Bispecific antibody combining BCMA and CD3 as well as preparation method and application of bispecific antibody

Abstract

The invention relates to the technical field of genetic engineering antibodies, in particular to a bispecific antibody combined with BCMA and CD3 as well as a preparation method and application of the bispecific antibody. The invention provides a bispecific antibody which can be combined with BCMA and CD3 at the same time, has a specific targeting effect and can efficiently excite oriented immune response. The bispecific antibody well retains the biological functions of an anti-CD3 antibody and an anti-BCMA antibody, realizes that one bispecific antibody molecule simultaneously has an excellent BCMA and CD3 targeted binding biological function, can build a bridge between tumor cells and immune effector cells, effectively activates the immune effector cells and oriented immune response, and has a good application prospect. The tumor cell killing effect of immune cells is remarkably enhanced, the target cell killing efficiency is high, the ADCC effect is reduced to the maximum degree, high safety is achieved, and good application prospects are achieved in tumor immunotherapy.

Description

Technical Field

[0001] This invention is a divisional application of a patent application with a filing date of June 24, 2021, application number 2021107042556, and invention name "Bispecific antibodies binding to BCMA and CD3, and preparation methods and applications thereof". The present invention relates to the field of genetic engineering antibody technology, and specifically to bispecific antibodies binding to BCMA and CD3, and preparation methods and applications thereof. Background Art

[0002] Monoclonal antibodies can be used clinically for tumor treatment, immune disease treatment, and anti-infection treatment. Among them, tumor treatment is currently the most widely used field for monoclonal antibodies. Monoclonal antibody treatment of tumors is an immunotherapy that targets specific targets of diseased cells to stimulate the immune system to kill target cells. On the basis of monoclonal antibodies, in order to further improve the efficacy and reduce toxic side effects, new antibody drugs based on monoclonal antibodies, including antibody drug conjugates (ADC), bispecific antibodies (bsAb), and CAR-T (chimeric antigen receptor T cells) and other new protein and cell drugs have developed rapidly. Among them, cell-bridging bispecific antibodies constructed based on bispecificity have significantly improved the killing activity and specificity due to their ability to connect immune T cells and cancer target cells, and have breakthrough clinical value, and have received special attention. For multiple myeloma, cell-bridging bispecific antibodies with the above properties targeting BCMA are one of the development directions for improving the therapeutic effect of antibodies, and have now become a hot spot in the field of antibody engineering research.

[0003] The CD3 molecule on the surface of T cells is composed of 4 subunits: δ, ε, γ, ζ, with molecular weights of 18.9k Da, 23.1kDa, 20.5kDa and 18.7kDa, respectively, and lengths of 171, 207, 182 and 164 amino acid residues, respectively. Together, they form 6 peptide chains, which often bind tightly to the T cell receptor (TCR) to form a TCR-CD3 complex containing 8 peptide chains. The schematic diagram of its structure is shown in Figure 1A. This complex has the functions of T cell activation, signal transduction and stabilization of TCR structure. The CD3 cytoplasmic segment contains an immunoreceptor tyrosine-based activation motif (ITAM). TCR recognizes and binds to antigenic peptides presented by MHC (major histo-compatibility complex) molecules, causing the tyrosine residues in the conserved sequence of CD3 ITAM to be phosphorylated by the tyrosine protein kinase p56lck in T cells, and then recruiting other tyrosine protein kinases containing SH2 (Scr homology 2) domains (such as ZAP-70). ITAM phosphorylation and binding to ZAP-70 are one of the important biochemical reactions in the early stages of the T cell activation signal transduction process. Therefore, the function of the CD3 molecule is to transduce the activation signal generated by TCR recognition of antigens.

[0004] 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 in differentiated plasma cells and is involved in B-cell maturation, growth and survival. BCMA was originally reported as an integral membrane protein in the Golgi apparatus of mature human B lymphocytes, i.e., as an intracellular protein, which suggests that BCMA appears to have an important role in B-cell development and homeostasis.

[0005] BCMA expression is restricted to the B-cell lineage and is primarily found on plasma cells and plasmablasts ( Figure 1 BCMA is expressed on multiple myeloma (MM) cells and to some extent on memory B-cells, but is virtually absent on peripheral B-cells. BCMA is also expressed on multiple myeloma (MM) cells. Together with its family members, transmembrane activator and cyclophilin ligand interactor (TACI) and B-cell activating factor of the TNF family receptor (BAFF-R), BCMA regulates different aspects of humoral immunity, B-cell development, and homeostasis. BCMA is a receptor for two ligands of the TNF superfamily ( Figure 1C): APRIL (proliferation-inducing ligand, CD256, TNFSF13), a high-affinity ligand of BCMA, and B cell activating factor BAFF (THANK, BlyS, B lymphocyte stimulating factor, TALL-1 and zTNF4), a low-affinity ligand of BCMA. APRIL and BAFF show structural similarities and overlapping but different receptor binding specificities. The negative regulator TACI also binds to both BAFF and APRIL. The coordination binding of APRIL and BAFF to BCMA and / or TACI activates the transcription factor NF-κB and increases the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1) and downregulates the expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc.), thereby inhibiting cell apoptosis and promoting survival. This combined action promotes B cell differentiation, proliferation, survival and antibody production. BCMA expression occurs at a later stage in B-cell differentiation and contributes to the long-term survival of plasmablasts and plasma cells in the bone marrow.

[0006] BCMA is an extremely important B cell biomarker that is widely present on the surface of MM cells and is a popular immunotherapy target for MM and other hematological malignancies. BCMA has been the focus of industry attention at the three consecutive ASCO annual meetings. The latest analysis by the American Cancer Research Institute (CRI) shows that BCMA has become the second most popular target for anticancer cell therapy after CD19. MM is a heterogeneous disease and most are caused by chromosomal translocations such as t(11;14), t(4;14), t(8;14), del(13), del(17) (among others). MM-affected patients may experience a variety of disease-related symptoms due to bone marrow infiltration, bone destruction, renal failure, immune deficiency, and the psychological burden of cancer diagnosis.

[0007] Treatments for MM, such as chemotherapy and stem cell transplantation, can improve survival, but are often accompanied by harmful side effects, and therefore MM remains difficult to treat. Despite great advances in chemotherapy, proteasome inhibitors, immunomodulatory agents thalidomide derivatives, and CD38-targeted antibodies, almost all patients eventually relapse. Therefore, the need for new drugs in this area remains urgent. To date, the two most common treatment options for patients with multiple myeloma are steroids, thalidomide, lenalidomide, bortezomib, or a combination of various cytotoxic agents, and high-dose chemotherapy combined with autologous stem cell transplantation for younger patients. Most transplants are autologous, using the patient's own cells. Such transplants, although not curative, have been shown to prolong the lives of selected patients. They can be performed as initial therapy in newly diagnosed patients or at relapse. Sometimes more than one transplant may be recommended for selected patients in order to adequately control the disease. The chemotherapeutic agents used to treat the disease are cyclophosphamide, doxorubicin, vincristine, and melphalan, and combination therapy with immunomodulators such as thalidomide, lenalidomide, bortezomib, and corticosteroids such as dexamethasone has become an important option for the treatment of myeloma in newly diagnosed patients and in patients with advanced disease who have failed chemotherapy or transplantation.

[0008] Current treatments for MM are generally non-curative. Stem cell transplantation may not be an option for many patients because of advanced age, the presence of other serious illnesses, or other physical limitations. Chemotherapy can only partially control multiple myeloma and rarely achieves complete remission. Therefore, new innovative treatments are urgently needed.

[0009] Antagonistic BCMA-specific antibodies can block NF-κB activation, which is involved in a potent pro-survival signaling pathway in normal and malignant B-cells.

[0010] Other approaches in combating hematogenous tumors or autoimmune disorders focus on the interaction between BAFF and APRIL (i.e., ligands of the TNF ligand superfamily) and their receptors TACI, BAFF-R, and BCMA activated by BAFF and / or APRIL. For example, atacicept (TACI-Ig) was generated by fusing the Fc-domain of human immunoglobulin with TACI from Zymogenetics to neutralize these two ligands and prevent receptor activation. Atacicept is currently in clinical trials for the treatment of systemic lupus erythematosus (SLE, Phase III), multiple sclerosis (MS, Phase II), and rheumatoid arthritis (RA, Phase II), as well as in Phase I clinical trials for the treatment of B-cell malignancies chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and MM. In preclinical studies, atacicept reduced the growth and survival of naive MM cells and MM cell lines in vitro and in vivo, indicating the relevance of the TACI ligand for MM cells. Since most MM cells and their derived cell lines express BCMA and TACI, the receptors for both may contribute to ligand-mediated growth and survival. These data suggest that antagonism of BCMA and TACI may be beneficial for the treatment of plasma cell disorders. In addition, BCMA-specific antibodies that cross-react with TACI have been described (WO02 / 066516).

[0011] Human Genome Sciences and GlaxoSmithKline have developed an antibody targeting BAFF called belimumab. Belimumab blocks the binding of soluble BAFF to its receptors BAFF-R, BCMA, and TACI on B cells. Belimumab does not bind directly to B cells, but by binding to BAFF, belimumab inhibits the survival of B cells, including autoreactive B cells, and reduces the differentiation of B cells into plasma cells that produce immunoglobulins.

[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 are amenable to therapeutic regimens in the fight against cancer and / or autoimmune disorders, there is still a need for other available options for the treatment of such medical conditions. Summary of the invention

[0013] The first object of the present invention is to provide a bispecific antibody that binds to BCMA and CD3 and an active fragment thereof.

[0014] The second object of the present invention is to provide a nucleic acid encoding the above-mentioned antibody or an active fragment thereof.

[0015] The third object of the present invention is to provide uses of the above-mentioned antibody or its active fragment.

[0016] Specifically, the present invention provides the following technical solutions: First, the present invention provides a bispecific antibody that binds to BCMA and CD3, the bispecific antibody comprising: The first domain binds to the B cell maturation antigen BCMA.

[0017] and, the second domain, which binds to the T cell surface antigen CD3,

[0018] The first domain and the second domain are connected by a connecting peptide.

[0019] The first domain includes a heavy chain variable region and a light chain variable region, and CDR1, CDR2, and CDR3 of the heavy chain variable region respectively have the amino acid sequences shown in SEQ ID NOs. 4-6, or respectively have amino acid sequences with the amino acid sequences shown in SEQ ID NOs. 4-6 as reference sequences and containing one or more of the following mutations: (1) The first S in the amino acid sequence shown in SEQ ID NO.4 is mutated to P or K; (2) The second Y in the amino acid sequence shown in SEQ ID NO.4 is mutated to H, D, G, M or S; (3) The third A in the amino acid sequence shown in SEQ ID NO.4 is mutated to N; (4) The 5th S in the amino acid sequence shown in SEQ ID NO.4 is mutated to H, N, A or M; (5) The first G in the amino acid sequence shown in SEQ ID NO.5 is mutated to V or T; (6) The 3rd position I of the amino acid sequence shown in SEQ ID NO.5 is mutated to H; (7) The 4th position I of the amino acid sequence shown in SEQ ID NO.5 is mutated to D or A; (8) The 5th F in the amino acid sequence shown in SEQ ID NO.5 is mutated to H; (9) The 7th T in the amino acid sequence shown in SEQ ID NO.5 is mutated to K; (10) the 4th position F of the amino acid sequence shown in SEQ ID NO.6 is mutated to L; The CDR1, CDR2 and CDR3 of the light chain variable region of the first domain respectively have the amino acid sequences shown in SEQ ID NO.1-3, or respectively have the amino acid sequences obtained by taking the amino acid sequences shown in SEQ ID NO.1-3 as reference sequences and containing one or more of the following mutations: (1) The 5th S in the amino acid sequence shown in SEQ ID NO.1 is mutated to N, R or T; (2) L at position 7 of the amino acid sequence shown in SEQ ID NO.3 is mutated to Q, V or A; (3) The 8th position I of the amino acid sequence shown in SEQ ID NO.3 is mutated to S, A, R or P; (4) The Y at position 10 of the amino acid sequence shown in SEQ ID NO.3 is mutated to M, L, W or T; (5) The V at position 11 of the amino acid sequence shown in SEQ ID NO.3 is mutated to L or Q.

[0020] The present invention screens out genetically engineered single-chain antibodies against BCMA from a fully synthetic single-chain human antibody library, obtains the variable region gene sequence of the antibody, constructs a mutation library using a point mutation kit, obtains clones with high affinity, mixes the DNA of these clones, assembles a single-chain antibody combinatorial library in a recombinant manner, and obtains BCMA antibodies with high affinity that bind to human BCMA after screening.

[0021] The antibody variable region amino acid sequence pattern provided by the present invention is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the present application, the regional division of FR and CDR is based on the Kabat naming system. Here, FR1-4 represents 4 framework regions, and CDR1-3 represents 3 hypervariable regions. FR1-4 can be separated from the constant region sequence (such as the most commonly used amino acids of human immunoglobulin light and heavy chains, subclasses or subfamilies), or can be separated from a single human antibody framework region or from a combination of different framework region genes.

[0022] After further screening, the present invention provides the following bispecific antibodies, the first domain of which exhibits higher affinity to BCMA: A bispecific antibody that binds to BCMA and CD3, comprising: The first domain binds to the B cell maturation antigen BCMA.

[0023] and, the second domain, which binds to the T cell surface antigen CD3,

[0024] The first domain and the second domain are connected by a connecting peptide.

[0025] The first domain includes a heavy chain variable region and a light chain variable region, wherein CDR1 of the heavy chain variable region 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; The CDR1 of the light chain variable region 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 any one of SEQ ID NOs.2, and the CDR3 has the amino acid sequence shown in any one of SEQ ID NOs.3, 13, 14, 15, and 16.

[0026] Furthermore, among the above antibodies that bind to BCMA, the following antibodies exhibit higher affinity to BCMA: The CDR region sequence of the heavy chain variable region is any of the following: (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; (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 any of the following: (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; (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; Furthermore, among the above antibodies that bind to BCMA, the following antibodies exhibited higher affinity to BCMA: The CDR of the heavy chain variable region and the CDR of the light chain variable region are any of the following: (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, 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.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; (4) 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.16.

[0027] Based on the above CDR sequences, antibodies with the following variable region sequences (first domain) exhibited higher affinity for BCMA: The heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs. 17, 19-21, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs. 18, 22-27; Based on the above-mentioned heavy chain variable region and light chain variable region, compared with their sequences, the amino acid sequences that meet at least one of the following two conditions: a) bind to the same antigenic epitope; b) have a sequence identity greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% are also within the scope of protection of the present invention.

[0028] Furthermore, antibodies with the following variable region sequences (first domain) exhibited superior affinity for BCMA: The heavy chain variable region 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 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 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 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 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.

[0029] In the bispecific antibody of the present invention, the first domain is an antibody capable of binding to BCMA, comprising two complete heavy chain-light chain pairs, wherein each pair of light chains and heavy chains are connected by a disulfide bond.

[0030] 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).

[0031] As one embodiment of the present invention, the Fc fragment of the heavy chain of the first domain is the Fc fragment of a human or humanized IgG4 antibody.

[0032] 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.

[0033] Alternatively, the heavy chain has the amino acid sequence shown in SEQ ID NO.49, and the light chain has the amino acid sequence shown in SEQ ID NO.42,

[0034] Alternatively, the heavy chain has the amino acid sequence shown in SEQ ID NO.50, and the light chain has the amino acid sequence shown in SEQ ID NO.44,

[0035] Alternatively, the heavy chain has the amino acid sequence shown in SEQ ID NO.51, and the light chain has the amino acid sequence shown in SEQ ID NO.46,

[0036] Alternatively, the heavy chain has the amino acid sequence shown in SEQ ID NO.52, and the light chain has the amino acid sequence shown in SEQ ID NO.48.

[0037] These BCMA-binding antibodies have a higher affinity for BCMA and can ensure that when forming bispecific antibodies with antibodies that bind to CD3, the functions of both are fully exerted.

[0038] Based on the above-mentioned heavy chain and light chain sequences, compared with the sequences, the amino acid sequences that meet at least one of the following two conditions: a) bind to the same antigenic epitope; b) have a sequence identity greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% are also within the scope of protection of the present invention.

[0039] The humanized anti-human BCMA antibody provided by the present invention binds to human BCMA with an affinity of 0.2 nM-10 nM. The antibody inhibits BCMA ligand binding to human BCMA. The antibody binds to cells expressing BCMA, and the cells may be human multiple myeloma cells or lymphoma cells.

[0040] For the second domain that binds to the CD3 antigen, its heavy chain variable region has the amino acid sequence shown in SEQ ID NO.28, and its light chain variable region has the amino acid sequence shown in SEQ ID NO.29.

[0041] Preferably, the light chain variable region and the heavy chain variable region of the second domain are connected via a connecting peptide to form a single chain antibody, and the single chain antibody has the amino acid sequence shown in SEQ ID NO.31.

[0042] The bispecific antibody of the present invention is preferably designed as follows: the first domain comprises two complete light chain-heavy chain pairs, and the second domain comprises two single-chain antibodies, which are connected in any of the following ways to form a symmetrical structure: (1) The C-termini of the two single-chain antibodies in the second domain are connected to the N-termini of the two heavy chains in the first domain through connecting peptides; (2) The N-termini of the two single-chain antibodies in the second domain are connected to the C-termini of the two heavy chains in the first domain via connecting peptides.

[0043] The present invention finds that for the sequences of the first domain and the second domain, the bispecific antibody with the above symmetrical structure can better retain the specific antigen binding ability of the original antibody of the first domain and the second domain compared with bispecific antibodies of other structures, and at the same time has excellent biological functions of binding to BCMA and CD3, and has obvious advantages in production process and pharmaceutical performance, etc. The present invention has developed a bispecific antibody binding to BCMA and CD3 with the above antibody molecular structure, which has a specific targeting effect and can efficiently stimulate a directed immune response to kill tumor cells.

[0044] The amino acid sequence of the connecting peptide used to connect the first domain and the second domain is preferably (GGGGX)n, wherein X is Gly or Ser, and n is a natural number of 1-4.

[0045] As a preferred embodiment of the present invention, the amino acid sequence of the connecting peptide is shown as SEQ ID NO.30.

[0046] As an example of a bispecific antibody having the above structure, the present invention provides a bispecific antibody against human CD3 and BCMA. In the structure and sequence of the heavy chain variable region and light chain variable region of the above single-chain antibody and the heavy chain and light chain of the monoclonal antibody, the present invention constructs two bispecific antibodies that can simultaneously bind to CD3 and BMCA, and the structure and sequence thereof are as follows: (1) The heavy chain of the first domain is connected 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 connected 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 and the second domain, after being connected via a connecting peptide, have the amino acid sequence shown in SEQ ID NO.41, and the light chain has the amino acid sequence shown in SEQ ID NO.42; (4) the heavy chain of the first domain and the second domain, after being connected via a connecting peptide, have the amino acid sequence shown in SEQ ID NO.43, and the light chain has the amino acid sequence shown in SEQ ID NO.44; (5) The heavy chain of the first domain and the second domain, after being connected via a connecting peptide, have the amino acid sequence shown in SEQ ID NO.45, and the light chain has the amino acid sequence shown in SEQ ID NO.46; (6) The heavy chain of the first domain and the second domain, after being connected via a connecting peptide, have the amino acid sequence shown in SEQ ID NO.47, and the light chain has the amino acid sequence shown in SEQ ID NO.48.

[0047] The sequences disclosed and claimed above as SEQ ID NO.1-52 include "conservative sequence modifications", i.e., nucleotide and amino acid sequence modifications that do not significantly affect and change the binding characteristics of the antibody or the antibody containing the amino acid sequence. The conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. Modifications can be introduced into SEQ ID NO.1-52 by standard techniques in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacement of amino acid residues by amino acid residues or other amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a non-essential amino acid residue in the human anti-BCMA antibody with another amino acid residue from the same side chain family.

[0048] Therefore, the antibodies disclosed above having the amino acid sequence and / or antibodies containing the amino acid sequence disclosed above, including antibodies substantially encoded by similar sequences modified by conservative sequences, or antibodies containing similar sequences modified by conservative sequences, should all be considered to be within the scope of the present invention.

[0049] The present invention also provides a nucleic acid encoding the bispecific antibody that binds to BCMA and CD3.

[0050] Taking into account the degeneracy of codons, the gene encoding the antibody of the present invention can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody. Those skilled in the art can artificially synthesize and modify genes according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0051] The present invention also provides a biological material containing a nucleic acid encoding the bispecific antibody that binds BCMA and CD3, wherein the biological material includes recombinant DNA, an expression cassette, a vector, a host cell, an engineered bacterium or a cell line.

[0052] The vector includes but is not limited to a cloning vector and an expression vector, and may be a plasmid vector, a viral vector, a transposon or the like.

[0053] The host cell or cell line may be a cell or cell line derived from a microorganism or an animal.

[0054] The present invention also provides a method for preparing a bispecific antibody that binds BCMA and CD3, the method comprising: constructing an expression vector containing the coding genes of 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 through separation and purification.

[0055] When preparing the bispecific antibody, those skilled in the art can select conventional host cells, expression vectors, methods for introducing expression vectors into host cells, and methods for separating and purifying antibodies according to their needs.

[0056] Based on the function of the bispecific antibody binding to BCMA and CD3 provided by the present invention, the present invention provides any of the following uses of the bispecific antibody binding to BCMA and CD3 or its encoding nucleic acid or a biomaterial containing the nucleic acid: (1) Use in the preparation of a drug for diagnosing, preventing or treating a BCMA-expressing B cell-related disease; preferably, the BCMA-expressing B cell-related disease includes a B cell-related tumor or an autoimmune disease caused by a B cell; more preferably, the B cell-related tumor is multiple myeloma, lymphoma, plasma cell leukemia, plasmacytoma, macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, hairy cell leukemia, myeloid leukemia, acute myeloid leukemia AML, acute lymphocytic leukemia ALL, chronic lymphocytic leukemia CLL or chronic myeloid leukemia CML; the autoimmune disease caused by a B cell is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, membranous nephropathy, chronic kidney disease, myasthenia gravis, ANCA vasculitis, neuromyelitis optica spectrum disorder, MOG-AD, POEMS syndrome or an IgG4-related disease; (2) Use in the preparation of drugs for diagnosing, preventing or treating diseases targeting BCMA; (3) Use in the preparation of a drug for killing BCMA-expressing cells; preferably, the BCMA-expressing cells include B cell-related tumor cells or B cell-induced autoimmune disease cells; more preferably, the BCMA-expressing B cell-related tumor is multiple myeloma, lymphoma, plasma cell leukemia, plasmacytoma, macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, hairy cell leukemia, myeloid leukemia, acute myeloid leukemia AML, acute lymphocytic leukemia ALL, chronic lymphocytic leukemia CLL or chronic myeloid leukemia CML; the autoimmune disease caused by B cells is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, membranous nephropathy, chronic kidney disease, myasthenia gravis, ANCA vasculitis, neuromyelitis optica spectrum disease, MOG-AD, POEMS syndrome or IgG4-related disease; (4) Application in the preparation of BCMA and / or CD3 detection reagents; (5) Application in the preparation of relevant reagents suitable for CAR-T therapy.

[0057] The above-mentioned BCMA-expressing B cell-related diseases are preferably malignant tumors and autoimmune diseases that highly express / overexpress BCMA.

[0058] The bispecific antibodies provided by the present invention can be used for treatment and diagnosis. Antibodies can be labeled, cross-linked or coupled and fused with other protein or polypeptide molecules to form complexes (such as cytotoxic substances, radioactive toxins and / or chemical molecules, etc.) for diagnosis and treatment.

[0059] The present invention also provides a multispecific antibody, a fusion protein, an immunotoxin, a drug or a detection reagent comprising the bispecific antibody that binds to BCMA and CD3.

[0060] The immunotoxins described above comprise the bispecific antibody linked to a cytotoxic agent in various forms.

[0061] The various connection forms are antibody labeling, in vitro cross-linking or molecular coupling. The cytotoxic agents include chemical molecules, radioisotopes, polypeptides, toxins and other substances that have the characteristics of killing cells or inducing cell death.

[0062] The above-mentioned fusion protein comprises a complex of the above-mentioned bispecific antibody provided by the present invention and other proteins or polypeptide molecules having certain functions.

[0063] Specifically, the fusion protein can be obtained by connecting an antibody gene with an immunotoxin or cytokine gene to construct a recombinant expression vector, and obtaining the recombinant fusion protein molecule through mammalian cells or other expression systems.

[0064] The drugs and detection reagents described above may also contain other active ingredients or excipients permitted in the pharmaceutical and detection reagent fields (eg, antibody components and pharmacologically acceptable delivery molecules or solutions. Among them, the therapeutic components are sterile and can be freeze-dried at low temperatures).

[0065] The bispecific antibodies of the present invention can inhibit one or more biological activities induced by BCMA. They can act by preventing BCMA from binding to its ligand, or by killing cells with high BCMA expression, or by internalizing the complex after binding to BCMA, thereby consuming BCMA on the cell surface. All interference functions of BCMA antagonists should be equally regarded as the purpose of the present invention.

[0066] The beneficial effects of the present invention are: The present invention screens specific antibodies against human B cell surface antigen BCMA from a single-chain antibody library of natural full-human sequences through genetic engineering and phage surface display library technology. Multiple clones with significantly improved binding ability are obtained through affinity maturation transformation, and their affinity for human BCMA is between 0.26 nM and 0.31 nM. The apparent affinity measured by flow cytometry is 61-97 times higher than that of the parent antibody, confirming that the anti-human BCMA antibody of the present invention and the optimized mutant have good ability to bind to BCMA.

[0067] On this basis, the present invention provides a bispecific antibody that can simultaneously bind to BCMA and CD3, has a specific targeting effect, and can efficiently stimulate a directed immune response. The bispecific antibody better retains the biological functions of anti-CD3 antibodies and anti-BCMA antibodies, and realizes that a bispecific antibody molecule has excellent biological functions of binding to BCMA and CD3 at the same time, can build a bridge between tumor cells and immune effector cells, effectively activate immune effector cells and directed immune responses, significantly enhance the efficacy of immune cells in killing tumor cells, and at the same time minimize the ADCC effect, with high safety.

[0068] The bispecific antibody provided by the present invention has good therapeutic application prospects, mainly manifested in specific binding activity with human BCMA and CD3. The antibody can specifically bind to BCMA on the surface of H929 and RPMI8226 cells and T cells through ELISA detection and flow cytometer detection, indicating good target specificity. The bispecific antibody has a high target cell killing efficiency through in vitro cell killing efficiency detection.

[0069] In addition, the bispecific antibody provided by the present invention has the characteristics of completely symmetrical structure. When expressed in the host, it will not produce protein isomers with other structures, thereby greatly reducing the difficulty of the extraction and purification process. It has the advantages of simple preparation and high yield, and has broad application prospects in tumor immunotherapy.

[0070] The present invention provides a bispecific antibody candidate molecule for the development of anti-tumor antibody drugs targeting BCMA, CAR-T reagent development, 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 bind to immune cells and tumor cells at the same time, guide T immune response, specifically and effectively kill tumor cells, and can be developed as an antibody drug for multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0072] Figure 1The expression of BCMA and the schematic diagram of TCR structure in the background technology of the present invention, wherein A is the expression of BCMA antigen during the development period of B cells; B is the ligand action signal channel of BCMA antigen, source: fimmu-09-01821-g001.jpg(1050×762) (frontiersin.org); C is the structure of the T cell receptor (TCR) complex and the composition of CD3 therein, source: https: / / www.researchgate.net / publication / 299549376.

[0073] Figure 2 This is a flow cytometer (FACS) analysis of the binding of the BCMA lead clone B10 in Example 2 of the present invention, wherein the two upper pictures: negative control (NC); the two middle pictures: positive control (PC); the two lower pictures: candidate clone B10 (B10).

[0074] Figure 3 The flow cytometer analysis of the binding of B10 and its affinity matured mutants in Example 3 of the present invention, wherein A, B, C, D, and E represent the binding of B10, B10.3, B10.4, B10.5, and B10.9 to H929 cells, and F, G, H, I, and J represent the binding of B10, B10.3, B10.4, B10.5, and B10.9 to RPMI8226 cells, respectively.

[0075] Figure 4 The ELISA and flow cytometric analysis in Example 3 of the present invention respectively compare the binding of J6, B10 and their mutants, wherein A is ELISA and B is FACS.

[0076] Figure 5 Schematic diagram of the structure of the anti-BCMA×CD3 bispecific antibody constructed based on the FIST platform in Example 4 of the present invention, wherein A is a bispecific antibody constructed by N-terminal fusion (nFIST): the anti-CD3 single-chain antibody is fused to the N-terminus of the anti-BCMA antibody VH, and B is a bispecific antibody constructed by C-terminal fusion (cFIST): the anti-CD3 single-chain antibody is fused to the C-terminus of the anti-BCMA antibody Fc.

[0077] Figure 6 The SDS-PAGE electrophoresis diagram of the bispecific antibodies K3B10.3 and B10.3K3 in Example 5 of the present invention, wherein A and C are reducing SDS-PAGE electrophoresis; B and D are non-reducing SDS-PAGE electrophoresis; A and B are the SDS-PAGE electrophoresis results of the K3B10.3 bispecific antibody; C and D are the SDS-PAGE electrophoresis results of the B10.3K3 bispecific antibody; lane M represents the protein molecular weight standard, and lane 1 is the target protein.

[0078] Figure 7 This is a peak graph of the HPLC-SEC purity analysis of the bispecific antibodies K3B10.3 and B10.3K3 purified in Example 5 of the present invention, wherein A is the bispecific antibody K3B10.3; B is the bispecific antibody B10.3K3.

[0079] Figure 8 The flow cytometer analysis of the binding of K3B10.3 and B10.3K3 to human Jurkat T cells and RPMI cells in Example 6 of the present invention, wherein A is a secondary antibody control; B is K3B10.3; C is the binding of B10.3K3 to CD3; D is a secondary antibody control; E is K3B10.3; and F is the binding of B10.3K3 to BCMA.

[0080] Fig. 9 The binding of cFIST bispecific antibodies to BCMA antigens in Example 7 of the present invention mediates T cell killing of BCMA-expressing cells RPMI8226, wherein A is the ELISA binding of B10 affinity-enhanced mutant bispecific antibodies to BCMA antigens, wherein the antibody coating is 200 ng / well, and biotin-BCMA is diluted half-fold starting at 250 ng / ml, and B is the mediated killing of BCMA-expressing cells by B10 affinity-enhanced mutant bispecific antibodies, wherein B10K3 was subjected to high and low concentration experiments respectively, and the low concentration (B10K3) could not obtain a standard S curve, and the high concentration (B10(high)K3) could obtain a standard S curve, and the EC50 value was calculated based on the curve.

[0081] Fig.10 This is a diagram of the BCMA dual antibody-mediated T cell killing of BCMA cells with different expression levels in Example 7 of the present invention, wherein A represents H929 cells and B represents RPMI8226 cells. DETAILED DESCRIPTION

[0082] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0083] Example 1 Screening of anti-human BCMA antibodies from a natural human antibody phage display library

[0084] Antibody library technology is a technology that clones all the variable region genes of antibodies of a certain animal (including humans) into plasmids or phages. After the latter infects E. coli, the antibody fragments are expressed on the surface of phage particles, or in the periplasm and cytoplasm of E. coli. Then the target antigen is used to screen clones carrying specific antibody genes from the antibody library to obtain the corresponding specific antibodies. Antibodies required for a variety of basic research and clinical development have been screened from the antibody library, such as tumor-associated membrane protein antigens, autoantigens associated with autoimmune diseases, and antibodies against viral antigens that resist viral diseases. These show the great application potential of antibody library technology in basic research and antibody drug development. In particular, fully human monoclonal antibodies obtained from human antibody libraries overcome the difficulty of obtaining human monoclonal antibodies using mouse hybridoma technology; due to the high conservation of human antibody sequences, these antibodies are much less immunogenic to the human immune system than antibodies derived from animals, and are therefore safer.

[0085] The non-immune human natural antibody library uses healthy people as the source of antibody genes, and these people are called donors. Since all natural human antibody sequences are used, the obtained antibodies have extremely low immunogenicity to the human immune system. Using molecular biology and DNA manipulation techniques, RNA extraction kits (such as QIAGEN's RNeasy Mini Kit, catalog number 74104) are used to extract all RNA from the donor's peripheral blood mononuclear cells (PBMC). Equal amounts are taken from each RNA sample and combined, and reverse transcription kits (such as ThermoFisher Scientific's cDNA synthesis kit (SuperScript™ IVReverse Transcriptase) is used to synthesize the cDNA of the light and heavy chain genes. Using these antibody cDNAs as templates, in the first round of amplification, PCR amplification is performed with the upstream primers and constant region primers specific to each subgroup to obtain all member genes of each subgroup. In the second round of PCR, the 3'-end primer set of the heavy chain variable region (VH) with a bridge and the 5'-end primer set of the light chain variable region (VL) with a bridge are respectively matched with the other end primers (containing specific restriction endonuclease sites) of each other, so that the obtained VH and VL can be linked to form single-chain antibody (scFv) genes. Using the specific restriction sites at the 5'-end and 3'-end of these single-chain antibody genes, these scFv genes are cloned into lysogenic phagemid (such as M13 phagemid) vectors to construct an antibody library. The size of the library reached 5 billion colony-forming units (cfu).

[0086] Biopanning refers to the process of obtaining specific clones from antibodies by screening with specific targets. In order to obtain human antibodies specific to human BCMA antigen, liquid phase screening was used for panning. The general steps of liquid phase panning are: first, the commercial BCMA antigen (BCMA-Fc fusion protein, Acrobiosystems, catalog number BC7-H5254) was modified with biotin, and each molecule was cross-linked with 3-5 biotin molecules to remove free biotin. An appropriate amount of biotin-labeled BCMA antigen was bound to streptavidin-coupled magnetic beads (such as Dynabeads™ M-280 Streptavidin, ThermoFisherScientific, catalog number 11205D). Thaw a human antibody library containing 10 billion phage particles expressing different antibodies. Both the magnetic beads and the antibody library were blocked with 4% skim milk powder solution (4% MPBS) to eliminate nonspecific action sites. Since there may be streptavidin on the magnetic beads that is not bound by avidin, and since the BCMA antigen molecule is a fusion protein composed of human Fc, it is necessary to add sufficient amount of streptavidin and human antibody Fc to the thawed antibody library (50-100 times the amount of target protein used) to remove the antibody clones that bind to them. The blocked antibody library solution and magnetic beads (total volume <1 ml) are added to a 1.5 ml Eppendorf tube, and rotated and mixed at room temperature for 2 hours to allow the BCMA-specific phage antibody to bind. After the incubation is completed, place the Eppendorf tube on the magnetic stand for 1 minute to separate the magnetic beads and the solution. Use a pipette to remove the solution as completely as possible, replace the tip with a new pipette, add 1 ml of washing solution PBST (Tween 20 with a final concentration of 0.05% is added to the phosphate buffer (PBS) solution) to the Eppendorf tube, away from the magnetic stand, gently suspend the magnetic beads with a pipette, and place it back on the magnetic stand to separate the magnetic beads and the solution. Remove the solution and repeat this three times. Then change the washing solution to PBS and wash the magnetic beads three times. After washing, most of the nonspecific and low-affinity phage antibodies are removed, and the specific phage antibodies remain on the magnetic beads. Add elution solution (10 mM Glycine, pH2.0) to the magnetic beads, resuspend the magnetic beads, let stand at room temperature for 10 minutes, separate the magnetic beads and solution with a magnetic rack, pipette the solution into a clean Eppendorf tube, and add 1 / 10 1M Tris solution (pH8.0) to neutralize the solution. This is the elution solution, which contains thousands of different phage antibodies that bind to the BCMA antigen, thus completing the first round of panning.

[0087] In order to further harvest specific antibody clones with relatively high affinity, more rounds of panning are required. To this end, the phage antibody solution eluted in the first round of panning is used to infect Escherichia coli (such as TG1 strain) in the logarithmic phase that can be infected by M13 phage to obtain the infection solution. A small amount is taken for a series of 10-fold gradient dilutions (usually diluted to one millionth of the original solution, and the last three gradients are taken for coating) to determine the titer of the first round of output elution (titer), which is also called the maximum diversity of the first round. Usually, the output titer after the first round of panning is below 10E6 cfu. Spread all the remaining infection liquid on a bacterial culture plate containing the corresponding antibiotics and culture overnight to obtain colonies; scrape the colony layer and resuspend it in culture medium, take enough resuspension containing the first round of output diversity to a shake flask containing enough liquid culture medium (2YT-CG, Carbenicillin and glucose are added to 2YT culture medium, the final concentrations are 100μg / ml and 2%, respectively), dilute the resuspension to below 0.1OD600 and start culturing until the logarithmic phase, that is, OD600 reaches about 0.5. In order to make these antibodies obtained in the first round of panning appear on the surface of phage particles again, take 10 ml of bacterial liquid, add helper phage M13K07, make the infection multiplicity 20:1, and stand at 37°C for 30 minutes (this stage is called phage rescue). Centrifuge and resuspend the cells in 50 ml expression medium (2YT-AK, Carbenicillin and Kanamycin were added to 2YT medium, the final concentrations were 100 µg / ml and 30 µg / ml respectively), and incubate at 30°C, 200 rpm overnight. The next day, harvest the culture supernatant by centrifugation, add 1 / 5 volume of PEG8000 / NaCl (PEG-800020%, NaCl2.5 M), mix well, and incubate on ice for 1 hour. Centrifuge at high speed (11500×g) for 30 minutes to harvest phage antibody particles. Resuspend the precipitate in 1 ml PBS solution and centrifuge at high speed again to remove bacterial debris. The supernatant is the amplification solution after the first round of panning, in which each antibody clone contained in it has been amplified more than ten thousand times. This amplification solution can be used for the second round of panning experiments. The second round of panning operations are exactly the same as the first round except that the number of times (6 / 6) is increased to 6 in PBST / PBS. In the third round, the number of washes can be further increased to 10 / 10. Multiple rounds of panning usually effectively enrich specific clones, with significantly reduced diversity but high affinity, which is convenient for subsequent single clone screening.

[0088] In order to obtain specific monoclonal antibodies, a monoclonal phage ELISA is required. To this end, the well-separated single colonies obtained in the second and / or third rounds of gradient dilution are inoculated individually into a 96-well culture plate containing 2YT-AG (93 colonies are inoculated per plate, and three wells are left as negative controls) and cultured overnight. This is the master plate. The bacterial solution in each well of the master plate is inoculated into a new culture plate and grown to the logarithmic phase. The above-mentioned phage rescue is performed to express the antibody of each clone on the phage surface. BCMA antigen (1µg / ml) is coated on a common 96-well ELISA plate, and another ELISA plate is coated with human Fc at the same concentration. Each individually expressed monoclonal phage antibody bacterial solution is added to the corresponding wells of the BCMA plate and the Fc plate, followed by a suitable secondary antibody and a tertiary antibody coupled to horseradish peroxidase (HRP), the substrate is used for color development, and the absorbance value (450nM) is read. The method for judging BCMA positive clones is: clones that are negative on the Fc plate (not more than 1.5 times the absorption value of the negative wells on the plate), positive on the BCMA plate (more than 3 times the absorption value of the negative wells on the plate), and whose well absorption value is higher than the absorption value of the corresponding well on the Fc plate. After analysis, clones corresponding to 10 wells showed positive only for BCMA antigen and negative for Fc, and these clones are collectively referred to as hits.

[0089] These hit bacterial solutions were inoculated into 3 ml 2YT-CG from the corresponding wells of the mother plate, and cultured overnight at 37°C and 200 rpm. The next day, phagemid DNA was extracted, and the sequence of the single-chain antibody region containing each hit was determined with specific primers. The coding region DNA sequence was translated into amino acid sequence, and multiple sequence comparisons were performed on them (CLUSTALW, website link https: / / www.genome.jp / tools-bin / clustalw) to determine the cloning specificity. After analysis, these ten hits belonged to two different clones in sequence, one of which was named B10, and the variable region sequence of the full human antibody against 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 NO.4-6, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO.1-3, 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 the single-chain antibody composed of the light chain and heavy chain variable regions of B10 is shown in SEQ ID NO.36.

[0090] Example 2 Antibody Function Verification

[0091] In order to verify whether the obtained BCMA antibody clone binds to the purified BCMA antigen and BCMA expressed on the cell membrane surface, the gene of the B10 single-chain antibody was cloned into the eukaryotic expression vector pFH (produced by Ecstar) to obtain the plasmid pFH-B10. In this vector, the scFv gene and the Fc gene of human IgG4 are fused to express a protein in the form of scFv-Fc, which can be affinity purified with Potein-A or detected with (HRP or fluorescein) labeled anti-human Fc antibody.

[0092] After obtaining the scFv-Fc protein of B10, the binding of this antibody to BCMA was detected using Octet RED, confirming the specific binding of B10 to BCMA.

[0093] The effect of B10 on BCMA-expressing cell line H929 (NCI-H929, ATCC ® CRL-9068 ™ ), indicating that B10 specifically binds to the BCMA antigen on the cell membrane surface (see Figure 2 ). In this FACS analysis, the percentage in the upper left corner of the quadrant (positive percentage) represents the binding strength of each single-chain antibody to the cell surface antigen. The positive percentage of clone B10 is 48.96%, which verifies the binding ability of the antibody against human BCMA antigen.

[0094] Example 3 Antibody Affinity Maturation

[0095] In vitro affinity maturation is a rapid directed molecular evolution operation: mutations are introduced at the DNA level and screened at the protein binding level. For the B10 antibody, in order to avoid introducing mutations that may enhance immunogenicity or significantly change the structure of the antibody, the mutation sites are limited to the sequence of the CDR region, and saturation mutations are performed at each position, and each mutation is expressed and tested separately.

[0096] The operation process of affinity maturation is as follows: First, the light and heavy chain variable region genes of B10 are cloned into the pUFL vector (pUFL is a plasmid that can express antibody Fab in E. coli, prepared by Ecstar) to obtain the Fab form of B10. A full set of single-point random mutation primers in the CDR region are designed, and each CDR position is PCR mutated using a point mutation kit (QuikChange LightningMulti Site-Directed Mutagenesis Kit, Agilent catalog number 210515), and BL21 (DE3) competent bacteria are transformed respectively to prepare monoclonal colony plates. These mutants are collectively referred to as single-point mutation libraries. At the same time, the Fab vector of the parent B10 is also transformed. Pick >30 colonies to prepare DNA for sequence analysis of the mutation region to evaluate the mutation efficiency. When the mutation efficiency is appropriate (greater than 80%), 92 colonies are picked according to the mutation of each CDR position to a 96-well culture plate containing the corresponding medium (2YT containing 100 µg / ml Carbenicillin + 0.1% glucose), and 3 parent colonies are picked, leaving three wells as blank controls. The plate is placed at 37°C, 300 rpm for 6 hours, IPTG is added to a final concentration of 1 mM, and transferred to 30°C, 300 rpm for overnight culture, and the Fab fragment is expressed and secreted into the culture medium.

[0097] Through preliminary experiments, the enzyme-linked reaction conditions were optimized so that the A450 absorption value of the B10 parent antibody Fab binding to the antigen BCMA-Fc was slightly higher than about 3 times the background signal. The BCMA-Fc antigen was coated on the enzyme-linked plate at 0.25 µg / ml one day in advance. The secreted mutant Fab was added to the wells the next day. The HRP-coupled anti-human Fab was used as the secondary antibody, the substrate was developed, and the A450 was read. The clones corresponding to the wells with a signal 1.5 times higher than the highest value of the parent well were obtained. These clones were called hits. All hits in the variable regions of the light and heavy chains were collected, and induced expression and enzyme-linked experiments were performed again to verify that they did have an affinity higher than that of the parent antibody. The plasmids of the clones with repeated positive results were sequenced in the mutant region to obtain the mutation information of the CDR amino acids. This process is called primary screening. In this way, all mutation information in the CDR region that is beneficial to improving affinity was obtained: the mutation sites that provide affinity in the heavy chain variable region CDR are shown in Table 1, and the mutation sites that provide affinity in the light chain variable region CDR are shown in Table 2.

[0098] Table 1 Mutations in the CDR of the B10 heavy chain variable region that are beneficial for improving affinity

[0099] Table 2 Mutations in the CDR of the light chain variable region of B10 that are beneficial for improving affinity

[0100] After obtaining all the single mutation information of the entire CDR region that is beneficial to affinity improvement from the primary library through enzyme-linked assay screening, a new multi-point mutation primer is redesigned to include the main beneficial mutations, and a mutation library is constructed again using a point mutation kit. This library is called a combinatorial library. In order to screen all mutation combinations in the light and heavy chain variable regions as much as possible, a combinatorial library of light and heavy chain variable regions containing multiple mutations of each is constructed and screened respectively. The top five clones with the highest affinity in the light chain variable region combinatorial library are obtained, and their light chain variable region amino acid sequences contain five mutation sequences, as shown in SEQ ID NO.22-26, and the CDR1 of the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO.1 and 12, CDR2 has an amino acid sequence shown in SEQ ID NO.2, and CDR3 has an amino acid sequence shown in any one of SEQ ID NO.3, 13, 14, 15, and 16.

[0101] Enzyme-linked screening was performed in the heavy chain variable region combinatorial library to obtain the amino acid sequences of the top three clones with the highest affinity in the heavy chain variable region combinatorial library. The amino acid sequences of their heavy chain variable regions contained three mutant sequences, as shown in sequences SEQ ID NO.19-21, respectively. The CDR1 of their heavy chain variable regions had an amino acid sequence shown in any one of SEQ ID NO.7, 8, and 9, CDR2 had an amino acid sequence shown in SEQ ID NO. 10, and CDR3 had an amino acid sequence shown in SEQ ID NO. 11.

[0102] The DNA of the top five clones with the highest affinity in the light and heavy chain variable regions were mixed and assembled into the final single-chain antibody combinatorial library by enzyme digestion and ligation recombination. This library was screened, and the top 10 with the highest signal were selected and their DNA sequences were analyzed to obtain four different single-chain antibody clones, and the corresponding amino acid sequences are shown in SEQ ID NO.37-40.

[0103] In order to test whether the affinity of the obtained mutants was improved, the B10 parent antibody and the fusion proteins of the four mutants B10.3, B10.4, B10.5, and B10.9 (amino acid sequences are shown in SEQ ID NO.37-40, respectively) and Fc (i.e., scFv-Fc) were expressed and purified. The purified products were detected by flow cytometry on two BCMA-positive cell lines H929 and RPMI8226 with different expression levels. Their binding percentages to H929 cells are: parent B10 is 75.31%, mutant B10.3 increases to 97.96%, mutant B10.4 increases to 97.89%, mutant B10.5 increases to 97.17%, mutant B10.9 increases to 91.61%; their binding percentages to RPMI8226 cells are: parent B10 is 5.54%, mutant B10.3 increases to 54.89%, mutant B10.4 increases to 57.51%, mutant B10.5 increases to 58.18%, mutant B10.9 increases to 42.89%. The results show that all four mutants have significantly improved their binding to BCMA compared to B10 (see Figure 3 ).

[0104] In order to understand the affinity of B10 and its derived antibodies, the B10 parent antibody and the four mutants obtained were compared with the BCMA antibody J6 (i.e., clone J6M0, according to the patent [WO2012163805A1], whose affinity is about 0.2nM) with known affinity by ELISA. After the different concentrations of antibodies were coated on the enzyme-linked plate, biotinylated BCMA antigen, streptavidin-coupled horseradish peroxidase (HRP) and colorimetric substrate were added step by step. The signal-antibody gradient curve was obtained as shown in Figure 4 The relative affinity change folds are shown in Table 3.

[0105] Table 3 ELISA detection of the binding of B10 and its affinity matured mutants to BCMA

[0106] The results showed that in terms of intrinsic affinity, since the binding of parent B10 to human BCMA was significantly weaker than that of J6, the S-curve could not be obtained, so its affinity value could not be generated. Others, B10.3 affinity is about 0.26 nM, B10.4 affinity is about 0.31nM, B10.5 affinity is about 0.27nM, and B10.9 affinity is about 0.29 nM.

[0107] In order to understand the apparent affinity of these antibodies for BCMA on the cell surface, the B10 parent antibody and the last four mutants were compared with J6 by flow cytometry binding to the BCMA-expressing cell line RPMI8226 (see Figure 4 B, Table 4). The results showed that at the cellular level, the EC50 of the parent B10 was 1.575 µg / ml, which was about 1.5 times higher than that of J6 (EC50 was 2.741 µg / ml). The EC50s 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. The EC50s of these affinity-enhanced mutants were increased by 97.13, 79.94, 67.46, and 60.72 folds relative to J6, respectively.

[0108] Table 4 FACS detection of the binding of B10 and its affinity matured mutants to RPMI8226 cell line

[0109] Example 4 BCMA×CD3 bispecific antibody design

[0110] In this example, a bispecific antibody was designed using the tumor cell surface antigen BCMA and the immune cell surface antigen CD3 as targets.

[0111] Combining protein structure design software and a large number of manual experimental screenings, the present invention screened and determined a bispecific antibody structure with a symmetrical structure including a single-chain antibody unit and a monoclonal antibody unit from a variety of bispecific antibody structures that bind to BCMA and CD3. The present invention refers to this technology platform as FIST (fusion of IgG and scFvtechnology). For example, the anti-BCMA monoclonal antibody unit can be used as an IgG antibody, including two complete light chain-heavy chain pairs (i.e., containing complete Fab and Fc domains, and the heavy chain and the light chain are connected by a disulfide bond), and the anti-CD3 can be used as a single-chain antibody unit, including two single-chain antibodies (ScFv). The single-chain antibody and the monoclonal antibody are connected by a connecting peptide (SEQ ID NO.30). For the connection method between the single-chain antibody and the monoclonal antibody, the following connection method is designed, thereby obtaining a bispecific antibody with a symmetrical structure ( Figure 5 ): The C-terminus of the single-chain antibody was connected to the N-terminus of the heavy chain variable region (VH) of the monoclonal antibody to obtain nFIST ( Figure 5 A). The single-chain antibody can also be fused to the C-terminus of IgG-Fc through a connecting peptide to obtain cFIST ( Figure 5 B).

[0112] In this example, the variable region sequence of the anti-CD3 single-chain antibody UCHT1 is from the literature (Beverley, PC & Callard, RE Distinctive 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 is 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 constructed in this way is named K3 (SEQ ID NO.31), which contains two cysteines (Cys) inserted in the heavy chain variable region and the light chain variable region, respectively, forming a pair of interchain disulfide bonds after folding. 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.

[0113] Example 5 Preparation of BCMA×CD3 bispecific antibody

[0114] According to Example 4, the bispecific antibody coding genes in the form of nFIST and cFIST were designed and cloned into the expression vector pG4HK to obtain two bispecific antibodies K3B10.3 and B10.3K3. Their heavy chain amino acid sequences are shown in SEQ ID NO.34 and 35 respectively (the heavy chain of the monoclonal antibody and the single-chain antibody are connected via a connecting peptide to form a fusion peptide); the 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 express and prepare the bispecific antibody proteins.

[0115] More anti-BCMA×CD3 bispecific antibodies were constructed in the cFIST format. The heavy chain variable region of the IgG unit that binds to BCMA is derived from SEQ ID NO.17-21; the light chain variable region of the IgG unit that binds to BCMA is derived from SEQ ID NO.18, 22, 25-27. The plasmids of these antibodies were transfected into 293F cells, and the preparation was carried out by transient expression and protein-A one-step purification to obtain B10K3 (heavy chain amino acid sequence is shown in SEQ ID NO.41; light chain amino acid sequence is shown in SEQ ID NO.42), B10.3K3, B10.4K3 (heavy chain amino acid sequence is shown in SEQ ID NO.43; light chain amino acid sequence is shown in SEQ ID NO.44), B10.5K3 (heavy chain amino acid sequence is shown in SEQ ID NO.45; light chain amino acid sequence is shown in SEQ ID NO.46), B10.9K3 (heavy chain amino acid sequence is shown in SEQ ID NO.47; light chain amino acid sequence is shown in SEQ ID NO.48) bispecific antibodies. SDS-PAGE purity analysis showed that the monomer was above 90%. Their binding to BCMA antigen was detected Fig. 9 A.

[0116] For the above-mentioned bispecific antibodies, in order to obtain high-purity bispecific antibodies, the following steps are followed. (1) Feed liquid pretreatment: The fermentation culture supernatant is centrifuged at 2000 rpm for 10 min, and then filtered with a 0.22µM filter membrane. (2) Affinity chromatography: The antibodies in the pretreated fermentation broth are captured using a MabselectSuRe affinity chromatography column (purchased from GE, catalog number 18-5438-02). After the column is fully equilibrated with an equilibrium buffer (10 mM PB, 0.1 M NaCl, pH 7.0), it is passed through the affinity chromatography column and eluted with an elution buffer (0.1 M citric acid, pH 3.0). (3) Cation exchange chromatography: The sample prepared by affinity chromatography is further purified by molecular sieve exchange chromatography, with a buffer (50 mM PBS, 0.2 ManNa2SO4, pH 6.7).

[0117] K3B10.3 and B10.3K3 were subjected to SDS-PAGE and HPLC-SEC detection. The results of SDS-PAGE are shown in Figure 6 Among them, the reduced SDS-PAGE electrophoresis detection results of K3B10.3 are shown in Figure 6 As shown in A, the results of non-reducing SDS-PAGE electrophoresis are as follows Figure 6 As shown in B; the results of reduced SDS-PAGE electrophoresis of B10.3K3 are shown in Figure 6 As shown in C, the results of non-reducing SDS-PAGE electrophoresis are as follows Figure 6 The HPLC-SEC test results are shown in D. Figure 7 Among them, the SEC test results of K3B10.3 are shown in Figure 7 As shown in A, the SEC test results of B10.3K3 are as follows Figure 7 As shown in B. The test results show that the bispecific antibodies K3B10.3 and B10.3K3 were successfully prepared through expression and purification, and the monomer purity of the purified bispecific antibodies was above 95%.

[0118] Example 6 Detection of the Binding Activity of Bispecific Antibodies to T Cells by Flow Cytometry

[0119] PBMC cells from healthy human donors were collected and T cells were purified at 1 × 10 6 Resuspend the cells / reaction tube in PBS, rinse the cells once with 1 ml binding buffer (PBS containing 0.5% w / v BSA + 2 mM EDTA), centrifuge (350 × g, 4°C, 5 min), and resuspend the cells in 200 µl binding buffer. Add the bispecific antibodies K3B10.3 and B10.3K3 to 5 µg / ml respectively and incubate on ice for 45 min. Rinse the cells once as above and resuspend the cells in 100 µl binding buffer. Add 5 µl fluorescently labeled secondary antibody (PE anti-human IgG Fc Antibody, Biolegend, 409304) to the sample tube and add isotype control (PE Mouse IgG2a, κ Isotype Ctrl (FC) Antibody, Biolegend, 400213) to the isotype control tube and incubate on ice for 15 min in the dark. Rinse the cells once as above. Resuspend the cells in 200 µl PBS and detect on a biotinylated instrument (Beckman).

[0120] Flow cytometry detection of the binding of K3B10.3 and B10.3K3 to human T cells Figure 8 As shown, the control test results are as follows Figure 8 A, K3B10.3 binding to T cells test results are as follows Figure 8 As shown in B, the detection results of B10.3K3 binding to T cells are as follows Figure 8 As shown in C. The results showed that both the bispecific antibodies K3B10.3 and B10.3K3 could specifically bind to T cells, that is, the bispecific antibody fusion protein retained the binding function of the single-chain antibody Anti-CD3.

[0121] To detect the binding of anti-BCMA cells to BCMA, the T cells in this experiment were replaced with RPMI8226 (ATCC® CCL-155) and a similar FACS experiment was performed. The control test results are as follows: Figure 8 The detection results of K3B10.3 and B10.3K3 are shown in D. Figure 8 E and F.

[0122] Example 7 Detection of in vitro cell killing efficiency mediated by bispecific antibodies

[0123] In this example, H929-luc and RPMI8226-Luc were used as target cells, and PBMCs were used as immune effector cells to detect the killing effect of target cells mediated by bispecific antibodies B10K3, B10.3K3, B10.4K3, B10.5K3, B10.9K3, and K3B10.3. The specific experimental steps are as follows: 1) Target cell preparation: Culture H929-luc and RPMI8226-Luc (luciferase-labeled H929 and RPMI8226 cells, prepared in our laboratory) cells, count the target cells after homogenization, centrifuge at 1000 rpm for 5 min, and wash once with PBS. After centrifugation and washing, adjust the density of target cells to 0.2×10 with GT-T551 medium. 6 / ml, add 50 µl to each well, then there are 10,000 cells in each well.

[0124] 2) PBMC preparation: PBMCs were used as effector cells. PBMCs frozen in liquid nitrogen tank were taken out (refer to cell freezing and recovery), thawed, added to a 15 ml centrifuge tube containing PBS or GT-T551 medium, centrifuged at 1000 rpm for 5 min, washed twice with PBS or GT-T551 medium, counted the cell number, activity and density, and adjusted the live cell density to 2×10 6 / ml, add 50 µl to each well, then there are 100,000 cells in each well.

[0125] 3) Antibody dilution: Use GT-T551 medium to dilute the bispecific B10K3, B10.3K3, B10.4K3, B10.5K3, B10.9K3, K3B10.3 and B10.3K3, and adjust the initial concentration of the antibody to 10 nM. Dilute them in a ratio of 1:5. Take 100µl of the diluted antibody and add it to the cells prepared above, mix well, put the 96-well plate back into the incubator, and detect the killing effect after 18 hours.

[0126] 4) Detection: Since H929 or RPMI8226 target cells carry the Luciferase gene, the LUMINEX method is used to detect the target cell killing efficiency.

[0127] Using Steady-GLO (Promega) as the substrate, thaw the buffer in the kit and add it to the substrate powder, mix well, and dispense into 5 ml or 10 ml tubes to complete the reconstruction of the Steady-GLO substrate. After blowing the co-cultured cells evenly, take 100 µl and transfer it to an opaque white plate, then add 100 µl of the reconstructed Steady-GLO substrate, tap to mix, and read the plate after leaving it for 5 minutes. The detection instrument is Synergy HT.

[0128] 5) Data processing: The calculation formula for the target cell killing ratio is as follows: Target cell killing ratio (percentage) = 100 × (value of only target cell wells - value of detection wells) / value of only target cell wells

[0129] The antibody concentration corresponding to the target cell killing ratio of all test wells was converted to log10, and a curve graph was prepared with this as the horizontal axis and the killing ratio as the vertical axis. The killing concentration-gradient curve of the cFIST-type dual antibody B10K3, B10.3K3, B10.4K3, B10.5K3, and B10.9K3 against the sensitive cell line RPMI8226 is shown in the figure Fig. 9 The killing percentages of each gradient are shown in Table 5.

[0130] The results of K3B10.3 and B10.3K3 are as follows Fig.10 As shown in Table 6; their killing percentages for RPMI8226 are shown in Table 6. Their killing percentages for H929 are shown in Table 7. The results show that the bispecific antibody B10.3K3 can effectively mediate PBMC killing of tumor cell lines H929 or RPMI8226, and that a single molecule of B10.3K3 has both the biological functions of anti-BCMA and anti-CD3 monoclonal antibodies. The potency of K3B10.3 in mediating killing of target cells RPMI8226 is significantly weaker than that of B10.3K3, especially in H929 cells, where there is almost no killing effect. The EC50 of target cell killing mediated by B10.9K3, B10.5K3, B10.4K3, B10.3K3, and B10K3 are 8.759pM, 12.87pM, 7.839pM, 5.833pM, and 281.7pM, respectively.

[0131] Table 5 Target cell killing ratio of RPMI8226 mediated by different doses of bispecific antibodies

[0132] Table 6 Target cell killing ratio of RPMI8226 mediated by different doses of bispecific antibodies K3B10.3 and B10.3K3

[0133] Table 7 Target cell killing ratio of H929 mediated by different doses of bispecific antibodies K3B10.3 and B10.3K3

[0134] In Tables 5, 6 and 7 above, the two columns of numerical values ​​for each bispecific antibody are the results of two duplicate experiments.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bispecific antibody that binds to BCMA and CD3, characterized in that include: The first domain binds to the B cell maturation antigen BCMA. and, the second domain, which binds to the T cell surface antigen CD3, The first domain and the second domain are connected by a connecting peptide, The first domain includes a heavy chain variable region and a light chain variable region, and CDR1, CDR2, and CDR3 of the heavy chain variable region respectively have the amino acid sequences shown in SEQ ID NOs. 4-6, or respectively have amino acid sequences with the amino acid sequences shown in SEQ ID NOs. 4-6 as reference sequences and containing one or more of the following mutations: (1) The first S in the amino acid sequence shown in SEQ ID NO.4 is mutated to P or K; (2) The second Y in the amino acid sequence shown in SEQ ID NO.4 is mutated to H, D, G, M or S; (3) The third A in the amino acid sequence shown in SEQ ID NO.4 is mutated to N; (4) The 5th S in the amino acid sequence shown in SEQ ID NO.4 is mutated to H, N, A or M; (5) The first G in the amino acid sequence shown in SEQ ID NO.5 is mutated to V or T; (6) The 3rd position I of the amino acid sequence shown in SEQ ID NO.5 is mutated to H; (7) The 4th position I of the amino acid sequence shown in SEQ ID NO.5 is mutated to D or A; (8) The 5th F in the amino acid sequence shown in SEQ ID NO.5 is mutated to H; (9) The 7th T in the amino acid sequence shown in SEQ ID NO.5 is mutated to K; (10) the 4th position F of the amino acid sequence shown in SEQ ID NO.6 is mutated to L; The CDR1, CDR2 and CDR3 of the light chain variable region respectively have the amino acid sequences shown in SEQ ID NO.1-3, or respectively have the amino acid sequences shown in SEQ ID NO.1-3 as reference sequences and contain one or more of the following mutations: (1) The 5th S in the amino acid sequence shown in SEQ ID NO.1 is mutated to N, R or T; (2) L at position 7 of the amino acid sequence shown in SEQ ID NO.3 is mutated to Q, V or A; (3) The 8th position I of the amino acid sequence shown in SEQ ID NO.3 is mutated to S, A, R or P; (4) The Y at position 10 of the amino acid sequence shown in SEQ ID NO.3 is mutated to M, L, W or T; (5) The V at position 11 of the amino acid sequence shown in SEQ ID NO.3 is mutated to L or Q.

2. Any of the following uses of the bispecific antibody binding to BCMA and CD3 according to claim 1, or a nucleic acid encoding the bispecific antibody, or a biological material containing the nucleic acid: (1) Use in the preparation of a drug for diagnosing, preventing or treating a BCMA-expressing B cell-related disease; preferably, the BCMA-expressing B cell-related disease includes a B cell-related tumor or an autoimmune disease caused by a B cell; more preferably, the B cell-related tumor is multiple myeloma, lymphoma, plasma cell leukemia, plasmacytoma, macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, hairy cell leukemia, myeloid leukemia, acute myeloid leukemia AML, acute lymphocytic leukemia ALL, chronic lymphocytic leukemia CLL or chronic myeloid leukemia CML; the autoimmune disease caused by a B cell is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, membranous nephropathy, chronic kidney disease, myasthenia gravis, ANCA vasculitis, neuromyelitis optica spectrum disorder, MOG-AD, POEMS syndrome or an IgG4-related disease; (2) Use in the preparation of drugs for diagnosing, preventing or treating diseases targeting BCMA; (3) Use in the preparation of a drug for killing BCMA-expressing cells; preferably, the BCMA-expressing cells include B cell-related tumor cells or B cell-induced autoimmune disease cells; more preferably, the BCMA-expressing B cell-related tumor is multiple myeloma, lymphoma, plasma cell leukemia, plasmacytoma, macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, hairy cell leukemia, myeloid leukemia, acute myeloid leukemia AML, acute lymphocytic leukemia ALL, chronic lymphocytic leukemia CLL or chronic myeloid leukemia CML; the autoimmune disease caused by B cells is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, membranous nephropathy, chronic kidney disease, myasthenia gravis, ANCA vasculitis, neuromyelitis optica spectrum disease, MOG-AD, POEMS syndrome or IgG4-related disease; (4) Application in the preparation of BCMA and / or CD3 detection reagents; (5) Application in the preparation of relevant reagents suitable for CAR-T therapy.

3. Any of the following uses of a bispecific antibody that binds to BCMA and CD3, or a nucleic acid encoding the bispecific antibody, or a biological material containing the nucleic acid: (1) Use in the preparation of a drug for diagnosing, preventing or treating a BCMA-expressing B cell-related disease; preferably, the BCMA-expressing B cell-related disease includes a B cell-related tumor or an autoimmune disease caused by a B cell; more preferably, the B cell-related tumor is multiple myeloma, lymphoma, plasma cell leukemia, plasmacytoma, macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, hairy cell leukemia, myeloid leukemia, acute myeloid leukemia AML, acute lymphocytic leukemia ALL, chronic lymphocytic leukemia CLL or chronic myeloid leukemia CML; the autoimmune disease caused by a B cell is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, membranous nephropathy, chronic kidney disease, myasthenia gravis, ANCA vasculitis, neuromyelitis optica spectrum disorder, MOG-AD, POEMS syndrome or an IgG4-related disease; (2) Use in the preparation of drugs for diagnosing, preventing or treating diseases targeting BCMA; (3) Use in the preparation of a drug for killing BCMA-expressing cells; preferably, the BCMA-expressing cells include B cell-related tumor cells or B cell-induced autoimmune disease cells; more preferably, the B cell-related tumor is multiple myeloma, lymphoma, plasma cell leukemia, plasmacytoma, macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, hairy cell leukemia, myeloid leukemia, acute myeloid leukemia AML, acute lymphocytic leukemia ALL, chronic lymphocytic leukemia CLL or chronic myeloid leukemia CML; the autoimmune disease caused by B cells is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, membranous nephropathy, chronic kidney disease, myasthenia gravis, ANCA vasculitis, neuromyelitis optica spectrum disease, MOG-AD, POEMS syndrome or IgG4-related disease; (4) Application in the preparation of relevant reagents suitable for CAR-T therapy; in, The bispecific antibody that binds to BCMA and CD3 includes: The first domain binds to the B cell maturation antigen BCMA. and, the second domain, which binds to the T cell surface antigen CD3, The first domain and the second domain are connected by a connecting peptide, The first domain comprises a heavy chain variable region and a light chain variable region, wherein the CDR1 of the heavy chain variable region of the first domain has an amino acid sequence as shown in any one of SEQ ID NOs. 4, 7, 8, and 9, the CDR2 has an amino acid sequence as shown in any one of SEQ ID NOs. 5 and 10, and the CDR3 has an amino acid sequence as shown in any one of SEQ ID NOs. 6 and 11; The CDR1 of the light chain variable region 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 any one of SEQ ID NOs. 2, and the CDR3 has the amino acid sequence shown in any one of SEQ ID NOs. 3, 13, 14, 15, and 16; Preferably, the CDR of the heavy chain variable region of the first domain is any one of the following: (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; (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 of the light chain variable region is any of the following: (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; (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 CDR of the heavy chain variable region and the CDR of the light chain variable region of the first domain are any of the following: (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, 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.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; (4) 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.

16.

4. The use according to claim 3, characterized in that: The heavy chain variable region of the first domain has an amino acid sequence shown in any one of SEQ ID NOs. 17, 19-21, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs. 18, 22-27; Alternatively, the heavy chain variable region and the light chain variable region satisfy at least one of the following two conditions compared with the aforementioned sequences: a) binding to the same antigenic epitope; b) an amino acid sequence with a sequence identity greater than 70%, 80%, 85%, 90%, 97%, 98% or 99%; 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 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.

26.

5. The use according to claim 3 or 4, characterized in that: The first domain is two complete light chain-heavy chain pairs connected by disulfide bonds; Preferably, 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.

6. The use according to claim 5, characterized in that: 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 has the amino acid sequence shown in SEQ ID NO.49, and the light chain has the amino acid sequence shown in SEQ ID NO.42, Alternatively, the heavy chain has the amino acid sequence shown in SEQ ID NO.50, and the light chain has the amino acid sequence shown in SEQ ID NO.44, Alternatively, the heavy chain has the amino acid sequence shown in SEQ ID NO.51, and the light chain has the amino acid sequence shown in SEQ ID NO.46, Alternatively, the heavy chain has the amino acid sequence shown in SEQ ID NO.52, and the light chain has the amino acid sequence shown in SEQ ID NO.48, Alternatively, the heavy chain and light chain have at least one of the following requirements compared to the aforementioned sequences: a) binding to the same antigenic epitope; b) an amino acid sequence with a sequence identity greater than 70%, 80%, 85%, 90%, 97%, 98% or 99%.

7. The use according to any one of claims 3 to 6, characterized in that: 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; Preferably, the light chain variable region and the heavy chain variable region of the second domain are connected to form a single-chain antibody via a connecting peptide, and the single-chain antibody has the amino acid sequence shown in SEQ ID NO.

31.

8. The use according to claim 7, characterized in that: The second domain comprises two single-chain antibodies, and the bispecific antibody is a symmetrical structure connected by any of the following methods: (1) The C-termini of the two single-chain antibodies of the second domain are respectively connected to the N-termini of the two heavy chains of the first domain through connecting peptides; (2) The N-termini of the two single-chain antibodies of the second domain are respectively connected to the C-termini of the two heavy chains of the first domain through connecting peptides; Preferably, the amino acid sequence of the connecting peptide is (GGGGX)n, wherein X is Gly or Ser, and n is a natural number of 1-4; More preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO.

30.

9. The use according to any one of claims 3 to 8, characterized in that: The heavy chain of the first domain and the second domain have the amino acid sequence shown in SEQ ID NO.34 or 35 after being connected via a connecting peptide, and the light chain has the amino acid sequence shown in SEQ ID NO.33, Alternatively, the heavy chain of the first domain and the second domain have the amino acid sequence shown in SEQ ID NO.41 after being connected via a connecting peptide, and the light chain has the amino acid sequence shown in SEQ ID NO.42, Alternatively, the heavy chain of the first domain and the second domain have an amino acid sequence as shown in SEQ ID NO.43 after being connected via a connecting peptide, and the light chain has an amino acid sequence as shown in SEQ ID NO.44, Alternatively, the heavy chain of the first domain and the second domain have the amino acid sequence shown in SEQ ID NO.45 after being connected via a connecting peptide, and the light chain has the amino acid sequence shown in SEQ ID NO.46, Alternatively, the heavy chain of the first domain and the second domain have the amino acid sequence shown in SEQ ID NO.47 after being connected via a connecting peptide, and the light chain has the amino acid sequence shown in SEQ ID NO.

48.

10. The use according to any one of claims 2 to 9, characterized in that: The biological material includes recombinant DNA, expression cassette, vector, host cell, engineered bacteria or cell line.

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