Bispecific antibody that binds to BCMA and CD3, method for producing the same, and use thereof

Abstract

The present invention relates to a gene 1689840495729_10 The present invention relates to the technical field of antibodies, specifically to a bispecific antibody that binds to BCMA and CD3, and the preparation method and use thereof. The present invention provides a bispecific antibody that can simultaneously bind to BCMA and CD3, has a specific targeting effect, and can efficiently induce directed immune responses. The bispecific antibody fully retains the biological functions of anti-CD3 antibody and anti-BCMA antibody, and realizes that one bispecific antibody molecule simultaneously has excellent biological functions of target binding to BCMA and CD3, can act as a bridge between tumor cells and immune effector cells, can effectively induce immune effector cells and directed immune responses, significantly improves the killing effect of immune cells against tumor cells, has high killing efficiency against target cells, minimizes the ADCC effect, has high safety, and has good prospects for use in tumor immunotherapy.

Description

cross reference

[0001] This application claims priority from Chinese Patent Application No. 202110704255.6, filed on June 24, 2021, entitled "Bispecific antibodies that bind to BCMA and CD3, and methods for preparing and using the same," the entire contents of which are incorporated herein by reference. [Technical field]

[0002] The present application relates to the technical field of recombinant antibodies, in particular to bispecific antibodies that bind BCMA and CD3, and methods for making and using same. [Background technology]

[0003] Monoclonal antibodies are used in clinical practice for tumor treatment, immune disease treatment, and anti-infection treatment. Among them, tumor treatment is currently the most widely used field for monoclonal antibodies, and tumor treatment with monoclonal antibodies is an immunotherapy that stimulates the immune system against specific targets of diseased cells to kill the target cells. Based on monoclonal antibodies, in order to further improve the efficacy and reduce toxicity and side effects, new antibody drugs based on monoclonal antibodies, such as antibody drug conjugates (ADCs), bispecific antibodies (bsAbs) and CAR-T (chimeric antigen receptor T cells), are being rapidly developed. Among them, cell-bridging bispecific antibodies constructed based on bispecificity have been particularly noted for their significantly improved killing activity and specificity due to their ability to link to immune T cells and target cancer cells, which has become of revolutionary clinical value. For multiple myeloma, cell-crosslinking bispecific antibodies with the above-mentioned properties targeting BCMA are one of the development targets for improving the therapeutic effect of antibodies, and have become a hotspot in the research field of antibody engineering.

[0004] The CD3 molecule on the surface of T cells is composed of four subunits, δ, ε, γ, and ζ, with molecular weights of 18.9 kDa, 23.1 kDa, 20.5 kDa, and 18.7 kDa, respectively, and lengths of 171, 207, 182, and 164 amino acid residues, respectively. They form six peptide chains and always bind closely to the T cell receptor (TCR) to form the TCR-CD3 complex containing eight peptide chains, the structure of which is shown in Figure 1A. This complex has functions such as T cell activation, signal transduction, and stabilization of the TCR structure. The CD3 cytoplasmic domain contains an immunoreceptor tyrosine-based activation motif (ITAM), and in order for TCR to recognize and bind to peptide antigens presented by major histo-compatibility complex (MHC) molecules, tyrosine residues within the conservative sequence of ITAM in CD3 are phosphorylated by the tyrosine protein kinase p56lck in T cells, which then recruits other tyrosine protein kinases containing SH2 (Scr homology 2) domains (e.g., ZAP-70). Phosphorylation of ITAM and binding to ZAP-70 are one of the important biochemical reactions at the early stage of the T cell activation signaling process. Thus, the function of the CD3 molecule is to transmit activation signals by antigen recognition via TCR.

[0005] BCMA (i.e. B-cell maturation antigen, TNFRSF17, CD269) is a non-glycosylated type I transmembrane protein belonging to the tumor necrosis (TNF) receptor superfamily that is preferentially expressed on differentiated plasma cells and is involved in B-cell maturation, growth, and survival. BCMA was first reported as an integral membrane protein, i.e. an intracellular protein, in the Golgi apparatus of human mature B lymphocytes, indicating that BCMA appears to play an important role in B-cell development and homeostasis.

[0006] BCMA expression is restricted to the B cell lineage and is present primarily in plasma cells and plasmablasts (Figure 1B), to some extent in memory B cells, but virtually absent from peripheral B cells. BCMA is also expressed on multiple myeloma (MM) cells. BCMA, along with its family members transmembrane activator and cyclophilin ligand interactor (TACI) and B cell activator of the TNF family receptor (BAFF-R), regulates humoral immunity, B cell development, and various aspects of homeostasis. BCMA is the receptor for two TNF superfamily ligands (Figure 1C), APRIL (proliferation-inducing ligand, CD256, TNFSF13), a high affinity ligand for BCMA, and BAFF (THANK, BlyS, B lymphocyte stimulatory factor, TALL-1, and zTNF4), a low affinity ligand for BCMA. APRIL and BAFF display structural similarities as well as overlapping but distinct receptor binding specificities. The negative regulator TACI also binds to both BAFF and APRIL. APRIL and BAFF bind to the BCMA and / or TACI coordinates to activate the transcription factor NF-κB, which in turn increases the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1) and decreases the expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc.), thereby inhibiting cell apoptosis and promoting survival. This combination promotes B cell differentiation, proliferation, survival, and antibody production. BCMA expression appears at a later stage of B cell differentiation and favors the long-term survival of plasmablasts and plasma cells in the bone marrow.

[0007] BCMA is a very important B cell biomarker, widely present on the surface of MM cells, and is a popular immunotherapy target for MM and other hematological malignancies. At three consecutive ASCO annual meetings, BCMA has been a hot focus of the industry. According to a recent analysis by the National Cancer Institute (CRI), BCMA is the second most popular target of anti-cancer cell therapy after CD19. MM is a heterogeneous disease, often caused by t(11;14), t(4;14), t(8;14), del(13), del(17) (except others) chromosomal translocations. MM patients may experience a variety of symptoms associated with the disease, such as bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and psychological burden of a cancer diagnosis.

[0008] In MM, chemotherapy and stem cell transplant treatments can improve survival rates, but MM remains difficult to treat because of frequent adverse side effects. Although there have been great advances in chemotherapy, protease inhibitors, immunomodulatory thalidomide derivatives, and CD38-targeted antibodies, almost all patients eventually relapse. Thus, there is still an urgent need for new drugs in this field. To date, the two most common treatment options for patients with multiple myeloma are the use of steroids, thalidomide, lenalidomide, bortezomib, or a combination of various cytotoxic agents, and high-dose chemotherapy for younger patients, in conjunction with autologous stem cell transplantation. Most transplants are autologous, i.e., using the patient's own cells. Although such transplants are not curative, they have been shown to extend the lifespan of selected patients. They can be used as initial therapy for newly diagnosed patients or in the event of relapse. To adequately manage the disease, patients may be advised to undergo one or more transplants. The chemotherapeutic agents used to treat this disease are cyclophosphamide, doxorubicin, vincristine, and melphalan, which in combination with immunomodulatory agents such as thalidomide, lenalidomide, bortezomib, and corticosteroids (e.g., dexamethasone) have already become important options for treating myeloma for newly diagnosed patients and for patients with advanced disease who have failed chemotherapy or transplantation.

[0009] Currently, therapies used to treat MM are usually non-curative. Stem cell transplantation is not an option for most patients due to advanced age, other serious illnesses, or other physical limitations. Chemotherapy can only partially control multiple myeloma and rarely achieves complete remission. Therefore, new innovative therapies are highly desirable. Antagonistic BCMA-specific antibodies can block NF-κB activation, which is associated with a potent pro-survival signaling pathway in normal and malignant B cells.

[0010] Other approaches to combat blood-borne tumors or autoimmune diseases focus on the interaction between BAFF and APRIL (i.e., ligands of the TNF ligand superfamily) and the receptors TACI, BAFF-R and BCMA that are activated by BAFF and / or APRIL. For example, the Fc-domain of human immunoglobulin has been fused to Zymogenetics' TACI to produce atacicept (TACI-Ig), which neutralizes these two ligands and inhibits receptor activation. Atacicept is currently in clinical trials to treat systemic lupus erythematosus (SLE, stage III), multiple sclerosis (MS, stage II) and rheumatoid arthritis (RA, stage II), as well as in phase I clinical trials to treat the B-cell malignancies chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and MM. In preclinical studies, atacicept reduced the proliferation and survival of naive MM cells and MM cell lines in vitro and in vivo, indicating the relevance of the TACI ligand to MM cells. As most MM cells and their derived cell lines express both BCMA and TACI, both receptors may promote ligand-mediated proliferation and survival. These data indicate that antagonism against BCMA and TACI may be beneficial for the treatment of plasma cell disorders. In addition, a BCMA-specific antibody that cross-reacts with TACI has been reported (WO02 / 066516).

[0011] Human Genome Sciences and GlaxoSmithKline have developed an antibody called belimumab that targets BAFF. Belimumab can block the binding of soluble BAFF and its receptors BAFF-R, BCMA, and TACI to B cells. Although belimumab does not bind directly to B cells, by binding to BAFF it can inhibit the survival of B cells, including autoreactive B cells, and reduce the differentiation of B cells into immunoglobulin-producing plasma cells.

[0012] However, despite the fact that BCMA, BAFF-R, and TACI (i.e., B cell receptors belonging to the TNF receptor superfamily) and their ligands BAFF and APRIL have application in therapeutic approaches to combat cancer and / or autoimmune diseases, there remains a need for other available options to treat this condition. Summary of the Invention

[0013] First, an object of the present invention is to provide a bispecific antibody that binds to BCMA and CD3, and an active fragment thereof. A second object of the present invention is to provide a nucleic acid encoding the above-mentioned antibody or an active fragment thereof.

[0014] A third object of the present invention is to provide use of the above-mentioned antibody or an active fragment thereof. Specifically, the present invention provides the following technical solutions:

[0015] First, the present invention provides 1. A bispecific antibody that binds to BCMA and CD3, a first domain that binds to the B cell maturation antigen BCMA and a second domain that binds to the T cell surface antigen CD3; The first domain and the second domain are linked via a linker peptide, The first domain is a heavy chain variable region in which CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID NOs: 4 to 6, respectively, or have amino acid sequences containing one type of mutation selected from the following mutations or a combination of multiple types of mutations, each of which is based on the amino acid sequences shown in SEQ ID NOs: 4 to 6 as a reference sequence; (1) Mutation of the first S in the amino acid sequence shown in SEQ ID NO:4 to P or K (2) A mutation from the second Y in the amino acid sequence shown in SEQ ID NO:4 to H, D, G, M, or S. (3) A to N mutation at the third position of the amino acid sequence shown in SEQ ID NO:4 (4) A mutation from S at the 5th position of the amino acid sequence shown in SEQ ID NO:4 to H, N, A, or M. (5) A mutation from G to V or T at the first position of the amino acid sequence shown in SEQ ID NO:5. (6) A mutation from I to H at the third position of the amino acid sequence shown in SEQ ID NO:5 (7) Mutation of the fourth I in the amino acid sequence shown in SEQ ID NO:5 to D or A (8) Mutation from F to H at the 5th position of the amino acid sequence shown in SEQ ID NO:5 (9) A mutation from T to K at the 7th position of the amino acid sequence shown in SEQ ID NO:5. (10) Mutation from F to L at the fourth position of the amino acid sequence shown in SEQ ID NO:6

[0016] a light chain variable region in which CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively, or have amino acid sequences containing one type of mutation selected from the following mutations or a combination of multiple types of mutations, each of which is based on the amino acid sequences shown in SEQ ID NOs: 1 to 3 as a reference sequence; (1) A mutation from S at the 5th position of the amino acid sequence shown in SEQ ID NO:1 to N, R, or T. (2) A mutation from L at position 7 of the amino acid sequence shown in SEQ ID NO:3 to Q, V, or A (3) A mutation from I to S, A, R, or P at the 8th position of the amino acid sequence shown in SEQ ID NO:3. (4) A mutation from Y at the 10th position of the amino acid sequence shown in SEQ ID NO:3 to M, L, W, or T. (5) A mutation from V to L or Q at position 11 of the amino acid sequence shown in SEQ ID NO:3. The present invention provides a bispecific antibody that binds to BCMA and CD3, comprising:

[0017] The present invention screens for recombinant anti-BCMA single-chain antibodies from a fully synthetic single-chain human antibody library, obtains the sequences of the variable region genes of the antibodies, constructs a mutant library using a point mutation kit, obtains clones with high affinity, mixes the DNA of these clones, and assembles a combinatorial library of single-chain antibodies by recombination, and then screens the library to obtain BCMA antibodies with high affinity that bind to human BCMA.

[0018] The amino acid sequence pattern of the antibody variable region provided by the present invention is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the present application, the FR and CDR region division is based on the Kabat naming system. Here, FR1-4 represent four framework regions, and CDR1-3 represent three hypervariable regions. FR1-4 may be isolated from the constant region sequence (e.g., the most commonly used amino acids for human immunoglobulin light and heavy chains, subclasses or subfamilies), isolated from a single human antibody framework region, or a combination of genes from different framework regions.

[0019] Through further screening, the present invention has 1. A bispecific antibody that binds to BCMA and CD3, a first domain that binds to the B cell maturation antigen BCMA and a second domain that binds to the T cell surface antigen CD3; The first domain and the second domain are linked via a linker peptide, The first domain is a heavy chain variable region in which CDR1 has an amino acid sequence shown in any one of SEQ ID NOs: 4, 7, 8, and 9, CDR2 has an amino acid sequence shown in any one of SEQ ID NOs: 5 and 10, and CDR3 has an amino acid sequence shown in any one of SEQ ID NOs: 6 and 11; and a light chain variable region, wherein CDR1 has the amino acid sequence shown in any one of SEQ ID NOs: 1 and 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in any one of SEQ ID NOs: 3, 13, 14, 15, and 16, wherein the first domain of the bispecific antibody binds to BCMA and CD3, and the first domain of the bispecific antibody exhibits a higher affinity for BCMA.

[0020] Additionally, among the above BCMA-binding antibodies, the following antibodies show higher affinity for BCMA: The CDR region sequence of the heavy chain variable region is (1) CDR1 has the amino acid sequence shown in SEQ ID NO: 4, CDR2 has the amino acid sequence shown in SEQ ID NO: 5, and CDR3 has the amino acid sequence shown in SEQ ID NO: 6; (2) CDR1 has the amino acid sequence shown in SEQ ID NO: 7, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11; (3) CDR1 has the amino acid sequence shown in SEQ ID NO: 8, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11; and (4) CDR1 has the amino acid sequence shown in SEQ ID NO: 9, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11; The CDR region sequence of the light chain variable region is (1) CDR1 has the amino acid sequence shown in SEQ ID NO:1, CDR2 has the amino acid sequence shown in SEQ ID NO:2, and CDR3 has the amino acid sequence shown in SEQ ID NO:3; (2) CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 13; (3) CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 14; (4) CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 15; and (5) An antibody which is any one of those in which CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 16.

[0021] Additionally, among the above BCMA-binding antibodies, the following antibodies show higher affinity for BCMA: The CDRs of the heavy chain variable region and the CDRs of the light chain variable region are (1) A heavy chain variable region in which CDR1 has the amino acid sequence shown in SEQ ID NO: 4, CDR2 has the amino acid sequence shown in SEQ ID NO: 5, and CDR3 has the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region in which CDR1 has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3; (2) A heavy chain variable region in which CDR1 has the amino acid sequence shown in SEQ ID NO: 8, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region in which CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 13; (3) A heavy chain variable region in which CDR1 has the amino acid sequence shown in SEQ ID NO: 9, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11, a light chain variable region in which CDR1 has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3, and (4) An antibody which is any one of the following: CDR1 of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 8, CDR2 having the amino acid sequence shown in SEQ ID NO: 10, CDR3 having the amino acid sequence shown in SEQ ID NO: 11, CDR1 of a light chain variable region having the amino acid sequence shown in SEQ ID NO: 12, CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and CDR3 having the amino acid sequence shown in SEQ ID NO: 16.

[0022] Based on the sequences of the above CDRs, antibodies having the following variable region sequences (first domain) show high affinity for BCMA: The heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 17, 19 to 21, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 18, 22 to 27.

[0023] Based on the above-mentioned heavy chain variable region and light chain variable region, amino acid sequences that satisfy at least one of the following conditions compared to the above-mentioned sequences: a) binding to the same antigen determinant; and b) having a sequence identity of greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% are also within the scope of the present invention.

[0024] Furthermore, antibodies having the following variable region sequences (first domain) show better affinity for BCMA: a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 18; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 19 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 27; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:21 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:25; Alternatively, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:20, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:26.

[0025] In the bispecific antibody of the present invention, the first domain is an antibody capable of binding to BCMA and comprises two complete heavy-light chain pairs, in which the light and heavy chains are linked via disulfide bonds.

[0026] The Fc fragment of the heavy chain of the first domain may be an Fc fragment of a human or humanized antibody (IgG1, IgG2, IgA, IgE, IgM, IgG4 or IgD).

[0027] In one embodiment of the invention, the Fc fragment of the heavy chain of the first domain is an Fc fragment of a human or humanized antibody IgG4.

[0028] As a preferred embodiment of the present invention, The heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 32, and the light chain has the amino acid sequence shown in SEQ ID NO: 33; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO: 49 and the light chain has the amino acid sequence set forth in SEQ ID NO: 42; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO: 50 and the light chain has the amino acid sequence set forth in SEQ ID NO: 44; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO:51 and the light chain has the amino acid sequence set forth in SEQ ID NO:46; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO:52 and the light chain has the amino acid sequence set forth in SEQ ID NO:48.

[0029] These BCMA-binding antibodies have high affinity for BCMA, and when they form a bispecific antibody with an antibody that binds to CD3, it is possible to ensure that the functions of both antibodies are fully exerted.

[0030] Based on the above-mentioned heavy chain sequence and light chain sequence, an amino acid sequence that satisfies at least one of the following conditions compared to the above sequence: a) binding to the same antigen determinant; and b) having a sequence identity of more than 70%, 80%, 85%, 90%, 97%, 98% or 99% is also within the scope of the present invention.

[0031] The anti-human BCMA antibodies of human origin provided by the present invention bind to human BCMA with an affinity of 0.2 nM to 10 nM. The antibodies inhibit the binding of BCMA ligands to human BCMA. The antibodies bind to cells expressing BCMA, which may be human multiple myeloma or lymphoma cells.

[0032] In the second domain that binds to the CD3 antigen, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:29.

[0033] It is preferable that the light chain variable region and heavy chain variable region of the second domain are linked via a linker peptide like a single chain antibody, and that the single chain antibody has the amino acid sequence shown in SEQ ID NO:31.

[0034] The bispecific antibodies of the invention are preferably designed to have the following structure: a first domain comprises two complete heavy-light chain pairs and a second domain comprises two single chain antibodies, said bispecific antibodies comprising: (1) The C-terminus of the two single-chain antibodies in the second domain is linked to the N-terminus of the two heavy chains in the first domain via a linker peptide, respectively; and (2) It has a symmetrical structure in which the N-terminus of the two single-chain antibodies in the second domain is linked to the C-terminus of the two heavy chains in the first domain via a linker peptide, respectively.

[0035] The present invention has found that, with respect to the sequences of the above first and second domains, the above bispecific antibody having a symmetric structure can better retain the specific antigen-binding ability of the primary antibody in the first and second domains compared to bispecific antibodies having other structures, and has excellent biological functions of binding to BCMA and CD3, and is clearly superior in terms of production process, pharmaceutical efficacy, etc. The present invention has developed a bispecific antibody that binds to BCMA and CD3 and has the above antibody molecular structure, which has a specific targeting effect, can efficiently induce a directional immune response, and can kill tumor cells.

[0036] The amino acid sequence of the linker peptide for linking the first domain and the second domain is preferably (GGGGX)n (wherein X is Gly or Ser, and n is a natural number from 1 to 4). In a preferred embodiment of the present invention, the amino acid sequence of the linker peptide is shown in SEQ ID NO:30.

[0037] As an example of a bispecific antibody having the above structure, the present invention provides a human CD3×BCMA bispecific antibody, which has the above structure and sequence of the heavy chain variable region and light chain variable region of the single chain antibody and the heavy chain and light chain of the monoclonal antibody, and the present invention has constructed two bispecific antibodies that can bind to CD3 and BMCA simultaneously, whose structures and sequences are as follows:

[0038] (1) The heavy chain of the first domain is linked to the second domain to form a fusion protein having the amino acid sequence shown in SEQ ID NO: 34, and the light chain of the first domain has the amino acid sequence shown in SEQ ID NO: 33. (2) The heavy chain of the first domain is linked to the second domain to form a fusion protein having the amino acid sequence shown in SEQ ID NO:35, and the light chain of the first domain has the amino acid sequence shown in SEQ ID NO:33. (3) The heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 41 after being linked to the second domain via a linker peptide, and the light chain has the amino acid sequence shown in SEQ ID NO: 42. (4) The heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 43 after being linked to the second domain via a linker peptide, and the light chain has the amino acid sequence shown in SEQ ID NO: 44. (5) The heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 45 after being linked to the second domain via a linker peptide, and the light chain has the amino acid sequence shown in SEQ ID NO: 46. (6) The heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 47 after being linked to the second domain via a linker peptide, and the light chain has the amino acid sequence shown in SEQ ID NO: 48. The sequences shown in SEQ ID NOs: 1 to 52 disclosed or to be protected above include "conservative sequence modifications", i.e., nucleotide and amino acid sequence modifications that do not significantly affect or alter the binding properties of the antibody or antibodies containing the amino acid sequences. The conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. Modifications can be introduced into SEQ ID NOs: 1 to 52 by standard techniques in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, and conservative amino acid substitutions include replacement of amino acid residues with amino acid residues having similar side chains or with other amino acid residues. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a non-essential amino acid residue in a human anti-BCMA antibody with another amino acid residue from the same side chain family.

[0039] Thus, antibodies having the amino acid sequences disclosed above and / or antibodies containing the amino acid sequences disclosed above also include antibodies encoded by similar sequences modified by substantially conservative sequences, or antibodies containing similar sequences modified by conservative sequences, all of which should be considered within the scope of the present invention. The present invention also provides a nucleic acid encoding the bispecific antibody that binds to BCMA and CD3.

[0040] Considering the degeneracy of codons, the gene encoding the antibody of the present invention can be obtained by modifying the gene sequence encoding the antibody, provided that the amino acid sequence in the coding region is not modified. Those skilled in the art can improve the expression efficiency of the antibody by artificially synthesizing and modifying the gene according to the codon bias of the host in which the antibody is expressed.

[0041] The present invention further provides a biological material containing a nucleic acid encoding said bispecific antibody that binds BCMA and CD3, the biological material including recombinant DNA, expression cassettes, vectors, host cells, recombinant bacteria or cell lines.

[0042] Here, the vector includes, but is not limited to, a clone vector and an expression vector, and may be a plasmid vector, a virus vector, a transposon vector, or the like.

[0043] The host cell or cell line may be derived from a microbial or animal cell or cell line.

[0044] The present invention also provides a method for producing a bispecific antibody that binds to BCMA and CD3, the method comprising: constructing an expression vector containing genes encoding the first domain and the second domain; introducing the expression vector into a host cell to obtain a host cell that expresses the bispecific antibody; culturing the host cell; and obtaining the bispecific antibody by separation and purification.

[0045] When preparing the bispecific antibody, those skilled in the art can select, as necessary, a host cell, an expression vector, a method for introducing the expression vector into a host cell, and a method for separating and purifying the antibody, which are conventional in the art.

[0046] Based on the function of the bispecific antibody that binds BCMA and CD3 provided by the present invention, the present invention provides any one of the following uses of a bispecific antibody that binds BCMA and CD3, a nucleic acid encoding same, or a biological material containing said nucleic acid:

[0047] (1) Use in the manufacture of a pharmaceutical for diagnosing, preventing, or treating a B cell-related disease in which BCMA is expressed (preferably, the B cell-related disease in which BCMA is expressed includes a B cell-related tumor (including, but not limited to, multiple myeloma or lymphoma), or an autoimmune disease mediated by B cells). (2) Use in the manufacture of a medicine for diagnosing, preventing or treating a disease targeting BCMA (wherein the medicine is preferably an anti-cancer drug or a medicine for treating an autoimmune disease). (3) Use in the manufacture of a medicine for killing cells expressing BCMA (4) Use in the manufacture of BCMA and / or CD3 detection reagents (5) Use in the manufacture of reagents related to CAR-T therapy

[0048] The above-mentioned B cell-related diseases in which BCMA is expressed are preferably malignant tumors and autoimmune diseases in which BCMA is highly or excessively expressed.

[0049] The bispecific antibodies provided by the present invention can be used for therapy and diagnosis by themselves, or can be labeled, crosslinked, or coupled, or fused and expressed with other proteins or peptide molecules to form conjugates (e.g., cytotoxic substances, radiotoxins, and / or chemical molecules, etc.) for use in diagnosis and therapy.

[0050] The invention also provides multispecific antibodies, including bispecific antibodies that bind BCMA and CD3, fusion proteins, immunotoxins, pharmaceuticals or detection reagents.

[0051] The immunotoxins described above include bispecific antibodies that are linked to cytotoxic agents in a variety of formats.

[0052] The various forms of attachment are antibody labeling, in vitro crosslinking, or molecular coupling. The cytotoxic agents include chemical molecules, radioisotopes, peptides, toxins, and other substances that have cell killing or cell death inducing properties.

[0053] The fusion proteins described above include conjugates of the bispecific antibodies provided by the present invention with other proteins or peptide molecules having a specific function.

[0054] Specifically, the fusion protein may be prepared by linking an antibody gene to an immunotoxin or cytokine gene to construct a recombinant expression vector to obtain a recombinant fusion protein molecule in mammalian cells or other expression systems.

[0055] The above-mentioned medicines and detection reagents may further contain other active ingredients or 1689840495729_0 (For example, the components of the antibody and a pharmacologically acceptable delivery molecule or solution. Here, the components used in the treatment may be sterile and lyophilized.)

[0056] The bispecific antibodies of the present invention are capable of inhibiting one or more of the biological activities induced by BCMA. They may exert their function by blocking the binding of BCMA to its ligands, but they may also exert their function by killing cells that highly express BCMA, or by internalization of BCMA-bound complexes resulting in consumption of BCMA on the cell surface. All interfering functions of BCMA antagonists should be considered equivalent for the purposes of the present invention. Effect of the Invention

[0057] The present invention uses recombinant gene and phage surface display library technology to screen for specific antibodies against the human B cell surface antigen BCMA from a single-chain antibody library of natural, fully human sequences. By improving affinity maturation, multiple clones with significantly improved binding ability were obtained, and their affinity to human BCMA was 0.26 nM to 0.31 nM. The apparent affinity measured by flow cytometry was 61 to 97 times higher than that of the parent antibody. This verified that both the anti-human BCMA antibody and the optimized mutant of the present invention have good binding ability to BCMA.

[0058] Based on this, the present invention provides a bispecific antibody that can simultaneously bind to BCMA and CD3, has a specific targeting effect, and can efficiently induce a directed immune response. The bispecific antibody fully retains the biological functions of anti-CD3 antibody and anti-BCMA antibody, and realizes that one bispecific antibody molecule has an excellent biological function of simultaneously binding to BCMA and CD3, and can act as a bridge between tumor cells and immune effector cells, efficiently induce immune effector cells and directed immune responses, significantly improve the killing effect of immune cells against tumor cells, minimize the ADCC effect, and have high safety.

[0059] The bispecific antibody provided by the present invention has good prospects for therapeutic application and is mainly expressed as having specific binding activity to human BCMA and CD3. ELISA detection and flow cytometry detection show that the antibody can specifically bind to BCMA on the surface of H929 and RPMI8226 cells and T cells, and therefore has good target specificity. In vitro cell killing efficiency detection shows that the bispecific antibody has high target cell killing efficiency.

[0060] In addition, the bispecific antibody provided by the present invention has the characteristic of being completely symmetrical in structure. Therefore, when expressed in a host, it will not produce protein isoforms with other structures, which can greatly reduce the difficulty of the extraction and purification process. It has the advantages of being simple to produce and having high yields, and has broad prospects for use in tumor immunotherapy.

[0061] The present invention provides a bispecific antibody candidate molecule for the development of antitumor antibody drugs targeting BCMA, the development of CAR-T reagents, and the prevention and treatment of other diseases such as B cell-related inflammation and autoimmune diseases. The bispecific antibody of the present invention can simultaneously bind to immune cells and tumor cells, induce directed T cell immune responses, and specifically and effectively kill tumor cells, and can be developed as an antibody drug for multiple myeloma. [Brief description of the drawings]

[0062] [Figure 1] FIG. 1 is a schematic diagram of BCMA expression and TCR structure described in the background art of the present invention, in which A shows the expression of BCMA antigen during B cell development, B shows the ligand-activated signaling channel of BCMA antigen (Source: fimmu-09-01821-g001.jpg(1050×762)(frontiersin.org)), and C shows the structure of the T cell receptor (TCR) complex and its CD3 composition (Source: https: / / www.researchgate.net / publication / 299549376). [Diagram 2] FIG. 2 shows the results of analyzing the binding between BCMA and lead clone B10 using a flow cytometer (FACS) in Example 2 of the present invention, in which the top two photographs are the analysis results of the negative control (NC), the middle two photographs are the analysis results of the positive control (PC), and the bottom two photographs are the analysis results of the candidate clone B10 (B10). [Diagram 3]FIG. 3 shows the results of analyzing the binding of B10 and its affinity matured mutants using a flow cytometer in Example 3 of the present invention, in which A, B, C, D, and E are the analytical results of the binding of B10, B10.3, B10.4, B10.5, and B10.9 to H929 cells, respectively, and F, G, H, I, and J are the analytical results of the binding of B10, B10.3, B10.4, B10.5, and B10.9 to RPMI8226 cells, respectively. [Figure 4] FIG. 4 shows the results of analyzing and comparing the binding of J6, B10 and their mutants using ELISA and a flow cytometer, respectively, in Example 3 of the present invention, where A is the result using ELISA and B is the result using FACS. [Diagram 5] FIG. 5 is a structural schematic diagram of an anti-BCMA x CD3 bispecific antibody constructed based on the FIST platform in Example 4 of the present invention, in which A is a structural schematic diagram of a bispecific antibody constructed by N-terminal fusion (nFIST) (in which an anti-CD3 single-chain antibody is fused to the N-terminus of an anti-BCMA antibody VH), and B is a structural schematic diagram of a bispecific antibody constructed by C-terminal fusion (cFIST) (in which an anti-CD3 single-chain antibody is fused to the C-terminus of an anti-BCMA antibody Fc). [Figure 6] FIG. 6 shows SDS-PAGE electrophoresis patterns of the bispecific antibodies K3B10.3 and B10.3K3 in Example 5 of the present invention, in which A and C are electrophoresis patterns of reduced SDS-PAGE, B and D are electrophoresis patterns of non-reduced SDS-PAGE, A and B are the electrophoresis results of the K3B10.3 bispecific antibody, C and D are the electrophoresis results of the B10.3K3 bispecific antibody, lane M represents the protein molecular weight standard, and lane 1 is the target protein. [Figure 7] FIG. 7 shows peak graphs obtained by HPLC-SEC purity analysis of the bispecific antibodies K3B10.3 and B10.3K3 purified in Example 5 of the present invention, in which A is the peak graph for the bispecific antibody K3B10.3 and B is the peak graph for the bispecific antibody B10.3K3. [Figure 8] Figure 8 shows the results of analyzing the binding of K3B10.3 and B10.3K3 to human Jurkat T cells and RPMI cells using a flow cytometer in Example 6 of the present invention, in which A is the analysis result of the secondary antibody control, B is the analysis result of K3B10.3 and C is the analysis result of B10.3K3 binding to CD3, D is the analysis result of the secondary antibody control, E is the analysis result of K3B10.3 and F is the analysis result of B10.3K3 binding to BCMA. [Figure 9] FIG. 9 shows the binding of the cFIST bibody of Example 7 of the present invention to the BCMA antigen and the killing of T cells expressing RPMI8226 mediated by them, in which A shows the ELISA measurement results of the binding of the affinity-improved mutant bibody of B10 to the BCMA antigen (wherein the antibody was coated at 200ng / well and biotin-BCMA was half-diluted from 250ng / ml), and B shows the killing of BCMA expressing cells mediated by the affinity-improved mutant bibody of B10 (wherein high and low concentrations of B10K3 were tested, and no standard S curve was obtained at the low concentration (B10K3), and a standard S curve was obtained at the high concentration (B10(high)K3), and the EC50 value was calculated based on the curve). [Figure 10] FIG. 10 shows the BCMA dual antibody-mediated T cell killing of cells with different BCMA expression levels in Example 7 of the present invention, in which A is H929 cells and B is RPMI8226 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] The following examples are intended to illustrate the present invention and are not intended to limit the scope of the invention.

[0064] Example 1 Screening for anti-human BCMA antibodies from a natural human antibody phage surface display library In antibody library technology, all antibody variable region genes of an animal (including human) are cloned into plasmids or phages, the latter are infected with E. coli, and antibody fragments are then ligated onto the surface of the phage particles or into the periplasm or endoplasmic reticulum of E. coli. 1689840495729_1 This technology involves expressing the human antibody in a human antibody library, then screening clones with specific antibody genes from the antibody library by the target antigen to obtain the corresponding specific antibody. The antibody library has been screened for many antibodies necessary for basic research and clinical development, such as membrane protein antigens related to tumors, autoantigens related to autoimmune diseases, and viral antigens for viral diseases. This shows that the antibody library technology has great potential for application in basic research and the development of antibody drugs. In particular, the fully human monoclonal antibodies obtained from the human antibody library have overcome the obstacle of the difficulty of obtaining human monoclonal antibodies by mouse hybridoma technology. Due to the high degree of conservatism of human antibody sequences, the immunogenicity of these antibodies to the human autoimmune system is much lower than that of animal-derived antibodies, making them safer.

[0065] The non-immune human natural antibody library uses healthy individuals as the source of antibody genes, and these individuals are called donors. Because natural, fully human antibody sequences are used, the resulting antibodies have extremely low immunogenicity to the human immune system. Through molecular biology and DNA manipulation techniques, total RNA is extracted from donor peripheral blood mononuclear cells (PBMCs) using an RNA extraction kit (e.g., QIAGEN's RNeasy Mini Kit, Cat. No.: 74104), and then equal amounts of RNA are taken from each RNA sample, combined, and then subjected to reverse transcription using a reverse transcription kit (e.g., Thermo Fisher Scientific's cDNA synthesis kit (SuperScript TMLight and heavy chain gene cDNAs were synthesized using PCR primers with PCR IV Reverse Transcriptase (RT-PCR). Using these antibody cDNAs as templates, PCR amplification was performed using subgroup-specific upstream primers and constant region primers in the first round to obtain genes of all members of each subgroup. In the second round of PCR, a cross-linked heavy chain variable region (VH) 3" end primer set and a cross-linked light chain variable region (VL) 5' end primer set were combined with their respective other end primers (containing specific restriction enzyme sites) so that the resulting VH and VL could be linked via a linker to form single-chain antibody (scFv) genes. Using the specific enzyme cleavage sites at the 5' and 3' ends of these single-chain antibody genes, these scFv genes were cloned into a lysogenic phagemid (e.g., M13 phagemid) vector to construct an antibody library. The size of the library reached 5 billion colony-forming units (cfu).

[0066] Biopanning refers to screening specific clones from antibodies by specific targets. To obtain human antibodies specific for human BCMA antigen, panning is performed by liquid-phase screening. The general procedure of the liquid-phase panning method is to first modify commercially available BCMA antigen (BCMA-Fc fusion protein, Acrobiosystems, catalog number BC7-H5254) with biotin, crosslink 3-5 biotin molecules to each molecule, and remove free biotin. An appropriate amount of biotin-marked BCMA antigen was bound to streptavidin-coupled magnetic beads (e.g., Dynabeads). TMM-280 Streptavidin, Thermo Fisher Scientific, Cat. No. 11205D). A portion of a human antibody library containing 10 billion phage particles expressing different antibodies was thawed. Both the magnetic beads and the antibody library were blocked with 4% nonfat dry milk solution (4% MPBS) to remove nonspecific sites of action. Because there may be streptavidin on the magnetic beads that is not bound to avidin, and because the BCMA antigen molecule is a fusion protein composed of human Fc, it is necessary to add a sufficient amount of streptavidin and human antibody Fc (50-100-fold excess over the target protein used) to the thawed antibody library to remove antibody clones that bind to them. The blocked antibody library solution was added to a 1.5 ml Eppendorf tube together with the magnetic beads (total volume <1 ml) and incubated with rotating mixing at room temperature for 2 hours to allow binding of the BCMA-specific phages and antibodies. After the incubation was completed, the Eppendorf tube was placed on a magnetic rack for 1 minute to separate the magnetic beads from the solution. The solution was removed as completely as possible with a pipette gun, replaced with a new tip, and 1 ml of washing solution PBST (phosphate buffer solution (PBS) with Tween 20 added to a final concentration of 0.05%) was added to the Eppendorf tube, which was then removed from the magnetic rack. The magnetic beads were gently suspended with a pipette gun and placed back on the magnetic rack to separate the magnetic beads from the solution. The solution was removed and this was repeated three times. Next, the washing solution was changed to PBS, and the magnetic beads were washed three times. By washing, most of the nonspecific and low affinity phage antibodies were removed, and the specific phage antibodies remained on the magnetic beads. The elution solution (10 mM glycine, pH 2.0) was added to the magnetic beads, the magnetic beads were resuspended, and left at room temperature for 10 minutes. The magnetic beads and solution were separated using a magnetic rack, and the solution was sucked into a clean Eppendorf tube. 1 / 10 1 M Tris solution (pH 8.0) was added to neutralize the solution. This resulted in the elution solution containing thousands of different phage antibodies that bind to the BCMA antigen, completing the first round of panning.

[0067] To obtain more specific antibody clones with high affinity, more panning is required. For this purpose, the phage antibody solution eluted from the first round of panning was used to infect M13 phage-infectable E. coli (e.g., TG1 strain) in the logarithmic phase to obtain an infection solution. A small amount of the infection solution was taken and subjected to a series of 10-fold gradient dilutions (usually diluted to 1 / 1,000,000 of the original solution, and the last three gradient dilutions were taken and coated) to measure the titer of the eluate produced in the first round. This titer is also called the maximum diversity of the first round, and the titer of the eluate panned in the first round is usually 10E6 cfu or less. The remaining infection solution was coated on a bacterial culture plate containing the appropriate antibiotic and cultured overnight to obtain colonies. The colonies were scraped and resuspended in medium, and a sufficient amount of the resuspension containing the diversity of the first round eluate (output) was placed in a shake flask containing a sufficient amount of liquid medium (2YT-CG, in which carbenicillin and glucose were added to final concentrations of 100 μg / ml and 2%, respectively) and the resuspension was diluted to 0.1 OD600 or less and cultured until the exponential phase, i.e., OD600 reached approximately 0.5. To redisplay the antibodies obtained by these first round panning on the surface of phage particles, 10 ml of the bacterial solution was taken, and the auxiliary phage M13K07 was added and incubated at 37°C for 30 minutes to achieve a multiplicity of infection of 20:1 (this step is called phage rescue). After centrifugation, the cells were resuspended in 50 ml of expression medium (2YT-AK, in which carbenicillin and kanamycin were added to 2YT medium to give final concentrations of 100 μg / ml and 30 μg / ml, respectively) and cultured overnight at 30°C at 200 rpm / min. The next day, the supernatant was collected by centrifugation, and 1 / 5 volume of PEG8000 / NaCl (PEG-8000 20%, NaCl 2.5 M) was added, mixed thoroughly, and incubated on ice for 1 hour. The phage antibody particles were obtained by high-speed centrifugation (11,500×g) for 30 minutes. The precipitate was resuspended in 1 ml of PBS solution and placed in a high-speed centrifuge again to remove bacterial debris.The supernatant is the amplified solution after the first round of panning, and each antibody clone contained therein has been amplified tens of thousands of times or more. This amplified solution can be used for the second round of panning experiments. The second round of panning procedures are exactly the same as the first round procedures, except that the washing with PBST / PBS is increased to six times (6 / 6). In the third round, the number of washings can be further increased to 10 / 10. Multiple rounds of panning usually effectively enrich specific clones, which have a greatly reduced diversity but have high affinity, which is convenient for subsequent monoclonal screening.

[0068] To obtain specific monoclonal antibodies, a monoclonal phage enzyme-linked assay (mono-phage ELISA) is required. For this, single colonies that could be sufficiently separated in the gradient dilution of the second and / or third rounds were individually inoculated into 96-well plates containing 2YT-AG (93 colonies were inoculated per plate, with three wells reserved as negative controls) and cultured overnight. This is the master plate. The bacterial solution of each well in the master plate was inoculated into a new culture plate, grown to logarithmic phase, and phage rescue was performed as described above so that the antibody of each clone was displayed on the phage surface. A regular 96-well enzyme-linked plate was coated with BCMA antigen (1 μg / ml) and another enzyme-linked plate was coated with human Fc at the same concentration. Bacterial solutions expressing each monoclonal phage antibody were added to the corresponding wells of the BCMA plate and the Fc plate, respectively, and then appropriate secondary antibodies and horseradish peroxidase (HRP)-coupled tertiary antibodies were added to develop the substrate, and the absorbance (450 nM) was read. The method for determining BCMA-positive clones is to determine whether a clone is BCMA-positive if it is negative on the Fc plate (absorbance is 1.5 times or less than that of the negative wells of the plate), but positive on the BCMA plate (absorbance is more than 3 times that of the negative wells of the plate), and the absorbance of the well is higher than that of the corresponding well on the Fc plate. Analysis showed that the clones corresponding to 10 wells were positive only for the BCMA antigen, but negative for Fc, and these clones were collectively referred to as hits.

[0069] These hit bacterial solutions were inoculated into 3 ml of 2YT-CG from the corresponding wells of the master plate, and incubated at 37°C, 200 rpm / min overnight. The next day, phagemid DNA was extracted and the sequence of the single-chain antibody region containing each hit was measured with specific primers. The DNA sequences of the coding regions were translated into amino acid sequences, and multiple sequences were compared against them (CLUSTALW, link to website https: / / www.genome.jp / tools-bin / clustalw) to determine the specificity of the clones. When analyzed, these 10 hits belonged to two different clones in terms of sequence, one of which was named B10, and thus the variable region sequence of a fully human antibody against the human BCMA antigen was obtained. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of B10 are shown in SEQ ID NOs: 4 to 6, respectively, the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 1 to 3, respectively, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18. The amino acid sequence of a single-chain antibody consisting of the light chain variable region and heavy chain variable region of B10 is shown in SEQ ID NO: 36.

[0070] Example 2: Verification of antibody function To verify whether the obtained BCMA antibody clone bound to purified BCMA antigen and BCMA expressed on the cell membrane surface, the gene of B10 single chain antibody was cloned into the eukaryotic expression vector pFH (Excyte) to obtain the plasmid pFH-B10. In this vector, the scFv gene is fused with the Fc gene of human IgG4 to express the protein in the form of scFv-Fc. It can be affinity purified by Protein A and detected by (HRP or fluorescein) labeled anti-human Fc antibody.

[0071] After obtaining the scFv-Fc protein of B10, the binding of the antibody to BCMA was detected by Octet RED, and specific binding of B10 to BCMA was confirmed.

[0072] Using a flow cytometer, the BCMA-expressing cell line H929 (NCI-H929, ATCC R CRT-9068 TM ), which showed that B10 specifically bound to the BCMA antigen on the cell membrane surface (see Figure 2). In this FACS analysis, the percentages in the upper left corners of the four compartments (positive percentages) represent the binding strength of each single-chain antibody to the cell surface antigen. The positive percentage of clone B10 was 48.96%, verifying the antibody binding ability of the anti-human BCMA antigen.

[0073] Example 3 Affinity Maturation of Antibodies In vitro affinity maturation is a rapid molecular evolution-oriented procedure that introduces mutations at the DNA level and screens at the protein binding level. In the case of the B10 antibody, in order to avoid introducing mutations that may increase immunogenicity or significantly modifying the antibody structure, the mutation sites are limited to the sequences of the CDR regions, and each position is subjected to saturation mutation separately, and each mutation is expressed and tested separately.

[0074] The operation process of affinity maturation is as follows. First, the light chain variable region gene and the heavy chain variable region gene of B10 were cloned onto the pUFL vector (pUFL is a plasmid capable of expressing antibody Fab in E. coli, manufactured by Excyte) respectively to obtain the Fab type of B10. A full set of single-site random mutation primers for the CDR region was designed, and each CDR site was PCR mutated using a point mutation kit (QuikChange Lightning Multi-Site Directed Mutagenesis Kit, Agilent Cat. No. 210515), respectively, transformed into BL21(DE3) competent cells, and respectively prepared monoclonal colony plates, and these mutants are collectively called the single-site mutant library. At the same time, the Fab vector of the parent antibody B10 was also transformed. More than 30 colonies were selected to prepare DNA, and the mutated regions were sequenced to evaluate the mutation rate. When the mutation rate was adequate (>80%), 92 colonies were selected according to the mutation at each CDR position and placed into a 96-well plate containing the corresponding medium (100 μg / ml carbenicillin + 0.1% glucose in 2YT), and three parent colonies were selected separately and three wells were reserved as blank controls. The plate was placed at 37°C and incubated at 300 rpm / min for 6 hours, IPTG was added to a final concentration of 1 mM, transferred to 30°C, and incubated at 300 rpm / min overnight, and the Fab fragments were expressed and secreted into the medium.

[0075] Preliminary experiments optimized the reaction conditions for enzyme binding so that the A450 absorbance of the B10 parent antibody Fab bound to the antigen BCMA-Fc was slightly higher than about 3 times the background signal. One day earlier, BCMA-Fc antigen was coated on the enzyme binding plate at 0.25 μg / ml, and the next day, the secreted and expressed mutant Fab was added to each well, anti-human Fab coupled with HRP was used as the secondary antibody, the substrate was developed, A450 was read, and clones corresponding to wells with signals higher than 1.5 times the maximum value of the parent well were obtained, and these clones were called hits. All hits in the light chain variable region and heavy chain variable region were collected, and by performing induction expression and enzyme binding experiments again, it was confirmed that they indeed had higher affinity than the parent antibody. The sequences of the mutation regions were analyzed for the plasmids of the clones that were repeatedly positive, and mutation information of the CDR amino acids was obtained. This process is called primary screening. This obtained mutation information that is useful for improving the affinity of all CDR regions. Mutation sites in the heavy chain variable region CDRs that confer affinity are shown in Table 1, and mutation sites in the light chain variable region CDRs that confer affinity are shown in Table 2.

[0076] [Table 1]

[0077] [Table 2]

[0078] After obtaining all the single mutation information beneficial to improve the affinity of the entire CDR region from the primary library by screening with an enzyme binding experiment, new primers for multiple point mutations were redesigned to include the major beneficial mutations, and a mutant library was constructed again using a point mutation kit. This library is called a combinatorial library. In order to screen all possible combinations of mutations in the light chain variable region and the heavy chain variable region, combinatorial libraries of the light chain variable region and the heavy chain variable region containing multiple mutations were constructed and screened separately. The top five clones with the highest affinity were obtained from the combinatorial library of the light chain variable region, which contains five mutant sequences whose amino acid sequences of the light chain variable region are shown in SEQ ID NOs: 22 to 26. The CDR1 of those light chain variable regions has the amino acid sequence shown in any one of SEQ ID NOs: 1 and 12, the CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and the CDR3 has the amino acid sequence shown in any one of SEQ ID NOs: 3, 13, 14, 15, and 16.

[0079] Enzyme binding screening was performed on a combinatorial library of heavy chain variable regions to obtain the top three clones with the highest affinity in the combinatorial library of heavy chain variable regions, the top three clones having the highest affinity in the combinatorial library of heavy chain variable regions, the heavy chain variable region amino acid sequences of which include three mutant sequences as shown in SEQ ID NOs: 19 to 21. CDR1 of the heavy chain variable regions has the amino acid sequence shown in any one of SEQ ID NOs: 7, 8, and 9, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11.

[0080] The DNA of the top five clones with the highest affinity among the light chain variable regions and heavy chain variable regions was mixed and assembled into a final single-chain antibody combinatorial library by recombination methods such as enzymatic cleavage and ligation. This library was screened to select the top ten clones with the highest signals, and their DNA sequences were analyzed to obtain four different single-chain antibody clones whose corresponding amino acid sequences are shown in SEQ ID NOs: 37 to 40.

[0081] To verify whether the affinity of the obtained mutants was improved, fusion proteins (i.e., scFv-Fc) of Fc with the parent antibody B10 and the four finally obtained mutants B10.3, B10.4, B10.5, and B10.9 (the amino acid sequences are shown in SEQ ID NOs: 37 to 40, respectively) were expressed and purified. The purified products were detected by flow cytometry on BCMA-positive cell lines H929 and RPMI8226 with two different expression levels. The percentages of binding to H929 cells were 75.31% for the parent antibody B10, improved to 97.96% for the mutant B10.3, improved to 97.89% for the mutant B10.4, improved to 97.17% for the mutant B10.5, and improved to 91.61% for the mutant B10.9, respectively. The percentage of binding to RPMI8226 cells was 5.54% for parent antibody B10, improved to 54.89% for mutant B10.3, 57.51% for mutant B10.4, 58.18% for mutant B10.5, and 42.89% for mutant B10.9. These results showed that all four mutants had significantly improved binding to BCMA compared to B10 (see Figure 3).

[0082] To know the affinity of the obtained B10 and its derivative antibodies, an experiment was carried out to compare the B10 parent antibody and the last four mutants obtained with the affinity-known BCMA antibody J6 (i.e., clone J6M0, whose affinity is about 0.2nM according to the description in patent [WO2012163805A1]) by ELISA. After coating an enzyme-linked plate with different concentrations of the antibody, the biotinylated BCMA antigen, streptavidin-coupled horseradish peroxidase (HRP), and color-developing substrate were added in sequence. The obtained signal-antibody slope curve is shown in Figure 4A, and the relative affinity fold change is shown in Table 3.

[0083] [Table 3]

[0084] The above results indicate that the parent antibody B10 binds to human BCMA significantly weaker than J6 in terms of intrinsic affinity, so no S-line curve could be obtained and a numerical affinity value could not be generated for it. Also, the affinity of B10.3 was about 0.26 nM, that of B10.4 was about 0.31 nM, that of B10.5 was about 0.27 nM, and that of B10.9 was about 0.29 nM.

[0085] To understand the apparent affinity of these antibodies to BCMA on the cell surface, a comparative experiment was performed by flow cytometry binding of the parental B10 antibody and the four final mutants with J6 to the BCMA-expressing cell line RPMI8226 (see Figure 4B, Table 4). The results showed that at the cellular level, the EC50 of the parental B10 was 1.575 μg / ml, which was about 1.5-fold improved over J6 (EC50 of 2.741 μg / ml). The EC50 of B10.3, B10.4, B10.5 and B10.9 were 0.02822 μg / ml, 0.03429 μg / ml, 0.04063 μg / ml and 0.04514 μg / ml, respectively. Compared to J6, the fold improvements in EC50 for these affinity-improved mutants were 97.13, 79.94, 67.46, and 60.72, respectively.

[0086] [Table 4]

[0087] Example 4 Design of BCMAxCD3 Bispecific Antibodies In this example, a bispecific antibody was designed to target the tumor cell surface antigen BCMA and the immune cell surface antigen CD3.

[0088] In the present invention, a bispecific antibody structure having a symmetric structure, including a single-chain antibody unit and a monoclonal antibody unit, was screened and determined from multiple bispecific antibody structures that bind to BCMA and CD3 by combining protein structure design software and screening by a large amount of artificial experiments. In the present invention, such a technology platform is called FIST (fusion of IgG and scFv technology). For example, an anti-BCMA monoclonal antibody unit includes two complete light chain-heavy chain pairs (i.e., including complete Fab and Fc domains, in which the light chain and the heavy chain are linked via a disulfide bond) as an IgG antibody, and an anti-CD3 can include two single chain antibodies (ScFv) as a single chain antibody unit. The single chain antibody and the monoclonal antibody are linked by a linker peptide (represented by SEQ ID NO: 30), and the following linking method was designed as a linking method between the single chain antibody and the monoclonal antibody to obtain a bispecific antibody having a symmetric structure (Figure 5).

[0089] The C-terminus of the single-chain antibody was linked to the N-terminus of the heavy chain variable region (VH) of a monoclonal antibody to obtain nFIST (Figure 5A). The single-chain antibody can also be fused to the C-terminus of IgG-Fc via a linker peptide to obtain cFIST (Figure 5B).

[0090] In this example, the variable region sequences of the anti-CD3 single chain antibody UCHT1 were derived from the literature (Beverley, PC & Callard, REDistinctive functional characteristics of human "T" lymphocytes defined by E rosetting or a monoclonal anti-T cell antibody (1981) Eur. J. Immunol. 11, 329-334) and were humanized (Shalaby et. al., Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. (1992) J Exp Med. Jan 1; 175(1): 217-25). The single-chain antibody thus constructed is designated K3 (SEQ ID NO: 31) and contains two cysteines (Cys) inserted into the heavy chain variable region and the light chain variable region, respectively, which form a pair of interchain disulfide bonds after folding, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29.

[0091] Example 5 Generation of BCMAxCD3 Bispecific Antibodies Two bispecific antibodies, K3B10.3 and B10.3K3, were obtained by coding genes according to the nFIST and cFIST format bispecific antibodies designed in Example 4 and cloning them into the expression vector pG4HK. Their heavy chain amino acid sequences are shown in SEQ ID NOs: 34 and 35, respectively (a fusion peptide in which the heavy chain of a monoclonal antibody and a single-chain antibody are linked via a linker peptide), and their light chain amino acid sequence is shown in SEQ ID NO: 33. The expression plasmids corresponding to K3B10.3 and B10.3K3 were stably transfected into CHO-K1, respectively, to produce bispecific antibody proteins.

[0092] More anti-BCMA×CD3 bispecific antibodies were constructed by cFIST format, among which the heavy chain variable regions of the IgG units that bind to BCMA were derived from those shown in SEQ ID NOs: 17-21, and the light chain variable regions of the IgG units that bind to BCMA were derived from those shown in SEQ ID NOs: 18, 22, and 25-27. Plasmids of these antibodies were transfected into 293F cells, and they were further purified by transient expression and protein A to obtain bispecific antibodies B10K3 (heavy chain amino acid sequence shown in SEQ ID NO: 41, light chain amino acid sequence shown in SEQ ID NO: 42), B10.3K3, B10.4K3 (heavy chain amino acid sequence shown in SEQ ID NO: 43, light chain amino acid sequence shown in SEQ ID NO: 44), B10.5K3 (heavy chain amino acid sequence shown in SEQ ID NO: 45, light chain amino acid sequence shown in SEQ ID NO: 46), and B10.9K3 (heavy chain amino acid sequence shown in SEQ ID NO: 47, light chain amino acid sequence shown in SEQ ID NO: 48). Purity analysis by SDS-PAGE showed that the monomers were greater than 90%. Their binding to the BCMA antigen is shown in Figure 9A.

[0093] The above bispecific antibodies were subjected to the following procedures to obtain high-purity bispecific antibodies. (1) Pretreatment of sample solution: The fermentation culture supernatant was centrifuged at 2000 rpm for 10 minutes, and then filtered through a 0.22 μM membrane filter. (2) Affinity chromatography: The antibody in the pretreated fermentation solution was captured using a Mabselect SuRe affinity chromatography column (purchased from GE, product number 18-5438-02). The chromatography column was sufficiently equilibrated with a buffer solution (10 mM PBS, 0.1 M NaCl, pH 7.0), and the sample solution was passed through the affinity chromatography column and eluted with an eluent (0.1 M citric acid, pH 3.0). (3) Cation exchange chromatography: The sample prepared by affinity chromatography was further purified by molecular sieve exchange chromatography and eluted with a buffer solution (50 mM PBS, 0.2 Man Na2SO4, pH 6.7).

[0094] K3B10.3 and B10.3K3 were subjected to SDS-PAGE and HPLC-SEC testing, and the results of SDS-PAGE are shown in FIG. 6. The results of reduced SDS-PAGE electrophoresis of K3B10.3 are shown in FIG. 6A, the results of non-reduced SDS-PAGE electrophoresis of B10.3K3 are shown in FIG. 6C, and the results of non-reduced SDS-PAGE electrophoresis of B10.3K3 are shown in FIG. 6D. The results of HPLC-SEC testing are shown in FIG. 7. The results of SEC testing of K3B10.3 are shown in FIG. 7A, and the results of SEC testing of B10.3K3 are shown in FIG. 7B. The results of the testing showed that the bispecific antibodies K3B10.3 and B10.3K3 were successfully produced by expression and purification. The purity of the purified bispecific antibody monomer is 95% or higher.

[0095] Example 6: Detection of binding activity between bispecific antibodies and T cells by flow cytometry Collect PBMC cells from healthy human donors to purify T cells and incubate at 1 x 10 6 The cells were resuspended in PBS at 1 cell / tube, washed once with 1 ml of binding buffer (PBS containing 0.5% w / v BSA + 2 mM EDTA), centrifuged (350 × g, 4 ° C, 5 min), and then resuspended in 200 μl of binding buffer. Bispecific antibodies K3B10.3 and B10.3K3 were added to 5 μg / ml, respectively, and incubated on ice for 45 min. The cells were washed once as above, and then resuspended in 100 μl of binding buffer. 5 μl of fluorescently labeled secondary antibody (PE anti-human IgG Fc Antibody, Biolegend, 409304) was added to the sample tube, and isotype control (PE Mouse IgG2a, κ Isotype Ctrl (FC) Antibody, Biolegend, 400213) was added to the isotype control tube, and incubated on ice for 15 min in the dark. The cells were washed once as above. The cells were resuspended in 200 μl of PBS and then measured using a machine (Beckman).

[0096] The binding assay results of K3B10.3 and B10.3K3 to human T cells by flow cytometry are shown in Figure 8, where the assay results of the control are shown in Figure 8A, the assay results of K3B10.3 to T cells are shown in Figure 8B, and the assay results of B10.3K3 to T cells are shown in Figure 8C. The results show that both bispecific antibodies K3B10.3 and B10.3K3 can specifically bind to T cells, that is, the bispecific antibody fusion protein retains the binding function of the single chain antibody anti-CD3.

[0097] To test for binding of the anti-BCMA antibody to BCMA, the T cells in this experiment were switched to RPMI8226 (ATCC® CCL-155) and a similar FACS experiment was performed. The results for the control are shown in Figure 8D, and the results for K3B10.3 and B10.3K3 are shown in Figures 8E and F.

[0098] Example 7: Determination of the efficiency of bispecific antibody-mediated cell killing in vitro In this example, H929-luc and RPMI8226-Luc were used as target cells, and PBMCs were used as immune effector cells to measure the target cell killing effect mediated by bispecific antibodies B10K3, B10.3K3, B10.4K3, B10.5K3, B10.9K3 and K3B10.3. The specific experimental procedure is as follows:

[0099] 1) Preparation of target cells: H929-luc and RPMI8226-Luc (luciferase-labeled H929 and RPMI8226 cells, produced in the Excyte lab) cells were cultured, pipetted up and down to mix the target cells evenly, then counted, centrifuged at 1000 rpm for 5 min, and washed once with PBS. After centrifuging and washing the target cells, they were cultured in GT-T551 medium at a density of 0.2 × 10 6 / ml and 50 μl was added per well, ie, 10,000 cells per well. 2) Preparation of PBMC: PBMC were used as effector cells. PBMC frozen and stored in a liquid nitrogen tank were removed and thawed (see Cryopreservation and Reculture of Cells), added to a 15 ml centrifuge tube containing PBS or GT-T551 medium, centrifuged at 1000 rpm for 5 minutes, washed twice with PBS or GT-T551 medium, and the cell number, viability and density were measured. The density of viable cells was 2 × 10 6 / ml and 50 μl was added per well, ie, 100,000 cells per well. 3) Antibody dilution: Bispecific B10K3, B10.3K3, B10.4K3, B10.5K3, B10.9K3, K3B10.3 and B10.3K3 were diluted in GT-T551 medium, respectively, and the initial antibody concentration was adjusted to 10 nM. The antibody was serially diluted at a ratio of 1:5. 100 μl of the diluted antibody was added to the cells prepared above, mixed evenly, and the 96-well plate was placed in an incubator, and the killing effect was measured after 18 hours. 4) Testing: Since the target cells H929 or RPMI8226 contain the luciferase gene, the killing efficiency of the target cells was tested by the LUMINEX method. Steady-GLO (Promega) was used as the substrate, and the buffer solution in the kit was thawed and melted, then added to the substrate powder and mixed evenly, and 5ml or 10ml was dispensed per tube to complete the reconstitution of the Steady-GLO substrate. The co-cultured cells were pipetted up and down to mix evenly, and 100μl was then taken and transferred to an opaque white plate, and 100μl of the reconstituted Steady-GLO substrate was added, tapped lightly to mix evenly, and left for 5 minutes before reading the plate. The detector was Synergy HT. 5) Data processing: The formula for calculating the killing rate of target cells is as follows: Killing rate (percentage) of target cells = 100 x (number of wells containing only target cells - number of test wells) / number of wells containing only target cells

[0100] The antibody concentrations corresponding to the killing rate of the target cells in all the test wells were converted to log10, and a graph was created with this as the horizontal axis and the killing rate as the vertical axis. The killing concentration-slope curves of the secondary antibodies B10K3, B10.3K3, B10.4K3, B10.5K3, and B10.9K3 in cFIST format against the sensitive cell line RPMI8226 are shown in Figure 9B, and the killing percentage of each slope is shown in Table 5.

[0101] The results for B10.3K3 and B10.3K3 are shown in Figure 10, their percentage killing against RPMI8226 is shown in Table 6, and their percentage killing against H929 is shown in Table 7. The results show that the bispecific antibody B10.3K3 can effectively mediate PBMC killing of the tumor cell lines H929 or RPMI8226, 1689840495729_6 The molecules of B10.3K3 have both the biological functions of anti-BCMA and anti-CD3 monoclonal antibodies at the same time. The killing effect of target cells RPMI8226 mediated by K3B10.3 is significantly weaker than that of B10.3K3, and in particular, there is almost no killing effect on H929 cells. The EC50 of target cell killing mediated by B10.9K3, B10.5K3, B10.4K3, B10.3K3, and B10K3 was 8.759pM, 12.87pM, 7.839pM, 5.833pM, and 281.7pM, respectively.

[0102] [Table 5]

[0103] [Table 6]

[0104] [Table 7]

[0105] In Tables 5, 6 and 7 above, the values ​​in the two columns for each bispecific antibody are the results of two replicate experiments (duplicates).

[0106] Although the present invention has been described in detail above through a general description and specific embodiments, it is obvious to those skilled in the art that some modifications and improvements can be made based on the present invention. Therefore, any modifications or improvements made within the scope of the present invention that do not deviate from the spirit of the present invention are within the scope of the present invention. [Industrial Applicability]

[0107] The present invention relates to a bispecific antibody that binds to BCMA and CD3, and a method for preparing and using the same. The present invention provides a bispecific antibody that simultaneously binds to BCMA and CD3, has a specific targeting effect, and can efficiently induce a directed immune response. The bispecific antibody fully retains the biological functions of anti-CD3 antibody and anti-BCMA antibody, and realizes that one bispecific antibody molecule simultaneously has excellent biological functions of target binding to BCMA and CD3, can act as a bridge between tumor cells and immune effector cells, can effectively induce immune effector cells and directed immune responses, significantly improve the killing effect of immune cells against tumor cells, has high killing efficiency against target cells, minimizes the ADCC effect, has high safety, and has good prospects for use in tumor immunotherapy.

Claims

1. A bispecific antibody that binds to BCMA and CD3, a first domain that binds to the B cell maturation antigen BCMA and a second domain that binds to the T cell surface antigen CD3; the first domain and the second domain are linked via a linker peptide, The first domain comprises: a heavy chain variable region in which CDR1, CDR2, and CDR3 have the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively, or have amino acid sequences containing one or a combination of mutations selected from the following mutations, each of which is based on the amino acid sequence set forth in SEQ ID NOs: 4 to 6 as a reference sequence; (1) a mutation from S at the first position of the amino acid sequence shown in SEQ ID NO: 4 to P or K; (2) a mutation from the second Y in the amino acid sequence shown in SEQ ID NO: 4 to H, D, G, M, or S; (3) A to N mutation at the third position of the amino acid sequence shown in SEQ ID NO: 4; (4) A mutation from S at the fifth position of the amino acid sequence shown in SEQ ID NO: 4 to H, N, A, or M; (5) a mutation from G to V or T at the first position of the amino acid sequence shown in SEQ ID NO: 5; (6) a mutation from I to H at the third position of the amino acid sequence shown in SEQ ID NO: 5; (7) A mutation from I to D or A at the fourth position of the amino acid sequence shown in SEQ ID NO: 5; (8) A mutation from F to H at the fifth position of the amino acid sequence shown in SEQ ID NO: 5; (9) A mutation from T to K at position 7 of the amino acid sequence shown in SEQ ID NO: 5; and (10) A mutation from F to L at the fourth position of the amino acid sequence shown in SEQ ID NO: 6; a light chain variable region in which CDR1, CDR2, and CDR3 have the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively, or have amino acid sequences containing one or a combination of mutations selected from the following mutations, with the amino acid sequences set forth in SEQ ID NOs: 1 to 3 as reference sequences; (1) a mutation from S at position 5 of the amino acid sequence shown in SEQ ID NO: 1 to N, R, or T; (2) a mutation from L to Q, V, or A at the 7th position of the amino acid sequence shown in SEQ ID NO: 3; (3) a mutation from I to S, A, R, or P at the 8th position of the amino acid sequence shown in SEQ ID NO: 3; (4) A mutation from Y at position 10 of the amino acid sequence shown in SEQ ID NO: 3 to M, L, W, or T; and (5) A mutation from V to L or Q at position 11 of the amino acid sequence represented by SEQ ID NO: 3; A bispecific antibody that binds to BCMA and CD3, comprising:

2. A bispecific antibody that binds to BCMA and CD3, a first domain that binds to the B cell maturation antigen BCMA and a second domain that binds to the T cell surface antigen CD3; the first domain and the second domain are linked via a linker peptide, The first domain comprises: a heavy chain variable region in which CDR1 has an amino acid sequence represented by any one of SEQ ID NOs: 4, 7, 8, and 9, CDR2 has an amino acid sequence represented by any one of SEQ ID NOs: 5 and 10, and CDR3 has an amino acid sequence represented by any one of SEQ ID NOs: 6 and 11; a light chain variable region in which CDR1 has the amino acid sequence set forth in any one of SEQ ID NOs: 1 and 12, CDR2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 has the amino acid sequence set forth in any one of SEQ ID NOs: 3, 13, 14, 15, and 16; Preferably, the CDRs of the heavy chain variable region of the first domain are: (1) CDR1 has the amino acid sequence shown in SEQ ID NO: 4, CDR2 has the amino acid sequence shown in SEQ ID NO: 5, and CDR3 has the amino acid sequence shown in SEQ ID NO: 6; (2) CDR1 has the amino acid sequence shown in SEQ ID NO: 7, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11; (3) CDR1 has the amino acid sequence shown in SEQ ID NO: 8, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11; and (4) CDR1 has the amino acid sequence shown in SEQ ID NO: 9, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11; The CDRs of the light chain variable region of the first domain are (1) CDR1 has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 13; (3) CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 14; (4) CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 15; and (5) CDR1 has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 16; More preferably, the CDRs of the heavy chain variable region and the CDRs of the light chain variable region of the first domain are (1) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 4, CDR2 has the amino acid sequence shown in SEQ ID NO: 5, and CDR3 has the amino acid sequence shown in SEQ ID NO: 6; CDR1 of the light chain variable region has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 8, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, and CDR3 has the amino acid sequence shown in SEQ ID NO: 11; CDR1 of the light chain variable region has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 13; (3) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 9, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, CDR3 has the amino acid sequence shown in SEQ ID NO: 11, CDR1 of the light chain variable region has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3; and (4) A bispecific antibody that binds to BCMA and CD3, characterized in that it is any one of the following: CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 8, CDR2 has the amino acid sequence shown in SEQ ID NO: 10, CDR3 has the amino acid sequence shown in SEQ ID NO: 11, CDR1 of the light chain variable region has the amino acid sequence shown in SEQ ID NO: 12, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO:

16.

3. the heavy chain variable region of the first domain has an amino acid sequence shown in any one of SEQ ID NOs: 17 and 19 to 21, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 18 and 22 to 27; Alternatively, the heavy chain variable region and the light chain variable region have at least one of the following two characteristics compared to the above sequences: a) they bind to the same antigenic determinant, and b) they have amino acid sequences with greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% sequence identity. Preferably, the heavy chain variable region of the first domain has the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 18, Alternatively, the heavy chain variable region of the first domain has the amino acid sequence shown in SEQ ID NO: 20, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 22, Alternatively, the heavy chain variable region of the first domain has the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 27, Alternatively, the heavy chain variable region of the first domain has the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 25, Alternatively, the bispecific antibody that binds to BCMA and CD3 according to claim 2, characterized in that the heavy chain variable region of the first domain has the amino acid sequence set forth in SEQ ID NO: 20 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:

26.

4. the first domain is two complete heavy-light chain pairs linked via disulfide bonds; The bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 3, characterized in that the Fc fragment of the heavy chain is an Fc fragment of a human or humanized antibody, and the human or humanized antibody is IgG1, IgG2, IgA, IgE, IgM, IgG4, or IgD.

5. The heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 32, and the light chain has the amino acid sequence shown in SEQ ID NO: 33; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO: 49, and the light chain has the amino acid sequence set forth in SEQ ID NO: 42; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO: 50 and the light chain has the amino acid sequence set forth in SEQ ID NO: 44; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO: 51 and the light chain has the amino acid sequence set forth in SEQ ID NO: 46; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO: 52 and the light chain has the amino acid sequence set forth in SEQ ID NO: 48; Alternatively, the bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 4, characterized in that, compared to the sequences, the heavy chains and light chains have at least one of the following two characteristics: a) they bind to the same antigenic determinant, and b) they have amino acid sequences with greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% sequence identity.

6. the heavy chain variable region of the second domain has the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 29; The bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 5, wherein the light chain variable region and heavy chain variable region of the second domain are preferably linked via a linker peptide to form a single-chain antibody, and the single-chain antibody has the amino acid sequence shown in SEQ ID NO:

31.

7. the second domain comprises two single chain antibodies; The bispecific antibody comprises: (1) The C-terminus of the two single-chain antibodies of the second domain is linked to the N-terminus of the two heavy chains of the first domain via a linker peptide, respectively; and (2) The N-terminus of the two single-chain antibodies in the second domain is linked to the C-terminus of the two heavy chains in the first domain via a linker peptide, respectively, to form a symmetric structure; Preferably, the amino acid sequence of the linker peptide is (GGGGX)n (wherein X is Gly or Ser, and n is a natural number from 1 to 4), The bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 6, wherein the amino acid sequence of the linker peptide is that shown in SEQ ID NO:

30.

8. The heavy chain of the first domain, after being linked to the second domain via a linker peptide, has the amino acid sequence shown in SEQ ID NO: 34 or 35, and the light chain has the amino acid sequence shown in SEQ ID NO: 33; Alternatively, the heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 41 after being linked to the second domain via a linker peptide, and the light chain has the amino acid sequence shown in SEQ ID NO: 42; Alternatively, the heavy chain of the first domain has the amino acid sequence set forth in SEQ ID NO: 43 after being linked to the second domain via a linker peptide, and the light chain has the amino acid sequence set forth in SEQ ID NO: 44; Alternatively, the heavy chain of the first domain, after being linked to the second domain via a linker peptide, has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain has the amino acid sequence set forth in SEQ ID NO: 46; Alternatively, the heavy chain of the first domain has the amino acid sequence shown in SEQ ID NO: 47 after being linked to the second domain via a linker peptide, and the light chain has the amino acid sequence shown in SEQ ID NO:

48. A bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 7, characterized in that

9. A nucleic acid encoding a bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 8.

10. 10. A nucleic acid-containing biomaterial according to claim 9, comprising recombinant DNA, an expression cassette, a vector, a host cell, a recombinant bacterium or a cell line.

11. 9. A method for producing a bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 8, comprising: constructing an expression vector containing genes encoding the first domain and the second domain; introducing the expression vector into host cells to obtain host cells that express the bispecific antibody; culturing the host cells; and obtaining the bispecific antibody by separation and purification.

12. Use of a bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 8, a nucleic acid according to claim 9, or a biomaterial according to claim 10, (1) Use in the manufacture of a pharmaceutical for diagnosing, preventing, or treating a B cell-related disorder in which BCMA is expressed; (2) Use in the manufacture of a pharmaceutical for diagnosing, preventing, or treating a disease that targets BCMA; (3) Use in the manufacture of a medicament for killing cells expressing BCMA. (4) Use in the manufacture of a detection reagent for BCMA and / or CD3; and (5) Use in the manufacture of related reagents applied to CAR-T therapy One of the following: Preferably, the B cell-related disease in which BCMA is expressed includes B cell-related tumors and B cell-mediated autoimmune diseases.

13. A multispecific antibody, fusion protein, immunotoxin, pharmaceutical or detection reagent comprising a bispecific antibody that binds to BCMA and CD3 according to any one of claims 1 to 8.

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