Monoclonal antibody against human bcma and hybridoma cell strain

Abstract

The application provides a monoclonal antibody against human BCMA and a hybridoma cell strain, the antibody has strong affinity with the BCMA antigen, the Kd value is 1.892*10 ‑8 M, the antibody can compete with a commercial BCMA antibody 19F2 to bind a target cell; the antibody can specifically bind to a BCMA positive cell line H929, and has no cross reaction with a BCMA negative cell line Raji and peripheral blood derived T cells; the monoclonal antibody 2D2 can be used for detecting cells expressing human BCMA, and can also be applied in tumor immunotherapy targeting human BCMA, and has diagnostic and therapeutic values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, the present application relates to a murine monoclonal antibody specifically binding to human B cell maturation antigen (BCMA). BACKGROUND

[0002] Multiple myeloma (MM) is a kind of hematological malignancies, which is manifested as abnormal malignant proliferation of bone marrow plasma cells, and is often seen in the elderly. In clinical practice, it often causes anemia, hypercalcemia, renal function damage, bone pain and other bone marrow tumor related organ function damage manifestations. Bone pain is the most common and most affecting patient quality of life symptom. B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily, which is a single transmembrane glycoprotein. It binds to the corresponding ligand B cell-activating factor (BAFF) and Aproliferation-inducing ligand (APRIL), activates the NF-kappaB signaling pathway, and regulates B cell survival, maturation and differentiation.

[0003] Because BCMA is only highly expressed on the surface of plasma cells and multiple myeloma cells, and is not expressed on the surface of other normal cells, and the expression level of BCMA on the surface of multiple myeloma cells is significantly higher than that of healthy plasma cells, which makes BCMA an important biomarker for MM diagnosis and a potential immunotherapy target. Therefore, high specificity is an important standard for the development of BCMA targeting antibodies. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a murine anti-human BCMA antibody or antigen binding fragment thereof, which has high affinity and strong specificity for BCMA and does not bind to BCMA negative cells. It can be used for the development of MM diagnostic antibodies, or for the construction of genetically engineered antibodies for tumor immunotherapy.

[0005] The technical scheme adopted by the present application is:

[0006] An isolated antibody or antigen binding fragment thereof, which specifically binds to human B cell maturation antigen (BCMA), the antibody or antigen binding fragment thereof comprising:

[0007] (a) the complementarity determining region (CDRH) of the heavy chain variable region: which is selected from at least one of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3; and / or

[0008] (b) a complementarity determining region (CDRL) of the light chain variable region: which is selected from at least one of the amino acid sequences as set forth in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDRH1 as set forth in SEQ ID No. 1, a heavy chain variable region CDRH2 as set forth in SEQ ID No. 2, and a heavy chain variable region CDRH3 as set forth in SEQ ID No. 3; and / or a light chain variable region CDRL1 as set forth in SEQ ID No. 4, a light chain variable region CDRL2 as set forth in SEQ ID No. 5, and a light chain variable region CDRL3 as set forth in SEQ ID No. 6.

[0010] In one embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDRH1 as set forth in SEQ ID No. 1, a heavy chain variable region CDRH2 as set forth in SEQ ID No. 2, and a heavy chain variable region CDRH3 as set forth in SEQ ID No. 3; and a light chain variable region CDRL1 as set forth in SEQ ID No. 4, a light chain variable region CDRL2 as set forth in SEQ ID No. 5, and a light chain variable region CDRL3 as set forth in SEQ ID No. 6.

[0011] In the present application, the above-mentioned antibody or antigen-binding fragment thereof is referred to as 2D2.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region as set forth in SEQ ID No. 7 and amino acid sequences having 90%, 95% or 99% or more identity thereto; and / or, a light chain variable region as set forth in SEQ ID No. 8 and amino acid sequences having 90%, 95% or 99% or more identity thereto.

[0013] In some embodiments, the antibody can be of mammalian origin, such as murine, etc. In other embodiments, the antibody can be a recombinant antibody, a humanized antibody or a fully human antibody.

[0014] In some embodiments, the antibody can be IgG, IgA, IgM, IgD or IgE. As preferred, in some embodiments, the type of the antibody can be IgG. Further, in some embodiments, the antibody can be one or several selected from IgGl, IgG2, IgG3 or IgG4. Preferably, the antibody can be of IgG2b subtype.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof is modified, the modification includes N-glycosylation modification, O-glycosylation modification, phosphorylation modification, methylation modification, acetylation modification, or label modification.

[0016] In some embodiments, the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, or single chain antibody scFv. Another aspect of the present application provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises the antibody or antigen-binding fragment thereof described above, and the intracellular signaling domain comprises one or more co-stimulatory signaling domains.

[0017] In some embodiments, the T cell comprises the chimeric antigen receptor described above.

[0018] Another aspect of the present application provides a multivalent antibody comprising the antibody or antigen-binding fragment thereof described above.

[0019] The multivalent antibody can be, for example, bivalent, trivalent, tetravalent, hexavalent, nonavalent, etc. The multivalent antibody can be prepared by using suitable methods in the art. Preferably, in some embodiments, the multivalent antibody is a bispecific antibody or a trispecific antibody.

[0020] Another aspect of the present application provides a multispecific antibody selectively binding to human BCMA, the multispecific antibody comprising the antibody or antigen-binding fragment thereof described above; the multispecific antibody is a monovalent antibody or a multivalent antibody.

[0021] Another aspect of the present application provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof described above, or encoding the multivalent antibody described above. Further, in some embodiments, the isolated nucleic acid encodes an amino acid sequence as shown in SEQ ID Nos. 1-8.

[0022] Another aspect of the present application provides a recombinant expression vector comprising the isolated nucleic acid described above.

[0023] Another aspect of the present application provides a host cell comprising the antibody or antigen-binding fragment thereof described above, the chimeric antigen receptor described above, the T cell modified by the chimeric antigen receptor described above, the multivalent antibody described above, the polynucleotide described above, or the vector described above. In some embodiments, the host cell can be any suitable host cell as a tool for producing target protein. For example, eukaryotic expression cell, prokaryotic expression cell.

[0024] In some embodiments, the host cell is preferably a eukaryotic cell, which is a CHO cell, a 293 cell, a 293T cell, a 293FT cell, a Per6 cell; more preferably, the host cell is a CHO cell.

[0025] Another aspect of the present application provides a method for producing an anti-human BCMA antibody or an antigen-binding fragment thereof, which comprises obtaining the anti-human BCMA antibody or the antigen-binding fragment thereof by performing protein expression on the above-mentioned cell.

[0026] Another aspect of the present application provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned chimeric antigen receptor, the above-mentioned chimeric antigen receptor modified T cell, the above-mentioned multivalent antibody, the above-mentioned multispecific antibody, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned cell, and a pharmaceutically acceptable excipient. In order to achieve better therapeutic effect, in some embodiments, the pharmaceutical composition can further comprise other therapeutic drugs.

[0027] Another aspect of the present application provides an immunoconjugate comprising:

[0028] a) the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned multivalent antibody; and

[0029] b) a therapeutic agent or a detectable label; and

[0030] c) a linker connecting the two parts of a) and b);

[0031] wherein the therapeutic agent comprises a drug, an enzyme, a toxin, a cytokine, or a radionuclide.

[0032] Another aspect of the present application provides the use of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned chimeric antigen receptor, the above-mentioned chimeric antigen receptor modified T cell, the above-mentioned multivalent antibody, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned cell, the above-mentioned pharmaceutical composition, or the above-mentioned immunoconjugate in the preparation of a medicament for treating and / or preventing a BCMA-mediated disease.

[0033] In some embodiments, the BCMA-mediated disease is a BCMA-positive tumor, an autoimmune disease, a graft-versus-host disease, or a light chain amyloidosis.

[0034] In some embodiments, the BCMA-positive tumor comprises multiple myeloma, diffuse large B-cell lymphoma, Waldenstrom macroglobulinemia, lymphoplasmacytic lymphoma, small lymphocytic lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin lymphoma, megakaryocytic leukemia.

[0035] In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome.

[0036] Another aspect of the present application provides a BCMA detection kit, which comprises the antibody or antigen-binding fragment thereof, the multivalent antibody, or the multispecific antibody.

[0037] Another aspect of the present application provides the use of the antibody or antigen-binding fragment thereof, the multivalent antibody, or the multispecific antibody in the preparation of a BCMA detection and diagnosis reagent.

[0038] The present application has the following beneficial effects:

[0039] The technical solution used in the present application is that, by using mouse hybridoma screening technology and RT-PCR method to clone Ig variable region genes, a hybridoma cell strain stably secreting specific anti-human BCMA antibody and the variable region sequence thereof are obtained, and the specificity and affinity activity of the antibody are identified by flow cytometry.

[0040] The anti-BCMA monoclonal antibody 2D2 provided by the present application has strong affinity with the BCMA antigen, and the Kd value is 1.892×10 -8 M; the light chain is a κ chain; can compete with the commercial BCMA antibody 19F2 for binding to target cells; can specifically bind to the BCMA positive cell line H929, and has no cross reaction with the BCMA negative cell line Raji and peripheral blood derived T cells. Due to these characteristics, the monoclonal antibody 2D2 can be used for detecting cells expressing human BCMA, and can be used alone or in combination with other methods in targeted tumor immunotherapy of human BCMA, and has diagnostic and therapeutic values. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Figure 2 is a subtype identification diagram of the 2D2 antibody;

[0042] Figure 2 Figure 4 is a SDS-PAGE diagram of the 2D2 pure antibody, wherein lane 1 is Non-reducing, lane 2 is Marker, and lane 3 is Reducing;

[0043] Figure 3 Figure 6 is an affinity constant analysis diagram of the PE-labeled antibody 2D2 and 3T3-BCMA cells;

[0044] Figure 4 Figure 8 is a diagram of the FACS method for detecting the binding of the antibody 2D2 and the human BCMA positive cell line H929, wherein a is a homologous control, and b is 2D2;

[0045] Figure 5FACS method for detecting cross-reaction of antibody 2D2 with human BCMA negative cell line Raji, wherein a is isotype control, b is BCMA commercial antibody 19F2, and c is 2D2;

[0046] Figure 6 FACS method for detecting cross-reaction of PE-labeled antibody 2D2 with human peripheral blood-derived T cells, wherein a is isotype control, b is BCMA commercial antibody 19F2, and c is 2D2;

[0047] Figure 7 FACS method for detecting competition of antibody 2D2 with BCMA commercial antibody 19F2 for binding to BCMA protein on the surface of 3T3-BCMA cells. DETAILED DESCRIPTION

[0048] The present application discloses a kind of anti-human BCMA monoclonal antibody and its application, and those skilled in the art can learn from the content of this paper, and realize by improving process parameters appropriately.It needs to be pointed out specially, all similar substitutions and changes are obvious to those skilled in the art, they are regarded as including in the present application, and relevant personnel obviously can change or change and combine appropriately described in this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0049] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, the experimental techniques herein use conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA.

[0050] Antibody or antigen-binding fragment thereof

[0051] In the present application, the term "BCMA" used herein is a member of the tumor necrosis factor receptor (TNFR) superfamily, which can bind to B-cell activating factor or B-lymphocyte stimulator and proliferation-inducing ligand. Multiple myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasma cells. BCMA RNA is commonly detected in MM cells, and BCMA protein can be detected on the surface of plasma cells in multiple myeloma patients.

[0052] In the present application, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2 and Fv. In some embodiments herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0053] An antibody refers to an immunoglobulin molecule that is composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. The light chains can be classified as kappa and lambda light chains. The heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a class of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antibody binding site. The term "antibody" is not limited by the method in which the antibody is produced. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody. In some embodiments, the antibody can be an IgG, IgA, IgM, IgD, or IgE. As a preference, in some embodiments, the type of the antibody can be IgG. Further, in some embodiments, the antibody can be one or more selected from IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody can be of IgG2b subtype.

[0054] The term "antigen binding fragment" in the present application refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as "antigen binding fragment". The antigen binding fragment of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, the antigen binding fragment includes Fab, Fab', F(ab')2, Fv, etc.

[0055] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy- chain variable domain and one light-chain variable domain connected by a pair of disulfide bonds. The three CDRs in each variable domain are from amino acid residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain and 26-32 (H1), 53- 56 (H2) and 96-101 (H3) in the heavy chain. While these are the only regions required to bind antigen, the framework regions, which often interact with the CDRs, can contribute to the antigen binding affinity and specificity as well. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0056] wherein the term "Fab fragment" means an antibody fragment consisting of a VL, a VH, a CL and a CH1 domain; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge over the hinge region. The term "Fd fragment" means an antibody fragment consisting of a VH and a CH1 domain; the term "Fv fragment" means an antibody fragment consisting of a VL and a VH domain of a single arm of an antibody.

[0057] "Fc region" (fragment, crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. In IgG antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of each of the antibody's two heavy chains.

[0058] Nucleic Acids, Expression and Chimeric Antigen Receptors

[0059] The term "isolated nucleic acid" in the present invention can also be used interchangeably as "nucleic acid" refers to a chain of nucleotides of any length and includes DNA or RNA. It can include any known nucleotide analog or modified nucleotide or base.

[0060] The present invention also provides polynucleotides encoding the various antibodies or fragments thereof described above. Polynucleotides encoding the heavy chain variable region, the light chain variable region, the heavy chain, the light chain, and each CDR are provided herein. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0061] Once the relevant sequence has been obtained, it can be obtained in large quantities using recombinant methods. This is usually done by cloning it into a vector, which is then transferred into a cell, and then isolated from the host cell after propagation by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in isolated form. At present, it is possible to obtain the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or as a vector) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0062] The term "expression vectors" in the present invention refers to vectors that have expression elements (such as promoters, RBS, terminators, etc.) added to the basic backbone of the cloning vector, so that the target gene can be expressed. The expression vector has four parts: the target gene, the promoter, the terminator, and the marker gene. The present invention includes but is not limited to prokaryotic cell expression vectors, eukaryotic cell expression vectors, or other cell expression vectors.

[0063] The term "lentivirus" used herein is a genus under the family of retroviruses, including 8 viruses that can infect humans and vertebrates, with lymphocytes and macrophages being the main cells of primary infection, and the infected individuals eventually developing the disease. Lentivirus species such as human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia (EIA), feline immunodeficiency virus (FIV). Lentivirus vectors have developed rapidly and have been studied in depth. This vector can effectively integrate foreign genes into the host chromosome, thereby achieving persistent expression. In terms of infection ability, it can effectively infect various types of cells such as neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells, thereby achieving good gene therapy effect. In addition, those skilled in the art can also select other suitable vectors in addition to lentivirus, which are within the scope of protection of the present invention.

[0064] Chimeric antigen receptor (CAR) is the core component of "chimeric antigen receptor T cell (CART)", which gives T cells the ability to recognize tumor antigens in a non-dependent manner, which makes CAR-modified T cells can recognize a wider range of targets compared to natural T cell surface receptors. The basic design of CAR includes a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.

[0065] Preparation of antibodies:

[0066] In some embodiments, the antibodies are produced using mammalian cells. For example, monoclonal antibodies are produced in mammalian cells using hybridoma technology. First, mice or other suitable host animals are immunized with an immunogen, if necessary with an adjuvant. The immunogen or adjuvant is usually injected subcutaneously in multiple sites or intraperitoneally. The adjuvant can be Freund's adjuvant (Freund's complete or incomplete adjuvant) or MPL-TDM, etc. After the animals are immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen are produced in the animals. The desired lymphocytes are collected and fused with myeloma cells using a suitable fusing agent, such as PEG4000, to form hybridoma cells. The hybridoma cells prepared as described above are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth of the unfused, parental myeloma cells.

[0067] The culture medium in which the hybridoma cells are grown is used to detect the production of monoclonal antibodies specific to the specific antigen. The following methods can be used to determine the binding specificity of the monoclonal antibodies produced by the hybridoma cells: immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0068] After the specificity, affinity and reactivity of the antibodies produced by the hybridoma are determined, the desired cell lines can be subcloned by limiting dilution. Suitable culture media can be DMEM or RPMI-1640, etc. In addition, the hybridoma cells can also be grown in the form of ascites tumors in animals.

[0069] The monoclonal antibodies secreted by the subcloned cells can be isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification methods, such as protein A sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, etc. to obtain the monoclonal antibodies.

[0070] In other embodiments, antibodies against human BCMA can also be produced by known recombinant methods, for example, by selecting a recombinant antibody library in phage or similar vectors.

[0071] Modification and modification of antibodies:

[0072] In some embodiments, the isolated antibodies can be humanized antibodies. Antibody humanization or can improve the affinity or other characteristics of the antibody.

[0073] In some embodiments, the antibody Fc (fragment, crystallizable region, Fc) is engineered to enhance its effector functions triggered by binding to Fc receptors or complement. These functions can include: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The above engineering can include: 1) engineering glycosylation, for example, the asparagine (N297) at position 297 of the Fc region can be modified by N-acetylglucosamine. Mutation of N297 to alanine (A), glutamine (Q), or glycine (G) will hinder the glycosylation of the antibody, thereby reducing the Fc-mediated effector function. After the antibody is deglycosylated, the ability to induce ADCC or CDC activity will decrease; sialic acid modification will reduce the binding to FcγRIIIa, thus leading to a decrease in CDC and ADCC activity.

[0074] In addition to the above functions, modification of the glycosylation of the antibody can also affect the conformation and stability of the antibody. For example, the sugar chain in the glycosylation modification can maintain the conformation of the antibody, preventing its aggregation or unfolding. At the same time, the glycosylation modification can also affect the binding of the antibody to the receptor on the cell membrane to form a complex, thus playing an important role in the signal transduction process. The regulation of this signal transduction is crucial for physiological processes such as cell proliferation, differentiation, and apoptosis.

[0075] The above engineering can also include: 2) point mutations, in some embodiments, the point mutations result in the substitution of some conservative amino acids, thereby obtaining a "conservative amino acid substitution variant". The changes result in the substitution of some amino acids by others that are similar in chemical properties and / or functions. The provision of conservative amino acid substitution tables that provide similar amino acids is well known in the art.

[0076] The above modification can also include: 3) phosphorylation modification. Phosphorylation modification refers to the process of adding phosphate groups to amino acids in proteins within cells. Phosphorylation antibodies can specifically recognize specific phosphorylation sites, thereby detecting the increase or decrease in the phosphorylation level of proteins when cells are stimulated. These antibodies play an important role in the fields of life science research such as cell signaling, apoptosis and cancer. 4) Methylation modification: Methylation modification is an important dynamic modification and biological phenomenon catalyzed by methyltransferase on specific residues of proteins. Methylation antibodies can specifically recognize specific methylation amino acid sites to distinguish between methylated and non-methylated forms of proteins. They have wide applications in research fields such as epigenetics, cancer, Alzheimer's disease and aging. 5) Acetylation modification. Acetylation is one of the most common types of acylation modification. Acetylation antibodies can specifically recognize the acetylated form of target proteins and specific acetylated amino acid sites to detect the activity level of the protein. These antibodies are widely used in the study of cell cycle regulation, signal transduction, neurodegenerative diseases, metabolic diseases and the development of cancer.

[0077] The above modification can also include: 6) marker modification. Antibodies can be cross-linked by different chemical reagents to be connected to enzymes, fluorescent dyes, biotin or colloidal gold and other substances to change their detection or analysis performance. For example, enzyme labeling: antibodies can be cross-linked to enzymes such as horseradish peroxidase (HRP), alkaline phosphatase, etc. Commonly used in immunohistochemistry, ELISA and other experiments, color reaction is generated through the catalytic action of enzymes to detect the presence of antibodies. Fluorescent dye labeling: antibodies can also be combined with fluorescent dyes (such as FTC, PE, APC, etc.) for flow cytometry, fluorescence microscopy and other detection methods. Fluorescently labeled antibodies can locate specific antigens in cells or tissue sections and determine the expression level of antigens by the strength of the fluorescent signal. Biotin labeling: biotin is a small molecule compound that can be combined with antibodies without affecting their antigen binding ability. Biotin-labeled antibodies can be combined with avidin (such as streptavidin) to amplify and detect signals. Commonly used in multiple immunolabeling experiments to detect multiple antigens at the same time.

[0078] In the present application, the modification and engineering of the antibody are usually performed on the Fc segment of the antibody and the framework regions (FRs) of the variable region of the antibody, but not on the complementarity-determining regions (CDRs) of the variable region of the antibody. The framework regions of the variable region of the antibody, whose amino acid sequences are relatively conserved, provide stable support for the structure of the hypervariable region and participate in maintaining the three-dimensional conformation of the antigen binding groove. Therefore, the engineering and modification thereof do not affect the binding ability of the antibody. In the present application, the term "having more than 90% identity" means that, under the above-mentioned engineering or modification, there is about 90%, about 91%, about 92%, about 93%, about 94, about 95%, about 96%, about 97%, about 98%, about 99% identity (identity) compared with the sequence shown as SEQ ID No. 1-8.

[0079] Multivalent antibodies and multispecific antibodies:

[0080] In some embodiments, the multivalent antibody comprises at least two antibodies or antigen-binding fragments described in the present application, which can competitively bind to the human BCMA molecule and produce different effects from the monovalent antibody, for example, by increasing the number of antigen binding sites, a tighter antigen-antibody complex can be formed, thereby enhancing the binding force and stability. This enhanced binding force helps to improve the affinity of the anti-human BCMA antibody to the antigen and enhances the interaction of the antigen with the cell surface receptor or other molecules. The multivalent antibody can be obtained by, for example, protein fusion, increasing the linker, covalent bond or non-covalent bond.

[0081] In some embodiments, the multispecific antibody, for example, bispecific antibody, trispecific antibody, comprises the antibody or antigen-binding fragment thereof described in the present application, which can bind to at least one other different site or target molecule in addition to competitively binding to the human BCMA molecule. The multispecific antibody can be linked together by direct connection or through a linker. The multispecific antibody can be expressed by recombinant method. In order to achieve better therapeutic effect, the multispecific antibody can be monovalent or multivalent. The above-mentioned multivalent antibody and multispecific antibody can be prepared by using conventional techniques in the art.

[0082] Pharmaceutical compositions, immunoconjugates and therapeutic uses thereof:

[0083] The term "pharmaceutical composition" in the present application refers to a composition comprising at least one other substance in addition to the antibody, the antigen binding fragment, the multivalent antibody, the polynucleotide, the vector, the host cell, or the multispecific antibody described in the present application. In some embodiments, the other substance can be, for example, a pharmaceutically acceptable carrier (a substance that does not affect the action of the antibody and has no effect on the physical condition of the patient, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and combinations thereof), an excipient, a stabilizer, a surfactant, a preservative, an isotonic agent, etc. It can also be other therapeutic agents, such as chemotherapeutic drugs: melphalan, doxorubicin, cyclophosphamide, vincristine, etc.; glucocorticoid drugs: prednisone, dexamethasone, betamethasone, etc.; immunomodulatory drugs: thalidomide, lenalidomide, pomalidomide, etc.; and biological agents against exemplary target points: CD33, CD123, CLEC12A, CD47, FLT-3, PDGFR, VEGFR, KIT, IDH1, IDH2, SMO, BCL-2, ALT, c-KIT, CD70, CD45, PD-1 / PD-L1, CTLA-4, TIM-3, TLR-2, CD3, CD4, IL-2, CD20, BAFF, TLR-4, TLR-7, TLR-8, TNF-α, IL-6, IL-6R, IL-17A, IL-17RA, IL-12, IL-23, IL-4, α4 integrin, cell adhesion molecule, complement factor D, JAK1, JAK2, JAK3, TYK2, IL-5, URAT1, TSLP, MASP-2, CSF1R, ROCK2. The above biological agents can be antibodies, small molecule inhibitors, or agonists against the target point.

[0084] In some embodiments, the above pharmaceutical composition can be prepared into any suitable preparation. For example, a pill, a tablet, a cream, a gelatin capsule, a capsule, a suppository, a soft gelatin capsule, a gel, a film, a tube, a solution, or a suspension.

[0085] In some embodiments, the immunoconjugate provided by the present application can be in any suitable form, such as an antibody-drug conjugate (ADC), a radionuclide drug conjugate (RDC), an antibody fusion protein, etc.

[0086] The antibody drug conjugates described above comprise an antibody, antigen binding fragment, multivalent antibody or multispecific antibody described herein, a linker, and a payload. Known linkers include, for example, N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), hydrazone linkers, Val-Cit dipeptide, tetrapeptide Gly-Gly-Phe-Gly, glucuronide-containing linkers, beta-galactosidase-containing linkers, and the like. Known payloads include, for example, haxan, maytansine derivatives, Tubulysins, Cryptomycins (CR), pyrrolo[2, 1-c][l,4]benzodiazepines (PBDs), duocarmycins, camptothecins (CPTs), calicheamicins, apoptosis inducers, thailanstatin A, amatoxins, nicotinamide phosphoribosyltransferase, a carmofur, and the like.

[0087] All aspects of the various antibodies described herein can be used to prepare a medicament for the prevention or treatment of the various conditions and diseases described herein, particularly conditions associated with BCMA-mediated diseases. In some embodiments, the BCMA-mediated disease is a BCMA-positive tumor, an autoimmune disease, graft versus host disease, or light chain amyloidosis, preferably multiple myeloma.

[0088] Diagnosis, detection, and kits

[0089] The antibodies of the present application, due to their high affinity for BCMA, can be used in assays, such as binding assays, to detect and / or quantify BCMA expressed in tissues or cells. The method of detecting BCMA is generally as follows: obtaining a sample of cells and / or tissue; detecting the level of BCMA in the sample

[0090] The anti-BCMA antibodies of the present application can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with BCMA. The present application provides for detecting the presence of BCMA in a sample using classical immunohistological methods known to those of skill in the art. Detection of BCMA can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of BCMA include ELISA, FACS, RIA, and the like.

[0091] Examples:

[0092] The present application is further described in connection with the following specific examples, which are not intended to limit the scope of the application.

[0093] Example 1. Screening of mouse hybridoma monoclonal antibodies

[0094] The gene encoding the extracellular domain of human BCMA was constructed into the multiple cloning site of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP. After lentiviral transfection, human BCMA protein was stably expressed on the cell membrane of 3T3 cells. After multiple positive sorting, a 3T3-BCMA cell line stably expressing BCMA protein was established. The Genbank number of BCMA in this example is 608.

[0095] 3T3-BCMA cell line was used as immunogen, and 1×10 7 3T3-BCMA cells / 500 μL PBS / mouse were used for booster immunization at 3 and 5 weeks after the initial immunization. On the 8th day after the booster immunization, 10 μL of tail blood was collected from the mice and placed in 90 μL PBS. The blood was allowed to stand at room temperature for 1 hour and then centrifuged at 12,000 rpm for 10 minutes at 4°C. The serum was collected and diluted with PBS to different concentrations: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600. 3T3 and 3T3-BCMA cells were collected, washed once with PBS, and the cells were counted. 100 μL of serum at different dilutions was added to 1×10 6 For each test cell, serum from an unimmunized mouse was used as a negative control, incubated at 37°C for 30 minutes, and washed twice with PBS. 0.2 μL of APC-conjugated anti-mouse IgG secondary antibody was added, and the cells were incubated at 37°C in the dark for 30 minutes. The cells were resuspended in 200 μL of PBS buffer, and the percentage of antibody binding to the cells and the fluorescence intensity in the serum were measured by flow cytometry. The effective titer was defined as the dilution at which the mean fluorescence intensity of 3T3-BCMA cells was at least twice that of 3T3 cells. Fusion was performed only when the titer was above 6400. Three days before fusion, the immunized mice were pulsed with the immunogen via tail vein injection.

[0096] Spleen cells from successfully immunized mice were isolated under sterile conditions and washed twice with serum-free 1640 medium at 1200 rpm for 6 minutes. Afterwards, cells were fused with myeloma SP2 / 0 cells in the logarithmic growth phase (at a ratio of 10:1). The fusion process was performed in a 37°C water bath. 1 mL of preheated 50% PEG was added dropwise over 1 minute to the mixed and supernatant-discarded spleen and myeloma cell pellets. Then, 10 mL of preheated serum-free 1640 medium was slowly added dropwise over 5 minutes. The cell suspension was washed twice by centrifugation at 800 rpm for 6 minutes, the supernatant was discarded, and the cells were resuspended in 1640 medium containing HAT and plated in 96-well plates (2.5×10 7 cells / plate) and cultured at 37°C in 5% CO2.

[0097] When the cell clones are large enough under microscope, 100 μL of supernatant of the corresponding well is taken, 3T3 and 3T3-BCMA cells are mixed at a ratio of 1:1 as detection cells, the supernatant is incubated with the detection cells, and detection is performed. When the average immunofluorescence intensity of 3T3-BCMA is higher than that of 3T3, the well is taken as a positive well for next step of expansion culture. The positive hybridoma clone is expanded from a 96-well plate to a 24-well plate for culture for 3-5 days, and supernatant screening detection is performed again. The clone with a positive detection result is taken for next step of subcloning, and the remaining cells are frozen. The hybridoma cells in the 24-well plate are collected, counted, and adjusted to a cell density of 10 / mL, and 100 μL is plated in each well of a 96-well plate, which is cultured at 37°C and 5% CO2 for about 10 days. Cloning can be observed, and only the wells with single clones are selected for culture supernatant detection, which is detected by the same method as before. The clone with a positive detection result is continuously expanded to a 24-well plate for culture, and after supernatant detection again, the clone with a positive detection result is subjected to a second round of subcloning culture. Generally, after multiple rounds of subcloning culture, all detection wells are positive, and a stable hybridoma cell strain is obtained. The positive hybridoma culture supernatant is subjected to antibody subtype detection by using an antibody subtype detection test paper to detect the antibody subtype. The monoclonal antibody is referred to as 2D2, and the results are shown in Table 1. The 2D2 antibody is of murine IgG2b subtype, and the light chain is κ chain. The specific sequences of the heavy and light chain variable regions are shown in Table 1. Figure 1 Table 1: Sequences of the heavy and light chain variable regions of the monoclonal antibody 2D2

[0098] Table 1: Sequences of the heavy and light chain variable regions of the monoclonal antibody 2D2

[0099]

[0100] Example 2: Preparation and purification of ascites

[0101] The hybridoma cells are washed with sterile PBS solution, and injected intraperitoneally into Balb / c mice pre-sensitized with liquid paraffin at a cell amount of 5×10 6 / 0.5 mL PBS per mouse. The ascites is collected after 7-10 days, centrifuged at 3000 rpm and room temperature for 10 min to obtain the middle layer. The antibody is subjected to crude purification with 45% saturated ammonium sulfate. Specifically, 1 part of ascites is added with 1 part of PBS, 1.64 parts of saturated ammonium sulfate is added dropwise while stirring, and the mixture is placed at 4°C overnight. The supernatant is removed by centrifugation at 10000 rpm for 10 min, and the precipitate is dissolved with a small amount of PBS. The solution is dialyzed against PBS at 4°C for 24 h, and the solution is replaced 3 times during the dialysis. The purified antibody is further purified by using an AKTA protein purification system according to the purification manual provided by GE company, and subjected to further purification by using a 1 mL ProteinG purification pre-packed column. The obtained antibody is detected by SDS-PAGE, and the results are shown in FIG. 2. Figure 2As shown, the antibody has a whole band at 150 kDa, and after reduction by mercaptoethanol, heavy chain and light chain bands appear at 50 kDa and 25 kDa, respectively.

[0102] Example 3: Detection of monoclonal antibody titer

[0103] The antibody was labeled with PE fluorescence. The labeled antibody was incubated with 2 x 10 5 3T3-BCMA cells at room temperature for 30 min, and light was avoided. The cells were centrifuged at 1800 rpm for 10 min, the supernatant was discarded, and the cells were washed with PBS three times. The cells were resuspended in 200 μL of PBS, and the fluorescence intensity was determined by FACS. The Mean value was calculated. The Kd value of the antibody was calculated using data analysis software GraphPad Prism 7. The results are shown in Figure 3. Figure 3 As shown, the Kd value of the 2D2 antibody was 1.892 x 10 -8 M.

[0104] Example 4: Cloning of Ig variable region gene by RT-PCR method

[0105] The total RNA was extracted, and the cDNA library was synthesized, as follows:

[0106] The total RNA of the 2D2 hybridoma cell line was extracted using Trizol reagent (purchased from Invitrogen), and the total RNA was reversely transcribed into a cDNA library using M-MLV reverse transcriptase (purchased from Invitrogen). The heavy chain (VH) and light chain (VL) variable region gene fragments of the anti-human BCMA antibody were amplified by RT-PCR. The primer sequences are shown in Table 2.

[0107] Table 2: Primer sequences for amplifying heavy chain and light chain variable regions

[0108]

[0109] The PCR reaction system (50 μL) was prepared as follows:

[0110] cDNA: 2 μL; upstream primer (10 μM): 2 μL; downstream primer (10 μM): 2 μL; dNTP mixture: 2 μL; pfu DNA polymerase (5 U / μL): 1 μL; 10 x pfu Buffer II: 5 μL; ddH2O: supplemented to 50 μL. Reaction conditions: 95 °C pre-denaturation for 5 min; repeat the following cycle for 35 times: 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min; finally, 72 °C extension for 10 min. The VL and VH fragments were separated by agarose gel electrophoresis and recovered. The recovered VL and VH fragments were ligated with pMD19-T (simple) vector (Takara) by T4 ligase (Takara), and the ligation system was as follows: 70 ng of VL PCR product and 70 ng of VH PCR product, 1 μL of pMD19-T (simple) vector, 5 μL of Solution I ligation reaction solution; supplemented with ddH2O to 10 μL, 4 °C ligation overnight. The ligation product was transformed into E. coli DH5α competent bacteria, which was cultured at 37 °C overnight, and then a single colony was picked and cultured at 37 °C for 2 hours, followed by bacterial liquid PCR identification, with the cDNA of the corresponding antibody as a positive control. The reaction system (25 μL) was prepared as follows: bacterial liquid: 1 μL, upstream primer (10 μM): 1 μL; downstream primer (10 μM): 1 μL; dNTP Mixture (2.5 Mm each): 2 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; 10 x Taq Buffer (Mg 2+ plus): 2.5 μL; supplemented with water to 25 μL. The reaction conditions were the same as before. The clones with positive bacterial liquid PCR were selected for expansion culture, and the positive clone plasmid was extracted by plasmid extraction kit (Takara), and sent for sequencing. At least 5 clone samples of each chain of each antibody were sent for sequencing, and at least three samples with the same sequencing results were selected. The variable region sequences of the heavy chain and light chain of 2D2 were successfully cloned, which were consistent with the typical antibody variable region sequence characteristics.

[0111] Example 5: Specific binding to H929 cells with high expression of BCMA

[0112] FACS detection of the binding of antibody 2D2 to H929 surface BCMA protein: 1 µL of antibody (200 µg / mL) was mixed with 1 x 10 6H929 cells were incubated at room temperature for 30 min, washed twice with PBS; 100 μL of the resuspended cells were added with 0.2 μL of APC-labeled anti-mouse IgG secondary antibody, incubated at room temperature for 30 min in the dark, and washed twice with PBS. At the same time, a homotypic negative control tube and a BCMA commercial antibody 19F2 (Biolegend: B258546) were set as a positive control tube, and the concentration was 0.2 μg / test. The cells were resuspended in 200 μL of PBS buffer, and FACS detection was performed. The results are shown in Figure 4 Figure 2, which shows that 2D2 can effectively bind to H929 with strong affinity.

[0113] Example 6: Cross-reaction with BCMA-negative cells

[0114] 1 μL of 2D2 antibody (200 μg / mL) was incubated with 2 x 10 5 Raji cells (BCMA-negative), and a BCMA commercial antibody 19F2 (Biolegend: B258546) was used as a control with a concentration of 0.2 μg / test. The cells were incubated at room temperature for 30 min, washed twice with PBS; 100 μL of the resuspended cells were added with 0.2 μL of APC-labeled anti-mouse IgG secondary antibody, incubated at room temperature for 30 min in the dark, and washed twice with PBS; and the cells were resuspended in 200 μL of PBS buffer, and FACS detection was performed. The results are shown in Figure 5 Figure 3, which shows that the antibody 2D2 has no cross-reaction with Raji cells.

[0115] Example 7: Cross-reaction of the antibody 2D2 with normal human peripheral blood-derived T cells

[0116] Normal human peripheral blood samples were taken, and human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation. T cells were obtained by sorting PBMCs with CD3-positive sorting magnetic beads. 2 x 10 5 T cells were incubated with 1 μL of PE-labeled 2D2 antibody (200 μg / mL) at room temperature for 30 min, and washed twice with PBS; and the cells were resuspended in 200 μL of PBS buffer, and FACS detection was performed. A BCMA commercial antibody 19F2 (Biolegend: B258546) was used as a control with a concentration of 0.2 μg / test. The results are shown in Figure 6 Figure 4, which shows that the 2D2 antibody has no non-specific binding to normal human peripheral blood-derived T cells.

[0117] Example 8: FACS detection of the competition relationship between the antibody 2D2 and the BCMA commercial antibody 19F2

[0118] 2D2 antibody 0.012 μg, 0.06 μg, 0.3 μg, 1.5 μg, respectively, while adding 0.2 μg of BCMA commercial antibody 19F2 (Biolegend: B258546) to each well, adjusting the final volume of each well to 200 μL with PBS, respectively, and incubating with 2 x 10 5 3T3-BCMA cells at room temperature in the dark for 30 min. Centrifugation at 1200 rpm for 6 min, discarding the supernatant, washing the cells with PBS, repeating three times, resuspending the cells in 200 μL of PBS, and measuring the fluorescence intensity by FACS. The results are shown in Figure 7 FIG. 2, with the fluorescence intensity of the binding of the commercial antibody 19F2 to the 3T3-BCMA cells in the same tube gradually weakening as the amount of 2D2 increases, i.e., the antibody 2D2 can effectively compete with the commercial BCMA antibody 19F2, and the competition is in a gradient-dependent manner.

[0119] The above only describes the preferred embodiments of the present application, and it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. An isolated antibody or antigen-binding fragment thereof, characterized in that The antibody or antigen-binding fragment thereof specifically binds to human B cell maturation antigen, and the antibody or antigen-binding fragment thereof comprises: The heavy chain variable region CDRH1 shown in SEQ ID No. 1, the heavy chain variable region CDRH2 shown in SEQ ID No. 2, and the heavy chain variable region CDRH3 shown in SEQ ID No. 3; and The light chain variable region CDRL1 is shown in SEQ ID No.4, the light chain variable region CDRL2 is shown in SEQ ID No.5, and the light chain variable region CDRL3 is shown in SEQ ID No.

6.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: The heavy chain variable region as shown in SEQ ID No. 7 or an amino acid sequence having more than 90% identity with the heavy chain variable region, wherein the difference in amino acids is a conservative substitution; and The light chain variable region is as shown in SEQ ID No. 8, or an amino acid sequence having more than 90% identity with the light chain variable region, and the different amino acids are conservative substitutions.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a recombinant antibody.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a humanized antibody.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is of IgG2b subtype.

6. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is modified, and the modification includes N-glycosylation modification, O-glycosylation modification, phosphorylation modification, methylation modification or acetylation modification.

7. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The antigen-binding fragment is Fab, Fab', F(ab')2, Fv fragment or single-chain antibody scFv.

8. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, characterized in that: The extracellular domain comprises the antigen-binding fragment according to any one of claims 1 to 7, and the intracellular signaling domain comprises one or more costimulatory signaling domains.

9. A chimeric antigen receptor modified T cell, characterized in that The T cell comprises the chimeric antigen receptor according to claim 8.

10. A multivalent antibody, characterized in that The multivalent antibody comprises the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

11. A multispecific antibody, characterized in that The multispecific antibody selectively binds to at least human BCMA, and the multispecific antibody comprises the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

12. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or encodes the multivalent antibody according to claim 10 or the multispecific antibody according to claim 11.

13. The isolated nucleic acid according to claim 12, characterized in that The isolated nucleic acid encodes the amino acid sequence shown in SEQ ID No. 1-8.

14. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 12 or 13.

15. A host cell, characterized in that The host cell comprises the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the multivalent antibody according to claim 10, the multispecific antibody according to claim 11, the isolated nucleic acid according to claim 12 or 13, or the recombinant expression vector according to claim 14.

16. A method for producing an anti-human BCMA antibody or an antigen-binding fragment thereof, characterized in that: After protein expression in the host cell according to claim 15, the anti-human BCMA antibody or antigen-binding fragment thereof is obtained.

17. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the chimeric antigen receptor according to claim 8, the chimeric antigen receptor-modified T cell according to claim 9, the multivalent antibody according to claim 10, the multispecific antibody according to claim 11, the isolated nucleic acid according to claim 12 or 13, the recombinant expression vector according to claim 14 or the host cell according to claim 15, and pharmaceutically acceptable excipients.

18. The pharmaceutical composition according to claim 17, characterized in that The pharmaceutical composition also contains other therapeutic drugs.

19. An immunoconjugate, characterized in that The immunoconjugate comprises: a) the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the multivalent antibody according to claim 10, or the multispecific antibody according to claim 11; and b) a therapeutic agent or a detectable marker; and c) a connector between parts a) and b) above; Wherein, the therapeutic agent includes a drug, an enzyme, a toxin, a cytokine or a radionuclide.

20. Use of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the chimeric antigen receptor according to claim 8, the chimeric antigen receptor-modified T cell according to claim 9, the multivalent antibody according to claim 10, the multispecific antibody according to claim 11, the isolated nucleic acid according to claim 12 or 13, the recombinant expression vector according to claim 14, the host cell according to claim 15, the pharmaceutical composition according to claim 17 or 18, or the immunoconjugate according to claim 19 in the preparation of a medicament for treating and / or preventing BCMA-mediated diseases; The BCMA-mediated disease is a BCMA-positive tumor.

21. The use according to claim 20, characterized in that The BCMA-positive tumors include multiple myeloma, diffuse large B-cell lymphoma, Waldenstrom's macroglobulinemia, lymphoplasmacytic lymphoma, small lymphocytic lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma or megakaryocytic leukemia.

22. A BCMA detection kit, characterized in that The BCMA detection kit comprises the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and / or the multivalent antibody according to claim 10 or the multispecific antibody according to claim 11.

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