3 monoclonal antibodies, and the CD3 protein includes human CD3 protein and monkey CD3 protein.

[0142] 2) The humanized antibody obtained by humanizing the mouse anti-CD3 monoclonal antibody also has higher CD3 protein binding activity than the existing CD3 monoclonal antibody. The CD3 protein includes human CD3 protein and monkey CD3 protein. The humanized antibody has lower immunogenicity, higher safety and longer efficacy.

[0143] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0144] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0145] Figure 1A The image shows the ELISA results of different concentrations of mouse CD3 antibody (m47B5) binding to human CD3EG protein according to an embodiment of the present invention.

[0146] Figure 1B The image shows the ELISA detection results of different concentrations of mouse CD3 antibody binding to human CD3ED protein according to an embodiment of the present invention.

[0147] Figure 1C Figure 1 shows the ELISA detection results of different concentrations of mouse CD3 antibody binding to monkey CD3EG protein according to an embodiment of the present invention.

[0148] Figure 1D Figure 1 shows the ELISA detection results of different concentrations of mouse CD3 antibody binding to monkey CD3ED protein according to an embodiment of the present invention.

[0149] Figure 2A This is a graph showing the detection results of different concentrations of human-mouse chimeric CD3 antibody ch47B5 binding to human PBMC cells according to an embodiment of the present invention;

[0150] Figure 2BThis is a graph showing the detection results of different concentrations of human-mouse chimeric CD3 antibody ch47B5 binding to monkey PBMC cells according to an embodiment of the present invention;

[0151] Figure 3A The graph shows the detection results of different concentrations of humanized CD3 antibody binding to human CD3 protein according to an embodiment of the present invention.

[0152] Figure 3B The graph shows the detection results of different concentrations of humanized CD3 antibody binding to monkey CD3 protein according to an embodiment of the present invention.

[0153] Figure 4A The graph shows the detection results of different concentrations of CD3×BCMA bispecific antibody binding to human CD3 protein according to an embodiment of the present invention.

[0154] Figure 4B The graph shows the detection results of different concentrations of CD3×BCMA bispecific antibody binding to human BCMA protein according to an embodiment of the present invention.

[0155] Figure 5 The graph shows the killing effect of different concentrations of CD3×BCMA bispecific antibody on NCI-H929 cells according to an embodiment of the present invention.

[0156] Figure 6A The graph shows the detection results of different concentrations of CD3×B7H6 bispecific antibody binding to human CD3 protein according to an embodiment of the present invention.

[0157] Figure 6B The graph shows the detection results of different concentrations of CD3×BCMA bispecific antibody binding to human B7H6 protein according to embodiments of the present invention; and

[0158] Figure 7 The graph shows the killing effect of different concentrations of CD3×BCMA bispecific antibody on HCT-15 cells according to an embodiment of the present invention. Detailed Implementation

[0159] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0160] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0161] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0162] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0163] The antibody or antigen-binding fragments described in this invention are typically prepared by biosynthetic methods. Based on the nucleotide sequence described in this invention, those skilled in the art can readily obtain the encoding nucleic acid of this invention using various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc., and specific methods can be found in J. Sambrook, *Molecular Cloning: A Laboratory Manual*. As one embodiment of this invention, the encoding nucleic acid sequence of this invention can be constructed by synthesizing nucleotide sequences in segments and then performing overlap extension PCR. The antibody or antigen fragments are numbered and defined using the Kabat numbering system.

[0164] In this article, "monoclonal antibody" refers to an antibody with a single antigen-binding site.

[0165] In this article, "dual antibody" refers to an antibody with two different antigen-binding sites.

[0166] In this document, the term "mutant" or "variant" may refer to a molecule obtained by mutating one or more nucleotides or amino acids into any naturally occurring or engineered molecule.

[0167] The terms "complementarity-determining region" or "CDR" or "CDR sequence" refer to the amino acid sequence in an antibody responsible for antigen binding. For example, it typically includes amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable region, and amino acid residues near 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)).

[0168] In this paper, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, see, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)). And the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, MountainView, California.

[0169] Without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody or its functional fragments. These are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are shown in N-terminus to C-terminus arrangement.

[0170] As previously described, the monoclonal antibodies of the present invention may be full-length antibodies or may contain only their functional fragments, or may be modified to affect function. The present invention includes anti-CD3 antibodies having modified glycosylation patterns. In some applications, modification to remove undesirable glycosylation sites may be useful, or to antibodies lacking a fucose moiety on the oligosaccharide chain to, for example, enhance antibody-dependent cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0171] In this document, the “full-length antibody” is a tetrapeptide chain structure consisting of two identical light chains and two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0172] As used herein, the term "functional fragment" specifically refers to antibody fragments such as CDR transplantation antibodies, Fab, F(ab')2, Fv, or scFv, nanobodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated, PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol), which have CD3 binding activity. Preferably, the functional fragment will consist of or contain a portion of the heavy chain variable region or light chain variable region of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, preferably at least 1 / 100 of the affinity for CD3, and more preferably at least 1 / 10. This functional fragment will contain at least 3 amino acids, preferably 5, 10, 15, 25, 50, and 100 consecutive amino acids from the antibody sequence from which it is derived.

[0173] In this invention, unless otherwise stated, the term "antigen-binding fragment" as used generally refers to an antigen-binding antibody fragment, which may include a portion of a complete antibody, typically an antigen-binding region or a variable region, such as, for example, CDR transplanted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.

[0174] In this paper, the term "CDR-transplanted antibody" refers to the transplantation of the CDR of a monoclonal antibody from one species into the variable region of an antibody from another species. For example, the CDR of a murine monoclonal antibody can be transplanted into the variable region of a human antibody to replace the human antibody's CDR, thereby giving the human antibody the antigen-binding specificity of the murine monoclonal antibody while reducing its heterologous nature.

[0175] In this article, the term "Fab antibody" or "Fab" generally refers to an antibody containing only Fab molecules, which consist of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0176] In this article, the term “F(ab')2 antibody” or “F(ab')2” has two antigen-binding F(ab) parts linked together by disulfide bonds.

[0177] In this paper, the term “nanobody” (single-domain antibody or VHH antibody), which was originally described as an antigen-binding immunoglobulin (variable) domain of “heavy chain antibody” (i.e., “antibody lacking light chain”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturallyoccurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)), contains only the heavy chain variable region (VH) and the conventional CH2 and CH3 regions, which bind specifically to the antigen through the heavy chain variable region.

[0178] In this article, the term "Fv antibody" generally refers to an antibody composed only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0179] In this paper, the term "single-chain antibody" or "scFv" refers to a fragment consisting of the variable regions of the antibody heavy chain and light chain linked by a short peptide.

[0180] In this paper, the "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, facilitating heavy chain interlocking and the formation of a heterodimer. For example, in this application, this is achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole"; and T366W, S354C mutations in the other chain, i.e., "knob"). The amino acid numbers here are based on the Kabat numbering system. For example, "T366S" means that the T amino acid at position 366 according to the Kabat numbering system is replaced by an S amino acid.

[0181] The sequence descriptions involved in this invention are detailed in Table 1.

[0182] Table 1:

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] The present invention will be described in detail below through examples. In the examples or test cases, experimental methods without specific conditions are performed under conventional conditions.

[0192] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0193] Example 1: Preparation of anti-human CD3 hybridoma monoclonal antibody

[0194] In this invention, anti-human CD3 monoclonal antibodies are generated by immunizing mice. Female, 6-week-old C57BL / 6 mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were used in the experiment. The immunizing antigens were human CD3E&D, human CD3E&G, monkey CD3E&D, and monkey CD3E&G extracellular protein (ACRO). For the initial immunization, 100 μg of the antigen was emulsified with Freund's complete adjuvant (Sigma, F5881) and administered intraperitoneally to each mouse. Subsequent immunizations were performed using the Ribi adjuvant system (Sigma, S6322) mixed with the antigen and administered intraperitoneally. Immunizations were repeated every 2 weeks after the initial immunization, for a total of 3 immunizations. Antibody titers were detected by ELISA using serum from the immunized mice, following standard procedures in the art.

[0195] Based on antibody titer results, mice with high serum antibody titers were selected for spleen cell fusion. 72 hours prior to fusion, the selected mice were immunized by sprinting with a mixture of Ribi adjuvant system and the aforementioned antigen via intraperitoneal injection. Using an optimized PEG-mediated fusion procedure, spleen lymphocytes were fused with myeloma Sp2 / 0 cells (ATCC, CRL-8287) to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HAT, and 1×OPI) and aliquoted into 96-well cell culture plates, incubated at 37°C and 5% CO2. On day 5 post-fusion, 50 μL of HAT complete medium was added per well. From day 7 to 8 post-fusion, the medium was completely replaced according to cell growth density, using 200 μL of HT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HT, and 1×OPI) per well.

[0196] On days 10-11 post-fusion, flow cytometry was performed to detect cell growth density. Positive wells were replaced with new medium, and the cells were promptly expanded to 24-well plates according to density. Cell lines transferred to 24-well plates were retested and then preserved for the first subcloning. Cells showing positive results in the first subcloning were preserved and subjected to a second subcloning. Cells showing positive results in the second subcloning were then preserved and subjected to protein expression. Subcloning and preservation were performed using standard techniques in the field. Antibodies were further prepared using serum-free cell culture, and the mouse antibodies were purified by protein G affinity chromatography for subsequent functional activity assays.

[0197] Example 2: Mouse CD3 Antibody ELISA Binding Assay

[0198] The ELISA assay was used to detect the binding characteristics of the murine CD3 antibody m47B5 obtained in Example 1. Human CD3ED protein (ACRO biosystems, CDD-H52W1), human CD3EG protein (ACRO biosystems, CDG-H52W5), monkey CD3ED protein (ACRO biosystems, CDD-C52W4), and monkey CD3EG protein (ACRO biosystems, CDG-H52W6) were diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 h. Wash three times with PBST. Add mouse antibody m47B5 to each well and incubate at room temperature for 1 hour. Wash three times with PBST. Add 100 μL of HRP-goat anti-mouse IgG secondary antibody (boster, catalog number BA1051) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2-5 minutes. Terminate the reaction with 2M sulfuric acid in each well. Finally, read the OD450 value using a microplate reader. Figure 1A This indicates that the murine m47B5 antibody of the present invention can bind to human CD3EG. Figure 1B This indicates that the murine m47B5 antibody of the present invention can also bind to human CD3ED. Figure 1C This indicates that the murine m47B5 antibody of the present invention can bind to monkey CD3EG. Figure 1D This indicates that the murine m47B5 antibody of the present invention can bind to monkey CD3ED.

[0199] Example 3: Hybridoma cell sequencing

[0200] The total number of m47B5 antibody candidate hybridoma cells selected through screening steps 1 and 2 was cultured to 10. 6Cells were collected by centrifugation at 800 rpm for 10 minutes, and total RNA was extracted using the Trizol kit (Invitrogen). Using the total RNA as a template, a cDNA library was synthesized by reverse transcription (Invitrogen). The cDNA was then used as a template for PCR amplification of the nucleic acid sequence of the variable region of the hybridoma cell corresponding to the m47B5 antibody. The primer sequences used in the PCR amplification reaction were complementary to the first frame region or signal peptide region and constant region of the antibody variable region (for specific sequences, refer to Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). PCR amplification was performed in a 50 μL reaction system, with the following components added: 2 μL cDNA, 5 μL 10× PCR buffer, 2 μL (5 μM) upstream and downstream primers, 2 μL dNTPs, 1 μL Taq enzyme (Takara, Ex Taq), and 38 μL H2O. The mixture was pre-denatured at 95℃ for 5 min, followed by temperature cycling for PCR amplification. The reaction conditions were: denaturation at 94℃ for 30 s, annealing at 58℃ for 45 s, extension at 72℃ for 50 s, for a total of 32 cycles, followed by a final extension at 72℃ for 7 min. Sequencing of the amplified products yielded the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) sequences of the mouse monoclonal antibody m47B5.

[0201] Example 4: Chimeric CD3 Antibody Flow Cytometry Combination Experiment

[0202] Human-mouse chimeric antibody (ch47B5) was constructed using the above-mentioned mouse monoclonal antibody m47B5, wherein the heavy chain constant region of the chimeric antibody uses the heavy chain constant region of human IgG1.

[0203] Human peripheral blood mononuclear cells (PBMCs) or monkey PBMCs were diluted with PBS to a concentration of 2 × 10⁻⁶. 6Cells were added in 100 μL / tube volume to 1.5 mL EP tubes, followed by 10 μL / tube of goat serum. The tubes were then blocked at 4°C for 30 min. A gradient concentration (5-fold dilution, with a final concentration up to 10 μg / mL) of the aforementioned human-mouse chimeric antibody ch47B5 was added, and the tubes were incubated at 4°C for 30 min. 1 mL of PBS was added to the EP tubes, and the tubes were centrifuged at 3500 rpm for 5 min at 4°C. The supernatant was discarded, and the cells were washed once more with PBS. After centrifugation, the supernatant was discarded again, and the cells were resuspended in 100 μL / tube of PBS. 0.1 μL / tube of Alexa-647-labeled rat anti-human antibody secondary antibody (Biolegend) was added, and the tubes were incubated at 4°C in the dark for 30 min. After incubation, the cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / tube of PBS, and then analyzed by flow cytometry, using BT062 antibody as a control antibody. Figure 2A and Figure 2B The results showed that the chimeric 47B5 antibody could bind to human PBMCs and monkey PBMCs, indicating that the chimeric CD3 antibody could bind to the CD3 protein on the cell surface.

[0204] Example 5: Humanization of Anti-human CD3 Monoclonal Antibody

[0205] 5.1 Selection and Reversion Mutations of the Variable Region Framework of CD3 Monoclonal Antibodies

[0206] Based on the chimeric antibody obtained in Example 4, by comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, heavy chain or light chain variable region germline genes with high homology to the murine monoclonal antibody m47B5 were selected as templates. The CDRs of the murine monoclonal antibody were then transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were determined and annotated using the Kabat numbering system.

[0207] To maintain the conformation of the CDR region, residues at the VL and VH binding interfaces, residues close to the CDR and embedded inside the protein, and residues that directly interact with the CDR are reverse-mutated to ensure that the activity of the variable region is not affected.

[0208] 5.2 Expression of CD3 humanized antibody

[0209] After selecting and reverting mutations to the variable region framework of the aforementioned CD3 monoclonal antibody, the heavy chain variable region sequence of the obtained humanized CD3 antibody h47B5 is shown in SEQ ID NO:27, and the light chain variable region sequence is shown in SEQ ID NO:28. The nucleotide sequences encoding the humanized antibody heavy chain (SEQ ID NO:43) and the nucleotide sequences encoding the humanized antibody light chain (SEQ ID NO:44) were then recombined into the pTT5 plasmid using molecular biology techniques.

[0210] Antibody was prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with the pTT5 vector carrying the humanized anti-human CD3 antibody h47B5 light and heavy chains. One day before transfection, the ExpiCHO-S cell density was adjusted to (3–4) × 10⁻⁶ cells / cells. 6 Incubate overnight at 37°C, 8% CO2, with shaking at 120 rpm. On the day of transfection, cells grew to 7 × 10⁹ / mL. 6 ~1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / mL using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6 The pTT5 plasmid carrying the humanized anti-human CD3 antibody h47B5 light and heavy chains was transfected into ExpiCHO-S cells at a 2:1 molar ratio using ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129). Cells were incubated at 37°C, 8% CO2, and 120 rpm with shaking. 18-22 h post-transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed and immediately added to the transfected cells. Cells were then incubated at 32°C, 5% CO2, and 120 rpm with shaking. On day 5 post-transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed, and the cells were cultured further. Cell count and viability were observed daily. Cells were harvested by centrifugation when viability dropped below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.22 μm filter membrane. Antibodies with Fc domains were captured from the expression supernatant using a Mabselect Prism A affinity chromatography column (GE, catalog number 17549854). After equilibrating the column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE and its purity was above 95%. The humanized m47B5 antibody (heavy chain as shown in SEQ ID NO:39, light chain as shown in SEQ ID NO:40) was finally obtained.

[0211] 5.3 Affinity verification of humanized antibody h47B5

[0212] The affinity of the humanized h47B5 antibody to the human CD3 antigen was tested using BIACORE, and the results are shown in Table 2. The obtained humanized antibody has good affinity to the CD3 antigen.

[0213] Table 2

[0214] Antibody antigen ka(1 / Ms) kd(1 / s) KD(M) h47B5 Human CD3 3.36E+05 1.84E-03 5.48E-09

[0215] Example 6: ELISA binding assay of humanized antibody h47B5

[0216] The ELISA assay was used to detect the binding characteristics of the humanized antibody h47B5, with wild-type human IgG1 antibody as a control. Human CD3 antigen (ACRO biosystems, CDD-H52W1) and monkey CD3 antigen (ACRO biosystems, CDD-C52W4) were diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 h. The plate was then washed three more times with PBST. A gradient concentration of humanized anti-human CD3 antibody h47B5 or human IgG1 antibody was added to each well, and the plate was incubated at room temperature for 1 h. Wash three times with PBST. Add 100 μL of HRP-goat anti-human IgG secondary antibody diluted with blocking buffer (Jackson ImmunoResearch, 109-036-170) to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2–5 minutes. Stop the reaction with 2M sulfuric acid in each well. Finally, read the OD450 value using a microplate reader. Figure 3A and Figure 3B This indicates that the humanized h47B5 antibody of the present invention can bind to the CD3 antigen in humans and monkeys.

[0217] Example 7: Preparation of bispecific antibodies against CD3×BCMA and detection of their binding activity and cell-killing ability.

[0218] Based on the experimental results of Examples 1-6, the inventors designed a bispecific antibody CD3×BCMA and tested its binding activity and cell-killing ability.

[0219] 7.1 Design and preparation of bispecific antibodies against CD3×BCMA

[0220] This antibody contains two monovalent units. One monovalent unit is an anti-CD3 scFv-Fc form, whose heavy and light chain variable region amino acid sequences are derived from the humanized antibody h47B5 obtained in Example 6 of this invention. The other monovalent unit is an anti-BCMA scFv-Fc form (sequence from US009273141B2). This bispecific antibody is named CD3×BCMA. This bispecific antibody contains two polypeptide chains: a heavy chain containing the CD3 single-chain antibody scFv (SEQ ID NO: 51) and a heavy chain containing the BCMA single-chain antibody scFv (SEQ ID NO: 52). The constant regions of the heavy chains of both chains are derived from the human antibody IgG1. Due to the molecule's unique asymmetric structure, different amino acid mutations are introduced into the constant regions of the two chains to reduce the formation of homodimers, forming a knock-in-hole structure. Simultaneously, to prevent cross-linking activation caused by the Fcγ receptor, a (L234A / L235A) mutation is also introduced into the constant region of the heavy chain.

[0221] The bispecific antibody was prepared according to the method described in Example 6.2, and the bispecific antibody was prepared by combining the plasmid ratio (1:1 or other ratio) and undergoing one-step affinity purification. The relevant sequence of the bispecific antibody CD3×BCMA is shown in Table 3.

[0222] Table 3

[0223] project CD3 Rechain BCMA heavy chain amino acid sequence SEQ ID NO: 51 SEQ ID NO: 52 nucleotide sequence SEQ ID NO: 58 SEQ ID NO: 59

[0224] 7.2 Assay for the binding activity of CD3×BCMA bispecific antibody to antigen (ELISA)

[0225] Dilute human BCMA (ACRO biosystems, BCA-H522y) or human CD3 antigen (ACRO biosystems, CDD-H52W1) to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), add 100 μL to each well of an ELISA plate, and incubate overnight at 4°C. Then wash three times with PBST (0.05% Tween 20-PBS, pH 7.2). Add 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) to the plate and incubate at room temperature for 2 h. Wash three more times with PBST. Add the corresponding bispecific antibody to each well and incubate at room temperature for 1 h. Wash the precipitate three more times with PBST. Add 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2–5 minutes. Terminate the reaction with 2M sulfuric acid in each well. Finally, read the OD450 value using a microplate reader. Figure 4A and 4B This invention demonstrates that CD3×BCMA can bind to both CD3 and BCMA antigens.

[0226] 7.3 Cell killing experiment

[0227] Multiple myeloma NCI-H929 cells were labeled with CellTrace Violet (thermo, C34557) and the cell density was adjusted to 1×10⁻⁶. 5 / mL. PBMCs were used to adjust cell density to 1×10⁹ / mL. 6 PBMCs and labeled NCI-H929 cells were mixed at a 1:1 ratio and added to 96-well round-bottom plates. Then, gradient concentrations of the aforementioned bispecific antibody or human IgG antibody (control group) were added and mixed thoroughly. After incubation at 37°C for 24 h, the mixture was transferred to flow cytometry tubes through a 200-mesh nylon mesh. 7AAD was added, and the cell killing efficiency was measured after 10 min. The ratio of CTV+7AAD+ cells was used to characterize the killing efficiency. Figure 5 The results showed that as the concentration of CD3×BCMA bispecific antibody increased, the killing efficiency of PBMC against NCI-H929 multiple myeloma cells gradually increased.

[0228] Example 8: Preparation of bispecific antibodies against CD3×B7H6 and detection of their binding activity and cell-killing ability.

[0229] Based on the experimental results of Examples 1-6, the inventors designed a bispecific antibody CD3×B7H6 and tested its binding activity and cell-killing ability.

[0230] 8.1 Design and preparation of bispecific antibodies against CD3×B7H6

[0231] This antibody contains two monovalent units. One monovalent unit is in the form of anti-CD3 scFv-Fc, where the light and heavy chain variable regions of the CD3 scFv are derived from the humanized sequence of Example 6 of this invention. The other monovalent unit is in the form of anti-B7H6 scFv-Fc (the B7H6 scFv sequence is derived from CN114395045A). This bispecific antibody is named CD3×B7H6. This bispecific antibody contains two polypeptide chains: a heavy chain containing the CD3 single-chain antibody scFv (SEQ ID NO: 51) and a heavy chain containing the B7H6 single-chain antibody scFv (SEQ ID NO: 53). Due to the molecule's unique asymmetric structure, different amino acid mutations are introduced into the constant regions of the two chains to reduce the formation of homodimers, forming a knock-in-hole structure. Simultaneously, to prevent cross-linking activation caused by the Fcγ receptor, a (L234A / L235A) mutation is also introduced into the constant region of the heavy chain.

[0232] The preparation method of the CD3×B7H6 bispecific antibody is as described in Example 6.2. The CD3×B7H6 bispecific antibody is prepared by combining the plasmid ratio (1:1 or other ratio) and undergoing one-step affinity purification. The relevant sequence of the CD3×B7H6 bispecific antibody is shown in Table 4.

[0233] Table 4

[0234] project CD3 Rechain B7H6 heavy chain amino acid sequence SEQ ID NO: 51 SEQ ID NO: 53 nucleotide sequence SEQ ID NO: 58 SEQ ID NO: 60

[0235] 8.2 Assay for the binding activity of bispecific antibodies to antigens (ELISA)

[0236] Human B7H6 (ACRO biosystems, B76-H52H8) or human CD3 antigen (ACRO biosystems, CDD-H52W) was diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6). 100 μL was added to each well of an ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 hours. The plate was then washed three more times with PBST. The corresponding bispecific antibody was added to each well and incubated at room temperature for 1 hour. The plate was then washed three more times with PBST. Add 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. After incubation, wash the precipitate three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2–5 minutes. Finally, stop the reaction with 2M sulfuric acid in each well and read the OD450 value using a microplate reader. Figure 6A and 6B The results show that the bispecific antibody CD3×B7H6 of the present invention can effectively bind to both CD3 and B7H6 antigens.

[0237] 8.3 Cell killing experiment

[0238] After digestion, HCT-15 colorectal cancer cells were counted, and the cell density was adjusted to 2 × 10⁻⁶. 5 / mL. Turn on the RTCA instrument (Agilent), select instrument type DP, select experimental mode, and fill in cell and drug information. Enter the schedule to set the experimental steps. Add 50μL of fresh culture medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin and streptomycin) to the plate and place it in the instrument. Close the instrument and click Start. After completion, remove the plate, add 100μL of cell suspension, incubate at room temperature for 15-30 minutes to prevent edge effects, place it in the instrument, and click Start. After growth reaches the logarithmic growth phase, pause and add 50μL of LPBMC (4×10⁻⁶). 6 Add the bispecific antibody CD3×B7H6 at a gradient concentration ( / mL), click start, and perform analysis after a period of time. Figure 7 The results showed that as the concentration of CD3×B7H6 bispecific antibody increased, the killing efficiency of PBMCs against colorectal cancer HCT-15 cells gradually increased, indicating that CD3×B7H6 can effectively promote the specific killing of B7H6 cells by PBMCs. + Tumor cells.

[0239] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0240] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-CD3 antibody or its antigen-binding fragment, characterized in that, include: The heavy chain variable region CDR1 shown in SEQ ID NO: 1, the heavy chain variable region CDR2 shown in SEQ ID NO: 2, the heavy chain variable region CDR3 shown in SEQ ID NO: 3, the light chain variable region CDR1 shown in SEQ ID NO: 4, the light chain variable region CDR2 shown in SEQ ID NO: 5, and the light chain variable region CDR3 shown in SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, It also includes a heavy chain FR region and a light chain FR region, wherein the heavy chain FR region and the light chain FR region are derived from at least one of primate-derived antibodies and mouse-derived antibodies.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, It also includes a heavy chain FR region and a light chain FR region, which are derived from human antibodies.

4. The antibody or its antigen-binding fragment according to claim 2 or 3, characterized in that, include: The heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO: 7-10 respectively; and the light chain frame regions LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO: 11-14 respectively; or The heavy chain frame regions HFR1, HFR2, HFR3 and HFR4 as shown in SEQ ID NO: 15~18 respectively; and the light chain frame regions LFR1, LFR2, LFR3 and LFR4 as shown in SEQ ID NO: 19~22 respectively.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, include: 1) The heavy chain variable region as shown in SEQ ID NO: 23, and the light chain variable region as shown in SEQ ID NO: 24; or 2) The heavy chain variable region as shown in SEQ ID NO:27, and the light chain variable region as shown in SEQ ID NO:

28.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a primate-derived antibody or a murine antibody.

7. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a human antibody.

8. The antibody or its antigen-binding fragment according to claim 6 or 7, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse IgG antibodies or human IgG antibodies.

9. The antibody or its antigen-binding fragment according to claim 6 or 7, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse IgG1 antibody or human IgG1 antibody.

10. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody has a heavy chain constant region as shown in SEQ ID NO:29 or 33 and / or a light chain constant region as shown in SEQ ID NO:30 or 34.

11. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has a heavy chain of amino acid sequences shown in any one of SEQ ID NO:35, 37 and 39 and a light chain of amino acid sequences shown in any one of SEQ ID NO:36, 38 and 40.

12. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:35 and a light chain of the amino acid sequence shown in SEQ ID NO:

36. The antibody or its antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:37 and a light chain of the amino acid sequence shown in SEQ ID NO:38; or The antibody or its antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:39 and a light chain of the amino acid sequence shown in SEQ ID NO:

40.

13. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes monoclonal antibodies.

14. The antibody or its antigen-binding fragment according to claim 13, characterized in that, The monoclonal antibody includes at least one of full-length antibody, Fv, single-chain antibody, and Fab.

15. A dual antibody, characterized in that, include: A first binding region, wherein the first binding region comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 14; and The second binding region has BCMA or B7H6 binding activity; Wherein, the antibody or its antigen-binding fragment is an anti-CD3 single-chain antibody, the anti-CD3 single-chain antibody includes an anti-CD3 antibody light chain variable region and an anti-CD3 antibody heavy chain variable region, the anti-CD3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or 27, and the anti-CD3 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 24 or 28; the first antigen-binding region further includes a first Fc peptide segment, the N-terminus of the first Fc peptide segment being linked to the C-terminus of the antibody or antigen-binding fragment; The second binding region includes an anti-BCMA single-chain antibody or an anti-B7H6 single-chain antibody. The anti-BCMA single-chain antibody includes a light chain variable region and a heavy chain variable region. The light chain variable region of the anti-BCMA antibody has the amino acid sequence shown in SEQ ID NO: 42, and the heavy chain variable region of the anti-BCMA antibody has the amino acid sequence shown in SEQ ID NO:

61. The anti-B7H6 single-chain antibody includes a light chain variable region and a heavy chain variable region. The heavy chain variable region of the anti-B7H6 antibody has the amino acid sequence shown in SEQ ID NO: 63, and the light chain variable region of the anti-B7H6 antibody has the amino acid sequence shown in SEQ ID NO:

62. The second antigen binding region further includes a second Fc peptide segment, the N-terminus of which is linked to the C-terminus of the anti-BCMA single-chain antibody or the anti-B7H6 single-chain antibody.

16. The dual antibody according to claim 15, characterized in that, The anti-CD3 single-chain antibody further includes a linker peptide 1, wherein the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the heavy chain of the anti-CD3 antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the light chain of the anti-CD3 antibody; or the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the light chain of the anti-CD3 antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the heavy chain of the anti-CD3 antibody.

17. The dual antibody according to claim 15, characterized in that, The anti-BCMA single-chain antibody further includes a linker peptide 2, wherein the N-terminus of the linker peptide 2 is connected to the C-terminus of the variable region of the heavy chain of the anti-BCMA antibody, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the variable region of the light chain of the anti-BCMA antibody; or the N-terminus of the linker peptide 2 is connected to the C-terminus of the variable region of the light chain of the anti-BCMA antibody, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the variable region of the heavy chain of the anti-BCMA antibody.

18. The dual antibody according to claim 15, characterized in that, The anti-B7H6 single-chain antibody further includes a linker peptide 3, wherein the N-terminus of the linker peptide 3 is connected to the C-terminus of the variable region of the heavy chain of the anti-B7H6 antibody, and the C-terminus of the linker peptide 3 is connected to the N-terminus of the variable region of the light chain of the anti-B7H6 antibody; or the N-terminus of the linker peptide 3 is connected to the C-terminus of the variable region of the light chain of the anti-B7H6 antibody, and the C-terminus of the linker peptide 3 is connected to the N-terminus of the variable region of the heavy chain of the anti-B7H6 antibody.

19. The dual antibody according to claim 16, characterized in that, The linker peptide 1 has an amino acid sequence (GGS)n, where n is an integer greater than or equal to 1.

20. The dual antibody according to claim 16, characterized in that, The linker peptide 1 has an amino acid sequence (GGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

21. The dual antibody according to claim 17 or 18, characterized in that, The linker peptide 2 and linker peptide 3 have an amino acid sequence (GGGGS)n, where n is an integer greater than or equal to 1.

22. The dual antibody according to claim 17 or 18, characterized in that, The linker peptides 2 and 3 have an amino acid sequence (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

23. The dual antibody according to claim 16, characterized in that, The linker peptide 1 has the amino acid sequence shown in SEQ ID NO:

48.

24. The dual antibody according to claim 17 or 18, characterized in that, The linker peptides 2 and 3 have the amino acid sequence shown in SEQ ID NO:

41.

25. The dual antibody according to claim 15, characterized in that, The anti-CD3 single-chain antibody has the amino acid sequence shown in SEQ ID NO:

45.

26. The dual antibody according to claim 15, characterized in that, The anti-BCMA single-chain antibody has the amino acid sequence shown in SEQ ID NO:

46.

27. The dual antibody according to claim 15, characterized in that, The anti-B7H6 single-chain antibody has the amino acid sequence shown in SEQ ID NO:

47.

28. The dual antibody according to claim 15, characterized in that, The first Fc peptide has the amino acid sequence shown in SEQ ID NO:

49.

29. The dual antibody according to claim 15, characterized in that, The second Fc peptide has the amino acid sequence shown in SEQ ID NO:

50.

30. The dual antibody according to claim 15, characterized in that, The first Fc peptide and the second Fc peptide are connected by a knock-into-hole structure.

31. The dual antibody according to claim 15, characterized in that, The first antigen-binding region has the amino acid sequence shown in SEQ ID NO:51, and the second antigen-binding region has the amino acid sequence shown in SEQ ID NO:52 or 53.

32. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment as described in any one of claims 1 to 14.

33. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific antibody as described in any one of claims 15 to 31.

34. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 32.

35. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 33.

36. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule of claim 32, the expression vector of claim 34, or an antibody or antigen-binding fragment of any one of claims 1 to 14, wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a mammalian cell.

37. The recombinant cell according to claim 36, characterized in that, The recombinant cells are obtained by introducing the expression vector of claim 34 into host cells.

38. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule of claim 33, the expression vector of claim 35, or the bispecific antibody of any one of claims 15 to 31, wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a mammalian cell.

39. The recombinant cell according to claim 38, characterized in that, The recombinant cells are obtained by introducing the expression vector of claim 35 into host cells.

40. A composition, characterized in that, It includes the antibody or its antigen-binding fragment as described in any one of claims 1 to 14, the nucleic acid molecule as described in claim 32, the expression vector as described in claim 34, or the recombinant cell as described in any one of claims 36 to 37.

41. A composition, characterized in that, It includes the bispecific antibody as described in any one of claims 15 to 31, the nucleic acid molecule as described in claim 33, the expression vector as described in claim 35, or the recombinant cell as described in any one of claims 38 to 39.

42. A drug, characterized in that, The invention includes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 14, the bispecific antibody as described in any one of claims 15 to 31, the nucleic acid molecule as described in any one of claims 32 to 33, the expression vector as described in any one of claims 34 to 35, the recombinant cell as described in any one of claims 36 to 39, or the composition as described in any one of claims 40 to 41.

43. A reagent kit, characterized in that, The kit contains the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 14, the bispecific antibody as described in any one of claims 15 to 31, the nucleic acid molecule as described in any one of claims 32 to 33, the expression vector as described in any one of claims 34 to 35, or the recombinant cell as described in any one of claims 36 to 39.

44. Use of the bispecific antibody according to any one of claims 15-31, the nucleic acid molecule according to claim 33, the expression vector according to claim 35, the recombinant cell according to any one of claims 38-39, or the composition according to claim 41 in the preparation of a medicament, wherein the medicament is used to treat CD3 and BCMA, or CD3 and B7H6-mediated diseases, wherein the CD3 and BCMA-mediated diseases are multiple myeloma, and the CD3 and B7H6-mediated diseases are colorectal cancer.

45. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 32, the expression vector according to claim 34, or the recombinant cell according to any one of claims 36 to 37 in the preparation of a kit for detecting CD3.

46. ​​Use of the bispecific antibody according to any one of claims 15 to 31, the nucleic acid molecule according to claim 33, the expression vector according to claim 35, or the recombinant cells according to any one of claims 38 to 39 in the preparation of a kit, wherein the kit is used to detect CD3 and / or BCMA, or CD3 and / or B7H6.

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