BCMA binding protein, bispecific antibody, preparation method, and use

Abstract

Provided are a BCMA binding protein, a bispecific antibody, a preparation method, and a use. The BCMA binding protein comprises a heavy chain variable region and a light chain variable region. The light chain variable region comprises LCDR1, LCDR2, and LCDR3; and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3. The BCMA binding protein of the present invention has good specific affinity, binds better to a tumor cell line than a control antibody, and has an internalization effect better than that of the control. The bispecific antibody effectively targets both human BCMA and human CD3.

Description

A BCMA binding protein, bispecific antibody and preparation method and application thereof

[0001] This application claims priority to Chinese Patent Application No. 2024109952136, filed on July 24, 2024. This application incorporates the entirety of the aforementioned Chinese Patent Application. TECHNICAL FIELD

[0002] The present application belongs to the field of biological macromolecules, and specifically relates to a BCMA binding protein, bispecific antibody and preparation method and application thereof. BACKGROUND

[0003] Multiple myeloma (MM) has always been characterized by uncontrolled proliferation of bone marrow-derived plasma cells, accompanied by abnormal secretion of immunoglobulin or free light chains, and is the second most common hematologic malignancy, accounting for about 10% of all hematologic tumors. In the United States, more than 30,000 new patients are diagnosed each year, and more than 12,000 people die. Although there are new targeted therapies such as proteasome inhibitors (Bortezomb), it is generally believed that it is incurable, so more efforts need to be made in drug development.

[0004] B-cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a specific antigen expressed only in the B-cell lineage, especially in terminally differentiated B cells. It is usually not present in naive and memory B cells and is highly expressed on multiple myeloma cells. When BCMA interacts with its ligands BAFF and APRIL, it can induce B-cell maturation, proliferation and survival through signal transduction of the NF-kB and JNK pathways. As a type I transmembrane protein, it plays an important role in humoral immunity and is an ideal target for antibody-derived immunotherapy.

[0005] However, the anti-BCMA antibodies in the prior art have the defects of weak internalization and weak binding to 293T-huBCMA cells, and therefore there is an urgent need for an anti-BCMA antibody that has both sufficient drugability affinity and excellent internalization. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects of the prior art, such as weak internalization of the existing anti-BCMA antibody and weak binding to cells expressing human BCMA, and to provide a BCMA binding protein and a preparation method and application thereof. To solve the above technical problems, the present application provides a BCMA binding protein, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:41 in the sequence table or a variant thereof, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:48 in the sequence table or a variant thereof, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:54-57 in the sequence table or a variant thereof; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:8-11 in the sequence table or a variant thereof, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:18-20 in the sequence table or a variant thereof, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:30-32 in the sequence table or a variant thereof. Wherein the variant is an amino acid sequence obtained by 3, 2 or 1 mutations based on the amino acid sequences shown in SEQ ID NO:8-11, 18-20, 30-32, 41, 48 and 54-57, respectively.

[0007] In the similar "with 3, 2 or 1 amino acid mutations", the "amino acid mutations" refer to the mutations of amino acids in the sequence of the variant compared with the original amino acid sequence, including the insertion, deletion or substitution of amino acids based on the original amino acid sequence. An exemplary explanation is that the mutation of CDR can contain 3, 2 or 1 amino acid mutations, and the same or different number of amino acid residues between these CDRs can be optionally selected for mutation, for example, 1 amino acid mutation to CDR1, and no amino acid mutation to CDR2 and CDR3.

[0008] The mutations in the present application can include mutations known to those skilled in the art at present, for example, some mutations that may be made to the antibody during the production or application of the antibody, such as mutations to the sites of potential post-translational modifications (PTMs) that may exist, especially in the CDR region, including mutations related to aggregation, asparagine deamidation (sites (NG, NS and / or NH, etc.), aspartate isomerization (DG, DP) sensitive sites, N-glycosylation (N-{P}S / T) sensitive sites and oxidation sensitive sites, etc.

[0009] In a preferred embodiment of the application, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences set forth in SEQ ID NO: 41, 48 and 54, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences set forth in SEQ ID NO: 8, 18 and 30, respectively; or

[0010] the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences set forth in SEQ ID NO: 41, 48, 55, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences set forth in SEQ ID NO: 9, 19, 31, respectively; or

[0011] the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences set forth in SEQ ID NO: 41, 48, 56, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences set forth in SEQ ID NO: 10, 19, 31, respectively; or

[0012] the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences set forth in SEQ ID NO: 41, 48 and 55, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences set forth in SEQ ID NO: 10, 19 and 31, respectively; or

[0013] In a preferred embodiment of the application, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:63 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:70 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:64 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:71 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:65 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:72 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:66 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:71 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:67 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:73 of the Sequence Listing.

[0014] In a preferred embodiment of the application, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:63 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:70 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:64 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:71 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:65 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:72 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:66 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:71 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:67 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:73 of the Sequence Listing.

[0015] In the present application, the amino acid sequences of the above listed CDRs are shown according to the Chothia definition rule (the sequences shown in the claims of the present application are also according to the Chothia definition rule). However, it is well known in the art that the CDRs of an antibody can be defined in various ways in the art, such as the Kabat definition rule based on sequence variability and the Chothia definition rule based on the position of structural loop regions. It is understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g. variable region) are understood to encompass the complementarity determining regions defined by any of the above known approaches described in the present application. Although the scope claimed in the claims of the present application is based on the sequences shown according to the Chothia definition rule, the amino acid sequences corresponding to the definition of other CDRs should also fall within the scope of protection of the present application.

[0016] The BCMA binding protein described in the present application preferably further comprises a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region is selected from hlgGl, hlgG2, hlgG3 or hlgG4 or a mutation thereof, and the light chain constant region is selected from a kappa chain or a lambda chain or a mutation thereof; wherein the kappa chain or the lambda chain is preferably a human antibody light chain kappa chain or a human antibody light chain lambda chain.

[0017] In a preferred embodiment of the present application, the heavy chain constant region is hlgGl, and the light chain constant region is a human antibody light chain kappa chain.

[0018] The BCMA binding protein described in the present application can be a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single region antibody, or a monoclonal antibody or polyclonal antibody prepared therefrom. The monoclonal antibody can be prepared by various approaches and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the mainstream is to prepare monoclonal antibody from wild type or transgenic mice by hybridoma technology.

[0019] In one embodiment of the present application, the BCMA binding protein is a full-length antibody, which comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 83; or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 77 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 84; or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 78 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 85; or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 79 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 84; or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 80 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 86.

[0020] The present application also provides a bispecific antibody comprising an antigen binding region A binding to BCMA and an antigen binding region B binding to CD3, which comprises three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; wherein the first polypeptide chain comprises, from N-terminus to C-terminus, in order, VH_A-CH1, the second polypeptide chain comprises, from N-terminus to C-terminus, in order, VK_A-VH_B-CH1, and the third polypeptide chain comprises, from N-terminus to C-terminus, in order, Vλ_B-Cλ.

[0021] In the present application, VH_A is the heavy chain variable region of the antigen binding region A, and VK_A is the light chain variable region of the antigen binding region A; VH_B is the heavy chain variable region of the antigen binding region B, and Vλ_B is the light chain variable region of the antigen binding region B; Cκ is the light chain constant region κ chain, and Cλ is the light chain constant region λ chain.

[0022] In one preferred embodiment of the present application, VK_A comprises LCDR1, LCDR2 and LCDR3, which have the amino acid sequences as set forth in SEQ ID NOs: 41, 48 and 54, respectively; VH_A comprises HCDR1, HCDR2 and HCDR3, which have the amino acid sequences as set forth in SEQ ID NOs: 8, 18 and 30, respectively; or

[0023] VK_A comprises LCDR1, LCDR2 and LCDR3, which have the amino acid sequences as set forth in SEQ ID NOs: 41, 48 and 55, respectively; VH_A comprises HCDR1, HCDR2 and HCDR3, which have the amino acid sequences as set forth in SEQ ID NOs: 9, 19 and 31, respectively; or

[0024] the VK_A comprises LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs:41, 48, and 55; and the VH_A comprises HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs: 10, 19, and 31; or

[0025] the VK_A comprises LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs:41, 48, and 55; and the VH_A comprises HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs: 10, 19, and 31; or

[0026] the VK_A comprises LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs:41, 48, and 57; and the VH_A comprises HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs: 11, 20, and 32.

[0027] In a preferred embodiment of the application, the VK_B comprises LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs:40, 47, and 53; and the VH_B comprises HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences set forth in SEQ ID NOs: 7, 17, and 29.

[0028] In a more preferred embodiment of the application, the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 94; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 93; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 82; or

[0029] the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 92; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 91; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 82; or

[0030] the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 88; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 87; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 82; or

[0031] the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 92; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 95; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 82; or

[0032] the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 90; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 89; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 82.

[0033] The present application also provides an isolated nucleic acid encoding the BCMA binding protein or the bispecific antibody as described above.

[0034] The present application also provides an expression vector comprising the isolated nucleic acid as described above.

[0035] The present application also provides a host cell comprising the expression vector as described above; preferably, the host cell is a prokaryotic cell or a eukaryotic cell. The host cell can be prepared by a conventional method in the art, for example, by transforming the expression vector as described above into the host cell. The host cell can be any conventional host cell in the art, as long as it can satisfy the requirements of stably replicating the expression vector as described above by itself and effectively expressing the nucleic acid carried by the expression vector. Preferably, the host cell is an E. coli TG1 or BL21 cell (for expressing a single-chain antibody or a Fab antibody), or a CHO-K1 cell (for expressing a full-length IgG antibody). The transformation method can be any conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0036] The present application also provides a method for preparing the BCMA binding protein or the bispecific antibody, comprising culturing the host cell as described above and obtaining the BCMA binding protein or the bispecific antibody from the culture.

[0037] The present application also provides an immunoconjugate comprising a cytotoxic agent and the BCMA binding protein or the bispecific antibody as described above.

[0038] The present application also provides a pharmaceutical composition comprising the BCMA binding protein as described above, the bispecific antibody as described above or the immunoconjugate as described above.

[0039] The present application also provides use of the BCMA binding protein as described above, the bispecific antibody as described above, the immunoconjugate as described above or the pharmaceutical composition as described above in the manufacture of a medicament for treating and / or preventing cancer; preferably, the cancer is myeloma, colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer or ovarian cancer.

[0040] The present application also provides a method for treating and / or preventing cancer, comprising administering to a patient in need thereof an effective amount of the BCMA binding protein as described above, the bispecific antibody as described above, the immunoconjugate as described above or the pharmaceutical composition as described above; preferably, the cancer is myeloma, colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer or ovarian cancer.

[0041] The present application also provides a BCMA binding protein as described above, a bispecific antibody as described above, an immunoconjugate as described above or a pharmaceutical composition as described above for use in treating and / or preventing cancer; preferably, the cancer is myeloma, colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer or ovarian cancer.

[0042] In addition, to solve the above technical problems, the present application also provides a kit combination comprising kit A and kit B; the kit A comprises the BCMA binding protein, the bispecific antibody, the host cell, the immunoconjugate or the pharmaceutical composition of the present application; the kit B comprises other targeted or same targeted antibodies, bispecific antibodies, genetically modified cells or pharmaceutical compositions. The kit A and kit B can be used in any order, or the kit A is used first and then the kit B is used, or the kit B is used first and then the kit A is used.

[0043] The BCMA binding protein, the bispecific antibody, the immunoconjugate, the pharmaceutical composition or the kit combination of the present application can be administered to patients for treating related tumors.

[0044] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. And, the cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory operation steps used herein are conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0045] The three-letter code and one-letter code of amino acids used in the present application are known to those skilled in the art, or described in J. Biol. Chem, 243, p3558 (1968).

[0046] As used herein, the terms "comprising" or "including," or "having" are intended to mean that the compositions and methods include the recited elements, but not excluding others.

[0047] The term "antibody" as used herein includes immunoglobulin (Ig), which is a four polypeptide chain structure connected by interchain disulfide bond, consisting of two identical heavy chains and two identical light chains. The antigenicity of immunoglobulin heavy chain constant region is different due to different amino acid composition and arrangement order. Accordingly, immunoglobulin can be divided into five types, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same type of Ig can be divided into different subtypes according to the difference of amino acid composition of hinge region and the number and position of heavy chain disulfide bond, such as IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain is divided into κ chain or λ chain through the constant region. Each type of Ig in the five types of Ig can have κ chain or λ chain.

[0048] In the present application, the antibody light chain variable region described in the present application can further comprise a light chain constant region comprising human κ, λ chain or a variant thereof. In the present application, the antibody heavy chain variable region described in the present application can further comprise a heavy chain constant region comprising human IgG1, 2, 3, 4 or a variant thereof.

[0049] The sequence of about 110 amino acids near the N-terminal of antibody heavy chain and light chain varies greatly, which is the variable region (V region); the remaining amino acid sequence near the C-terminal is relatively stable, which is the constant region (C region). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 complementarity determining regions (CDRs) and 4 framework regions (FWRs), and the sequence arranged from amino terminal to carboxyl terminal is: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The 3 CDRs of light chain are LCDR1, LCDR2 and LCDR3; the 3 CDRs of heavy chain are HCDR1, HCDR2 and HCDR3.

[0050] The term "mutation" includes substitution, addition and / or deletion of amino acid or nucleotide, and "substitution of amino acid" is the substitution of an amino acid residue with another amino acid residue and the substitution of an amino acid residue with an amino acid residue having a similar side chain.

[0051] As used herein, the term "vector" or "expression vector" refers to a composition containing isolated nucleic acids and capable of delivering the isolated nucleic acids into cells. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0052] The term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved through a variety of techniques known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0053] The preparation method of the immunoconjugate can be conventional in the art, and preferably follows the method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124. Preferably, the preparation method produces an antibody-drug conjugate having a minimum low conjugation fraction (LCF) of less than 10%. The immunoconjugate can exist in any physical form known in the art, preferably as a clear solution.

[0054] As used in this invention, the terms “cancer,” “tumor,” and “carcinoma” are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of their histopathological type or stage of invasiveness. Examples include, but are not limited to, solid tumors, hematologic malignancies, soft tissue tumors, and metastatic lesions.

[0055] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0056] The reagents and raw materials used in this invention are all commercially available.

[0057] The positive and progressive effects of this invention are as follows:

[0058] This invention uses immunoassay to identify several fully human antibodies with good specific affinity for BCMA, which bind better to tumor cell lines than control antibodies. These antibodies exhibit internalization effects superior to controls, thus they can be further developed as ADC candidates. ELISA assays show that all antibodies possess partial blocking function, potentially offering additional therapeutic benefits when used as monoclonal antibodies, bispecific antibodies, or in cell therapy. Attached Figure Description

[0059] FIG. 1A and FIG. 1B show the binding activity of the recombinant antibody to the cell line HEK293T / human BCMA expressing human BCMA.

[0060] FIG. 2A and FIG. 2B show the binding activity of the recombinant antibody to the cell line HEK293T / cyno BCMA expressing cynomolgus monkey BCMA.

[0061] FIG. 3 shows the binding activity of the recombinant antibody to the tumor cell line NCI-H929 expressing human BCMA.

[0062] FIG. 4 shows the blocking of the binding of BAFF to BCMA by the recombinant antibody.

[0063] FIG. 5A and FIG. 5B show the activity of the recombinant antibody in internalizing into NCI-H929 cells.

[0064] FIG. 6 shows a bispecific antibody targeting BCMA and CD3 with a "κ / λ" Fab-Fab tandem bispecific antibody structure produced in the specific embodiment. It has three polypeptide chains, a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises, from N-terminus to C-terminus, VH_A-CH1, the second polypeptide chain comprises, from N-terminus to C-terminus, VK_A-CK-VH_B-CH1, and the third polypeptide chain comprises, from N-terminus to C-terminus, Vλ_B-Cλ. Wherein the N-terminal part of the first polypeptide chain and the second polypeptide chain constitutes a Fab (Fab-A) targeting BCMA; the C-terminal part of the third polypeptide chain and the second polypeptide chain constitutes a Fab (Fab-B) targeting CD3.

[0065] FIG. 7A to FIG. 7D show that the BCMAxCD3 "κ / λ" Fab-Fab bispecific antibody can effectively kill NCI-H929 cells with high expression of BCMA. DETAILED DESCRIPTION

[0066] The present application is further illustrated by the following examples without thereby limiting the present application to the examples described. The experimental methods in the following examples, where no specific conditions are indicated, are selected in accordance with the conventional methods and conditions, or in accordance with the instructions of the commercial product.

[0067] The positive control (also known as control antibody PR000274 (Ref)) used in the following examples is the ADC drug GSK2857916 (i.e. CA8 J6M0 humanized antibody in US patent application US9273141B2). The amino acid sequences of each functional region of the control antibody are shown in Table 1 below (according to Chothia numbering rules):

[0068] Table 1

[0069] Example 1: Antigen preparation, mouse immunization and hybridoma preparation

[0070] 1. Antigen preparation

[0071] The antigens used in this example are shown in Table 2.

[0072] Table 2

[0073] 2. Immunization

[0074] Fully human anti-BCMA antibodies were identified from hybridomas generated from H2L2 mice immunized with huBCMA-ECD-Fc protein (and platinum therapeutics, EP2379727B1). The first injection of 50 μg of the above fusion protein was performed with CFA as the immunization adjuvant, followed by 7 additional boosts of 25 μg protein and Ribi adjuvant (Sigma-Aldrich; Sigma Adjuvant System; Catalog Number S6322) on days 15, 29, 43, 57, 71 and 86. Blood was collected on days 50, 78 and 107 for testing, and the binding affinity of the mouse serum was tested by FACS using HEK293T cells expressing human BCMA (HEK293T / human BCMA, purchased from Kangyuan Bocheng) or tumor cell line NCI-H929 cells (purchased from ATCC), while BCMA-His protein was used for ELISA testing in parallel. According to the results of the detection of the serum titer of the immunized mice, the mice were selected for hybridoma fusion, and 3 days before fusion, i.e. on day 132, a final boost of 25 μg protein and Ribi adjuvant was performed.

[0075] 3. Fusion

[0076] The hybridomas were generated and cloned by conventional methods, i.e. by the method of electrofusion, extraction of the mouse spleen and lymph nodes, extraction of single cells by grinding, cracking and washing, and mixing with sp2 / 0 cells. The cell suspension was placed in an electrofusion tank for electric shock fusion, and after standing, it was replaced with 20% FBS HT medium, and then HAT medium was used for culture.

[0077] The collected spleen B cells were mixed with mouse myeloma cell line Sp2 / 0 at a ratio of 2:1 (cell number ratio), and the mixed cells were subjected to cell fusion using an electrofusion instrument (BTX ECM2001). The fused cells were plated in a 96-well cell culture plate, and after 10 days of culture at 37°C in a carbon dioxide incubator, primary screening of the hybridoma was performed. After overnight recovery, the fused cells were inoculated into a 96-well plate using limited dilution, and were screened using hypoxanthine-aminopterin-thymidine. The presence of anti-BCMA antibodies in the hybridoma culture supernatant was detected by ELISA test and flow cytometry.

[0078] Example 2: Antibody screening and sequencing

[0079] 1. ELISA screening

[0080] In a 96-well plate (Corning 9018), freshly prepared hBCMA ECD-Fc protein or hFc at 1 pg / ml in PBS was added and coated at 4°C overnight, then discarded and washed 3 times with PBST. The plate was blocked with 5% milk at room temperature for 2 hours, and washed 3 times with PBST. 100 pl / well of hybridoma supernatant was added and incubated at room temperature for 1 hour, then washed 3 times with PBST. 100 pl / well of secondary antibody was added and incubated at room temperature for 1 hour, followed by washing. 100 pl / well of TMB was added to the plate and incubated at room temperature for 15 min, then stopped and read.

[0081] 2. FACS screening

[0082] For flow cytometry screening, adherent cells were digested with TypLE enzyme (CAT#12605010, Gibco) at 37°C for 3 minutes, and then the digestion was terminated with complete medium containing 10% FBS. The cells were washed with FACS buffer (CAT#14190250, Gibco) and counted, then diluted to a density of 3-5 x 10 6 / ml. The cells were added to a 96-well plate (Corning 3894) at 100 pl / well. After blocking for 3-4 minutes, 100 pl / well of hybridoma supernatant was added and incubated at 4°C for 1 hour. After washing, secondary antibody was added and incubated at 4°C for one hour. Then the cells were washed and subjected to FACS analysis.

[0083] 3. Subcloning and screening

[0084] Blocking test was performed using limited dilution method by ELISA.

[0085] Blocking screening:

[0086] For the blocking enzyme-linked immunosorbent assay (ELISA), plates were coated with 2 μg / ml of huBCMA-ECD-FC in PBS overnight, washed, blocked and incubated as described above. Then 6 ng / ml of biotinylated BAFF was added to the plates and incubated for 1 hour at room temperature. The second antibody incubation and subsequent detection steps were the same as above.

[0087] 4. Sequencing

[0088] Positive monoclonal antibodies were selected and total RNA was extracted. RT-PCR produced cDNA, followed by PCR amplification of heavy and light chains separately (RT-PCR from Qiagen, Cat#: 74134, see product manual for details. PCR used NEB's high fidelity DNA polymerase, Cat#: M0530L, see product manual for details). The PCR products were then constructed into T vectors and sequenced (Shanghai Branch of Beijing Engenco Biotechnology Co., Ltd.), and antibody isotype determination was performed.

[0089] 5. Antibody sequence information

[0090] The sequencing results of the hybridoma were analyzed to obtain the amino acid sequence of the antibody, and the corresponding sequence numbers are shown in Table 3 below (CDRs are defined according to Chothia numbering rules):

[0091] Table 3

[0092] Example 3: Production and purification of recombinant antibodies

[0093] After obtaining the sequences of the light and heavy chain variable domains of the antibody molecule, the light and heavy chain variable domain sequences and the corresponding human antibody light and heavy chain constant domain sequences can be fused and expressed using conventional recombinant DNA technology to obtain a recombinant antibody molecule. In this example, the antibody heavy chain variable domain sequence (VH) is synthesized by gene and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the full-length heavy chain of the IgG1 antibody. The antibody light chain variable domain sequence (VL) is synthesized by gene and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig kappa light chain constant domain sequence to encode the full-length light chain of the antibody. In this example, since the sequence of the variable domain of the monoclonal antibody molecule obtained from the immunized Harbour H2L2 mouse is a human antibody sequence, this example also obtains a fully human anti-BCMA recombinant IgG1 antibody.

[0094] The amino acid sequence of the humanized antibody of the present application is shown in Table 4 below (CDRs are defined according to Chothia numbering rules):

[0095] Table 4

[0096] The plasmid encoding the antibody heavy chain (Genscript US) and the plasmid encoding the antibody light chain (Genscript US) are transfected into mammalian host cells (e.g. human embryonic kidney cells HEK293) simultaneously, and using conventional recombinant protein expression and purification techniques, a purified recombinant antibody with correct pairing assembly of light and heavy chains can be obtained. Specifically, the HEK293 cells are expanded in FreeStyle TM Expression Medium (Thermo, Cat#: A1383504). Before the start of transient transfection, the cell concentration is adjusted to 6-8 x 10 5 cells / ml, and cultured at 37°C in an 8% CO2 incubator for 24 hours, and the cell concentration is adjusted to 1.2 x 10 6 cells / ml. 30 ml of the prepared cells are used. The above-mentioned plasmid encoding the antibody heavy chain and the plasmid encoding the antibody light chain are mixed in a ratio of 2:3 (mass ratio) to a total of 30 μg of plasmid, dissolved in 1.5 ml of Opti-MEM reduced serum medium (Thermo, Cat#: 31985088), and filtered with a 0.22 μm filter to remove bacteria. Then, 1.5 ml of Opti-MEM is added to 1 mg / ml PEI (Polysciences, Cat#: 23966-2) 120 μl, and allowed to stand for 5 minutes. The PEI is slowly added to the plasmid, and incubated at room temperature for 10 minutes. The plasmid-PEI mixed solution is slowly added dropwise to the culture bottle while shaking, and cultured at 37°C in an 8% CO2 incubator for 5 days. After 5 days, the cell viability is observed. The culture is collected, centrifuged at 3300 g for 10 minutes, and the supernatant is collected. Then, the supernatant is centrifuged at high speed to remove impurities. The MabSelect TM (GE Healthcare Life Science, Cat#: 71-5020-91AE) equilibrated with PBS (pH 7.4) is used, and 2-5 times the column volume is used for washing. The supernatant sample is passed through the column, and the column is washed with 5-10 times the column volume of PBS. The target protein is eluted with 0.1 M glycine at pH 3.5, and then adjusted to neutral with Tris-HCl at pH 8.0. Finally, the purified antibody solution is obtained by concentrating and replacing the solution with PBS buffer using an ultrafiltration tube (Millipore, Cat#: UFC901024). Finally, the purified antibody solution is analyzed using a NanoDrop (Thermo Scientific TMNanoDrop TM One) assay concentration, aliquot, store for future use.

[0097] Take the purified sample above, take an appropriate amount and load it into the analytical SEC column TSKgel G3000SWxl (HPLC instrument model: Agilent 1260 Infinity II) respectively, detect the purity of the sample, and ensure that the purity of the sample is above 95%. The mobile phase of this method is 1xPBS, pH 7.4 (Shanghai Yingyee Biotech Co., Ltd., Cat#: E607016), room temperature, flow rate 1.0 ml / min, sample concentration 1 mg / ml, injection volume 20 μl, detection wavelength 280 nm. After collection, use ChemStation software to integrate the chromatogram and calculate the relevant data.

[0098] Example 4: Binding to BCMA

[0099] This example studies the binding activity of the BCMA-targeting fully human recombinant antibodies (PR000897, PR000903, PR000905, PR000907, PR000909) prepared in Example 3 to BCMA.

[0100] Flow cytometry FACS was used to test the binding ability of the recombinant antibodies to HEK293T cell strains HEK293T / human BCMA (Kang Yuan Bochuang, KC-0233) highly expressing human BCMA and HEK293T cell strains HEK293T / cyno BCMA (Kang Yuan Bochuang, KC-0979) highly expressing cynomolgus monkey BCMA, and tumor cell lines NCI-H929 (ATCC, CRL-3580) highly expressing human BCMA.

[0101] The recombinant antibodies to be tested were diluted to 9 different concentrations in turn, and incubated with HEK293T / human BCMA, HEK293T / cyno BCMA or NCI-H929 target cells for 1 hour, then fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fc gamma Fragment Specific, Jackson ImmunoResearch, #109-605-098) was added, and incubated at 4°C in the dark for 1 hour. The fluorescence signal value was read using ACEA NovoCyte flow cytometer or BD FACS CANTOII flow cytometer, and the data was processed and analyzed using FlowJo v10 software (FlowJo, LLC). The data was processed and plotted using GraphPad Prism 8 software, and the binding curve and EC50 value of the antibody to the target cells were obtained by four-parameter nonlinear fitting.

[0102] FIG. 1A and FIG. 1B show the binding activity of the full human recombinant antibodies targeting BCMA to the cell line HEK293T / human BCMA expressing human BCMA, and the relevant curve fitting parameters are listed in Table 5 below. The results show that the binding ability of RP000903, RP000905, RP000907 and RP000905 is better than that of the control antibody PR000274.

[0103] FIG. 2A and FIG. 2B show the binding activity of the full human recombinant antibodies targeting BCMA to the cell line HEK293T / cyno BCMA expressing cynomolgus monkey BCMA, and the relevant curve fitting parameters are listed in Table 5 below. The results show that the binding ability of RP000903, RP000905, RP000907 and RP000905 is comparable to that of the control antibody PR000274.

[0104] FIG. 3 shows the binding activity of the full human recombinant antibodies targeting BCMA to the tumor cell line NCI-H929 expressing human BCMA, and the relevant curve fitting parameters are listed in Table 5 below. The results show that the binding ability of the full human recombinant antibodies is comparable to that of the control antibody PR000274; although the EC50 value of PR000897 is larger, it has a higher MFI signal reading value compared with the control antibody.

[0105] Table 5

[0106] Example 5: Blocking the binding of BAFF to BCMA

[0107] BAFF is a ligand of BCMA; when BCMA interacts with its BAFF, it induces B cell maturation, proliferation and survival through signal transduction, and is an important factor for the proliferation of multiple myeloma cells. Therefore, antibodies with the function of blocking the binding of BAFF to BCMA have better therapeutic effect.

[0108] This example used ELISA method to study the function of blocking the binding of BAFF to BCMA of the BCMA-targeted fully human recombinant antibodies (PR000897, PR000903, PR000905, PR000907, PR000909) prepared in Example 3.

[0109] The recombinant antibodies to be tested were sequentially diluted to 7 different concentrations; the plates were coated with 2 μg / ml of human BCMA recombinant protein (huBCMA-ECD-FC) overnight, washed, blocked and incubated; then 6 ng / ml of biotinylated BAFF protein was added to the plates, and incubated at room temperature for 1 hour; then, the secondary antibody was added and incubated. After adding the stop solution, the plates were placed into the plate reader to read the optical absorption value.

[0110] Figure 4 and Table 6 below show that the BCMA-targeted fully human recombinant antibodies all have the ability to block the binding of BAFF to BCMA.

[0111] Table 6

[0112] Example 6: Internalization of BCMA

[0113] This example studied the antibody internalization mediated killing of target cells after the binding of the BCMA-targeted fully human recombinant antibodies (PR000897, PR000903, PR000905, PR000907, PR000909) prepared in Example 3 to BCMA.

[0114] Specifically, NCI-H929 cells highly expressing human BCMA were digested, and the cells were resuspended with complete medium, counted and inoculated at 5000 or 10000 cells per well into 96-well black-walled transparent bottom plates (Perkin Elmer, #6005225). The recombinant antibodies were serially diluted to 9 different concentrations and added, with the final concentration starting from 100 nM. a-hFc-MMAF (Moradec, #AH-102-AF) was added to make the final concentration 1 μg / ml. The plates were incubated at 37°C, 5% CO2 for 72 hours, then lysed with CTG kit (Promega, #G7573), and Enspire TMMultimode plate reader (PerkinElmer, Inc.) was used to detect luminescence. GraphPad Prism 8 was used to process and analyze the data, and the binding curve of the antibody to the target cell and the EC50 value and the maximum killing rate were obtained by four-parameter nonlinear fitting.

[0115] Figures 5A and 5B and Table 7 show that the full human recombinant antibody targeting BCMA can mediate killing of NCI-H929 cells through internalization, and the killing activity is superior to the control antibody PR000274 (the full human recombinant antibody has a smaller EC50 value).

[0116] Table 7

[0117] Example 7: BCMAxCD3 bispecific antibody with “κ / λ” Fab-Fab structure

[0118] This example uses the antigen binding fragment sequences of the full human recombinant antibody targeting BCMA (PR000897, PR000903, PR000905, PR000907, PR000909) of the present application and the antigen binding fragment sequences of the antibody PR000512 (humanized variant of clone SP34; WO2021063330) targeting CD3 to construct a BCMAxCD3 bispecific antibody with “κ / λ” Fab-Fab structure, and to verify the target cell killing ability of the bispecific antibody.

[0119] 1. Molecular design of BCMAxCD3 bispecific antibody

[0120] As shown in Figure 6, the “κ / λ” Fab-Fab bispecific antibody structure has three polypeptide chains, namely a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first polypeptide chain comprises VH_A-CH1 in order from N-terminus to C-terminus, the second polypeptide chain comprises VK_A-CK-VH_B-CH1 in order from N-terminus to C-terminus, and the third polypeptide chain comprises Vλ_B-Cλ in order from N-terminus to C-terminus. Wherein A refers to the full human recombinant antibody targeting BCMA of the present application, and B refers to the antibody targeting CD3 used in this example; therefore, the N-terminal part of the first polypeptide chain and the second polypeptide chain constitutes a Fab (Fab-A) targeting BCMA; the C-terminal part of the third polypeptide chain and the second polypeptide chain constitutes a Fab (Fab-B) targeting CD3. The full human recombinant antibody targeting BCMA of the present application all has a kappa light chain structure; while the CD3 antibody PR000512 has a lambda light chain structure, the sequence of PR000512 is shown in Table 8 below (CDRs are defined according to Chothia numbering rules):

[0121] Table 8

[0122] The sequence of the fully human recombinant antibody targeting BCMA and the sequence of the CD3 antibody PR000512 of the present invention were used to design a BCMA×CD3 bispecific antibody according to the structure shown in Figure 6. The amino acid sequence of its polypeptide chain and the corresponding sequence number are shown in Tables 9 and 10 below.

[0123] Table 9

[0124] Table 10

[0125] The sequence numbers of the CDRs of each antigen-targeting Fab in the BCMA×CD3 bispecific antibody constructed in this invention are shown in Table 11 below.

[0126] Table 11

[0127] 2. Expression and purification of bispecific antibodies

[0128] Molecular cloning

[0129] The amino acid sequences of the three polypeptide chains of the “κ / λ”Fab-Fab protein were converted into nucleotide sequences using codon optimization methods; the encoded nucleotide sequences were synthesized and cloned into an expression vector compatible with host cells.

[0130] Transient transfection expression using the 293-F expression system

[0131] FreeStyle TM 293-F cells (Thermo, #R79007) in FreeStyle TM Expand the cells in F17 Expression Medium (Thermo, #A1383504). Adjust the cell density to 5 × 10⁶ cells / year before transient transfection. 5 Cells / ml were cultured at 37°C in an 8% CO2 shaker for 24 hours, and then the cell density was adjusted to 1×10⁻⁶. 6Cells / ml. Prepare 30 ml of culture of cells. Mix plasmids encoding three polypeptide chains in a certain ratio (e.g., 1:1:1) for a total of 30 pg of plasmids (1 pg of plasmid: 1 ml of cells) dissolved in 1.5 ml of Opti-MEM reduced serum medium (Thermo, #31985088) and filter-sterilized with a 0.22 pm filter. Take 1.5 ml of Opti-MEM and dissolve 1 mg / ml of PEI (Polysciences, #23966-2) in 120 pl for a total of 120 pg of PEI, and let stand for 5 minutes. Slowly add the PEI to the plasmids (PEI to plasmid ratio of about 4:1), incubate at room temperature for 15 minutes, and slowly drip the plasmid-PEI mixture into the culture bottle while shaking. Incubate at 37°C in a 8% CO2 shaker for 5 days. After 5 days, observe the cell viability, and when the cell viability is less than 70%, collect the culture.

[0132] Protein purification using KappaSelect

[0133] After centrifugation of the cell culture at 4000 g for 40 minutes, remove the cell debris and impurities by filtration to obtain the supernatant. Pre-equilibrate the KappaSelect packing (Cytiva, #17545801) with PBS buffer. Add the supernatant to a gravity column (Bio-Rad, #7311550) containing KappaSelect; rinse the column with 20 column volumes of PBS buffer; elute the target protein with 5-10 column volumes of 0.1 M glycine buffer at pH 2.5-3.0; and then adjust to neutral with Tris-HCl at pH 8.0.

[0134] Quantification and storage

[0135] Concentrate the exchange solution to PBS buffer or buffer containing other components with ultrafiltration tubes (Millipore, #UFC901024) to obtain the purified protein solution. Measure the concentration with a NanoDrop (Thermo, NanoDrop One). Finally, aliquot and store for future use. TM

[0136] SEC-HPLC analysis of protein purity and aggregation

[0137] ​An analytical column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 pm, 7.8 mm x 30 cm) was connected to a high pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 pg) was filtered with a 0.22 pm filter membrane before being injected into the system, and the HPLC program was set as follows: the sample was flowed through the column with PBS buffer at a flow rate of 1.0 ml / min for a maximum time of 25 min. The HPLC would generate an analysis report, which reported the retention time of different molecular size components in the sample.

[0138] The yield and purity of the BCMAxCD3 bispecific antibodies constructed in the present application after expression and purification are shown in Table 12. Almost all bispecific antibody molecules obtained high-purity protein samples.

[0139] Table 12

[0140] 3. T cell-mediated target cell killing

[0141] The activation of effector cells mediated by the BCMAxCD3 bispecific antibodies constructed in the present application and the killing of target cells were studied by the LDH method. Here, the effector cells were T cells isolated from human peripheral blood mononuclear cells PBMC, and the target cells were NCI-H929 cells (ATCC, #CRL-9068) that highly expressed BCMA.

[0142] Generally, the effector cells and the specific target cells are adjusted to the appropriate cell density with culture medium to adjust to the desired effector to target ratio (E:T); each of the two cell suspensions is seeded at 90 pL cells / well in a 96-well plate (Corning, #3799). Then, 20 pL / well of the pre-diluted test antibody (10-fold gradient dilution of the final concentration) is added. Two replicates are set up for each sample. The wells with the test antibody are called ER (experimental wells, containing the test antibody sample and effector cells and target cells); at the same time, different control groups are set up according to the following formula: ESR (effector cell spontaneous release wells, only effector cells and culture medium); TSR (target cell spontaneous release wells, only target cells and culture medium); CMB (culture medium reference wells, only culture medium); TMR (target cell maximum release wells, only target cells and culture medium); VCC (volume reference wells, only culture medium). The 96-well plate is incubated in a 37°C carbon dioxide incubator for 24 hours. Then, 10 pL of lysis solution is added to the TMR wells and the VCC wells, and incubation is continued for 30 minutes. After incubation, 50 pL / well of supernatant is taken, added to a 96-well plate (Corning, #3599), and 50 pL / well of a cytotoxicity detection reagent (CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit, Promega, #G1780) is added. After incubation at room temperature for 30 minutes, 50 pL of reaction termination solution is added to terminate the reaction. Finally, the optical absorption value is read at 490 nM using an Enspire Multifunctional Plate Reader (Perkin Elmer, Inc.), and the target cell killing rate is calculated according to the following formula. The software GraphPad Prism 8 is used for data processing and graphical analysis. Non-Radioactive Cytotoxicity Assay Kit, Promega, #G1780). After incubation at room temperature for 30 minutes, 50 pL of reaction termination solution is added to terminate the reaction. Finally, the optical absorption value is read at 490 nM using an Enspire Multifunctional Plate Reader (Perkin Elmer, Inc.), and the target cell killing rate is calculated according to the following formula. The software GraphPad Prism 8 is used for data processing and graphical analysis.

[0143] Killing rate = ((ER - CMB) - (ESR - CMB) - (TSR - CMB)) / (TMR - VCC) x 100%

[0144] wherein:

[0145] ER = experimental wells, sample + effector cells + target cells

[0146] ESR = effector cell spontaneous release wells, effector cells + culture medium

[0147] TSR = target cell spontaneous release wells, target cells + culture medium

[0148] TMR = target cell maximum release wells, target cells + culture medium + lysis solution

[0149] VCC = volume reference wells, culture medium + lysis solution

[0150] CMB = culture medium reference wells, culture medium

[0151] Further specifically, in NCI-H929 cell killing, NCI-H929 cells and isolated human T cells were seeded in 96-well plates at a ratio of 1 :4, and the test BCMAxCD3 antibodies were diluted from the highest final concentration of 1 nM into a total of three concentrations (1 nM, 0.05 nM, 2.5 pM) of samples in 20-fold gradient. In this experiment, PBMC (donor 1 and donor 2) isolated T cells from two different donors were used to perform two independent cell killing tests, and the results are shown in Figures 7A to 7D.

[0152] Figures 7A to 7D show that BCMAxCD3 bispecific antibodies can effectively kill NCI-H929 cells that highly express BCMA, and the trend is consistent in two independent experiments from different donor effector cells. At a 1 nM antibody concentration, all BCMAxCD3 bispecific antibodies can produce strong killing of target cells; at a 0.05 nM antibody concentration, the remaining four bispecific antibodies except PR001790 can also produce strong killing of target cells, and the killing ability is comparable to that at a 1 nM concentration.

Claims

1. A BCMA binding protein, characterized in that, comprises a heavy chain variable region and a light chain variable region, wherein the antibody is selected from any one of the following groups: the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 41, 48 and 54, respectively; and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 8, 18 and 30, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 41, 48 and 55, respectively; and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 9, 19 and 31, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 41, 48 and 56, respectively; and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 10, 19 and 31, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 41, 48 and 55, respectively; and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 10, 19 and 31, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 41, 48 and 57, respectively; and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 11, 20 and 32, respectively.

2. The BCMA binding protein of claim 1, wherein, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 70 or having at least 85% sequence identity to SEQ ID NO: 70; and the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 63 or having at least 85% sequence identity to SEQ ID NO: 63; or, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 71 or having at least 85% sequence identity to SEQ ID NO: 71; and the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 64 or having at least 85% sequence identity to SEQ ID NO: 64; or, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 72 or having at least 85% sequence identity to SEQ ID NO: 72; and the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 65 or having at least 85% sequence identity to SEQ ID NO: 65; or, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 72 or having at least 85% sequence identity to SEQ ID NO: 72; and the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 65 or having at least 85% sequence identity to SEQ ID NO: 65; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 71 or having at least 85% sequence identity to SEQ ID NO: 71; and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 66 or having at least 85% sequence identity to SEQ ID NO: 66; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 73 or having at least 85% sequence identity to SEQ ID NO: 73; and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 67 or having at least 85% sequence identity to SEQ ID NO:

67.

3. The BCMA binding protein of claim 1 or 2, wherein, Further comprising a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of hlgGl, hlgG2, hlgG3 and hlgG4 and mutants thereof, and the light chain constant region is selected from the group consisting of a Kappa chain, a Lambda chain and mutants thereof.

4. The BCMA binding protein of any one of claims 1-3, wherein, It is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, single-domain antibody or single-region antibody, or a monoclonal antibody or polyclonal antibody prepared from the above-mentioned antibodies.

5. The BCMA-binding protein of claim 4, wherein, the full-length antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 83; or, the full-length antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 77 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 84; or, the full-length antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 78 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 85; or, the full-length antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 79 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 84; or, the full-length antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 80 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO:

86.

6. A bispecific antibody comprising an antigen binding region A that binds BCMA and an antigen binding region B that binds CD3, the bispecific antibody comprising three polypeptide chains: a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein, the first polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable region (VH_A) of antigen binding region A and CH1, the second polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable region (Vκ_A)-Cκ of antigen binding region A, a heavy chain variable region (VH_B) of antigen binding region B and CH1, and the third polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable region (Vλ)_B of antigen binding region B and Cλ; wherein Cκ is a light chain constant region Kappa chain and Cλ is a light chain constant region Lambda chain. VH_A comprises HCDR1, HCDR2 and HCDR3, respectively has an amino acid sequence as shown in SEQ ID NO: 8, 18 and 30; VH_A comprises HCDR1, HCDR2 and HCDR3, respectively has an amino acid sequence as shown in SEQ ID NO: 8, 18 and 30; VH_A comprises HCDR1, HCDR2 and HCDR3, respectively has an amino acid sequence as shown in SEQ ID NO: 8, 18 and 30; VH_A comprises HCDR1, HCDR2 and HCDR3, respectively has an amino acid sequence as shown in SEQ ID NO: 8, 18 and 30; or VH_A comprises HCDR1, HCDR2 and HCDR3, respectively has an amino acid sequence as shown in SEQ ID NO: 8, 18 and 30; 7. The bispecific antibody of claim 6, wherein, VH_A comprises HCDR1, HCDR2 and HCDR3, respectively has an amino acid sequence as shown in SEQ ID NO: 8, 18 and 30.

8. The bispecific antibody of claim 6 or 7, wherein, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 94; the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 93; and the third polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 82; the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 92; the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 91; and the third polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 82; the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 88; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 87; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 82; the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 92; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 95; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 82; or the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 90; the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 89; and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO:

82.

9. An isolated nucleic acid encoding the BCMA binding protein of any one of claims 1-5 or the bispecific antibody of any one of claims 6-8.

10. An expression vector comprising the isolated nucleic acid of claim 9.

11. A host cell comprising the expression vector of claim 10; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

12. A method of producing a BCMA binding protein or a bispecific antibody, comprising culturing the host cell of claim 9, and obtaining the BCMA binding protein or the bispecific antibody from the culture.

13. An immunoconjugate comprising a cytotoxic agent, and the BCMA binding protein of any one of claims 1-5 or the bispecific antibody of any one of claims 6-8.

14. A pharmaceutical composition comprising the BCMA binding protein of any one of claims 1-5, the bispecific antibody of any one of claims 6-8, or the immunoconjugate of claim 13.

15. Use of the BCMA binding protein of any one of claims 1-5, the bispecific antibody of any one of claims 6-8, the immunoconjugate of claim 13, or the pharmaceutical composition of claim 14 in the manufacture of a medicament for treating and / or preventing a cancer; preferably, the cancer is myeloma, colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer, or ovarian cancer.

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