A bispecific antibody that simultaneously targets human BCMA and human CD3

Abstract

The application discloses a bispecific antibody simultaneously targeting human BCMA and human CD3, which comprises: a) an antigen binding part specifically binding to human BCMA; b) an antigen binding part specifically binding to human CD3; and c) a linker between the antigen binding parts; wherein the antigen binding part specifically binding to human BCMA comprises amino acid sequences of light chain variable region CDR1, CDR2 and CDR3 regions shown in SEQ ID No. 1-3, and amino acid sequences of heavy chain variable region CDR1, CDR2 and CDR3 regions shown in SEQ ID No. 4-6, or sequences homologous to the amino acid sequences with more than 80%. The application mediates PBMCs to produce antigen-specific activation, proliferation and target cell-specific killing by simultaneously targeting human BCMA and CD3. In a mouse subcutaneous myeloma model, the tumor has obvious growth inhibition.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to a bispecific antibody that simultaneously targets human BCMA and human CD3. Background Technology

[0002] In recent years, immunotherapy has received widespread attention due to its specific targeting, and bispecific T-cell binders (BiTEs) have achieved good results as a novel immunotherapy approach. BiTEs consist of two single-cell fungi targeting tumor cell-associated antigens (SCVAs) and CD3 on the surface of T cells, connected by a linker. BiTEs can recruit T cells to tumor cells, activating them and releasing cytotoxic factors to kill target cells. In 2014, blinatumomab became the first BiTE drug approved by the FDA for the treatment of Philadelphia chromosome-negative precursor B-cell acute lymphoblastic leukemia. To reduce side effects and expand the therapeutic window, BiTEs require highly specific expression of the target antigen on the surface of target cells, while expressing it at low levels or not at all on other normal cells. B-cell maturation antigen (BCMA), which is highly specifically expressed on the surface of plasma cells and multiple myeloma cells, is undoubtedly an ideal target. BCMA is mainly expressed on the surface of plasma cells and MM cells, but not on most B cells and normal cells, making it a popular therapeutic target for multiple myeloma and other hematologic malignancies. BI 836909 (AMG 420) is the first BiTE targeting BCMA. It has shown good results in phase I / II clinical trials for the treatment of relapsed / refractory multiple myeloma. However, due to the small molecular weight of BiTE, only 55kD, its short half-life limits its clinical application.

[0003] Multiple myeloma is a malignant proliferative disease of plasma cells. Its characteristic feature is the unlimited proliferation of plasma cells in the bone marrow, resembling tumor cells, accompanied by the secretion of monoclonal immunoglobulins, ultimately leading to organ and tissue damage. Over the past 20 years, significant progress has been made in the treatment of multiple myeloma. The intervention of drugs such as proteasome inhibitors and immunomodulatory agents has greatly increased the remission rate and prolonged patient survival. However, for most patients, multiple myeloma remains difficult to cure, with minimal residual disease persisting. Patients often relapse after achieving remission through treatment, and each relapse exacerbates the disease progression. Therefore, there is an urgent need for more effective new therapies.

[0004] Tandab is a form of genetically engineered bispecific antibody. These antibody fragments are typically composed of two peptide chains that pair in reverse, forming a tetravalent bispecific antibody fragment with two antigen-binding sites. Its molecular weight is twice that of 55kD BiTE, resulting in a longer half-life, which can overcome the limitations of clinical use and shows promising clinical application prospects in tumor immunotherapy. Its basic principle is that VH and VL in the same peptide chain cannot pair due to steric hindrance caused by the short linker (usually only 5-12 amino acids). Instead, they can only pair with VH and VL on another peptide chain, thus forming a dimer.

[0005] Currently, there is a lack of bispecific antibodies that simultaneously target human BCMA and human CD3. Furthermore, existing antigen conjugates targeting both human BCMA and CD3 exhibit weak affinity, and the resulting tandab also suffers from structural instability. To address these issues, there is an urgent need to find a novel bispecific antibody that simultaneously targets human BCMA and human CD3. Summary of the Invention

[0006] One aspect of this invention addresses the lack of bispecific antibodies that simultaneously target human BCMA and human CD3 in the prior art, and the fact that existing antigen conjugates targeting human BCMA and human CD3 have weak affinity and the resulting Tandab is structurally unstable. This invention provides a novel bispecific antibody, the tetravalent bispecific antibody Tandab (CD3 / BCMA), that can simultaneously target human B-cell maturation antigen (BCMA) and human T-cell surface CD3. This bispecific antibody utilizes dual targeting to recruit T cells expressing CD3 antigen to the vicinity of multiple myeloma cells that highly express BCMA, thereby reshaping the killing effect of T cells on multiple myeloma cells.

[0007] The technical solution provided by this invention is as follows:

[0008] A bispecific antibody that simultaneously targets human BCMA and human CD3, comprising:

[0009] a) Specifically binds to the antigen-binding portion of human BCMA;

[0010] b) Specifically binds to the antigen-binding portion of human CD3; and

[0011] c) Connectors between the antigen-binding portions;

[0012] The antigen-binding portion that specifically binds to human BCMA includes amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID No. 1-3, and amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID No. 4-6, or sequences having more than 80% homology with the amino acid sequences.

[0013] In this invention, the above-mentioned homology of more than 80% refers to having more than 80% homology with the nucleotide or amino acid sequence described in this invention. This homology can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The nucleotide or amino acid sequence described in this invention can be randomly or engineered to undergo point mutations in a suitable manner. The purpose can be, for example, to obtain better expression levels, affinity, and / or dissociation properties. All of these mutated nucleotide or amino acid sequences are included within the scope of protection of this invention.

[0014] Preferably, in one embodiment of the present invention, the antigen-binding portion that specifically binds to human BCMA comprises an amino acid sequence of the light chain variable region as shown in SEQ ID No. 7 and an amino acid sequence of the heavy chain variable region as shown in SEQ ID No. 8, or a sequence having more than 80% homology with the amino acid sequence.

[0015] In the bispecific antibody of the present invention, the antigen-binding portion specifically binding to human BCMA can be linked to any suitable antigen-binding portion specifically binding to human CD3 via a linker / hinge region. One function of the antigen-binding portion specifically binding to human CD3 is to recruit cytotoxic T lymphocytes. Preferably, in one embodiment of the present invention, the antigen-binding portion specifically binding to human CD3 comprises the amino acid sequence of the light chain variable region as shown in SEQ ID No. 9 and the amino acid sequence of the heavy chain variable region as shown in SEQ ID No. 10, or a sequence having more than 80% homology with said amino acid sequences.

[0016] In this invention, the light chain variable region sequence, heavy chain variable region sequence in the antigen-binding portion specifically binding to human BCMA, and the light chain variable region sequence, heavy chain variable region sequence in the antigen-binding portion specifically binding to human CD3 can be linked by a linker / hinge region in any suitable primary structural order to form a bivalent or multivalent bispecific antibody. For example, a bivalent bispecific antibody can be linked in the order VL3-VH3-VHBCMA-VLBCMA. As another example, in one embodiment of this invention, the tetravalent bispecific antibody simultaneously targeting human BCMA and human CD3 formed by the above sequences is linked in the following manner: VL3-VHBCMA-VLBCMA-VH3 (e.g., ...). Figure 1 (As shown).

[0017] Preferably, in embodiments of the present invention, the aforementioned bispecific antibody is a human, humanized, or chimeric antibody. The aforementioned humanized or chimeric antibody can be prepared using methods in the prior art.

[0018] The bispecific antibody of the present invention may also contain other functional peptide chains, such as a leader signal peptide with the amino acid sequence METDTLLLWVLLLWVPGSTGD; a His tag with the amino acid sequence HHHHHH, etc.

[0019] In the bispecific antibody of the present invention, the linker can be any suitable oligopeptide or polypeptide. Preferably, in an embodiment of the present invention, the linker is (GGGGS). n , where n is an integer and 1≤n≤3. More preferably, in one embodiment of the present invention, the linker amino acid sequence between VL3 and VHBCMA, and between VLBCMA and VH3, is GGGGS; the linker amino acid sequence between VHBCMA and VLBCMA is (GGGGS)3.

[0020] In this invention, the bispecific antibody (BiTE) containing the antigen-binding moieties that specifically bind to human BCMA and human CD3 exhibits strong affinity for tumor cell surface antigens and cytotoxic activity. To further improve the half-life of the bispecific antibody of this invention, the inventors further designed and constructed a tetravalent bispecific antibody (Tandab). The bispecific tetravalent antibody (Tandab) designed and constructed in this invention is a miniature bifunctional antibody expressed in single-chain form. Each single chain consists of two antibody VL and VH fragments tandemly connected, possessing two antigen-binding sites. The two peptide chains are joined head-to-tail by intermolecular forces to form a dimer, selectively recruiting effector cells to the vicinity of target cells and mediating specific cytotoxicity.

[0021] Preferably, in one embodiment of the present invention, the primary structure of the above-mentioned tetravalent bispecific antibody is arranged from the N-terminus to the C-terminus in the order of the light chain variable region sequence in the antigen-binding portion specifically binding to human CD3 - the heavy chain variable region sequence in the antigen-binding portion specifically binding to human BCMA - the light chain variable region sequence in the antigen-binding portion specifically binding to human BCMA - the heavy chain variable region sequence in the antigen-binding portion specifically binding to human CD3.

[0022] In this invention, to improve the stability of Tandab, the inventors introduced a mutation at position NO. 100 of the light chain of the anti-human CD3 parental antibody, changing the original serine to cysteine, i.e., changing the base from TCG to TGT. This allows a disulfide bond to form between the two peptide chains, increasing the in vitro and in vivo stability of Tandab, rather than relying solely on intermolecular forces to form a dimer. Therefore, as a preferred embodiment of this invention, the 100th serine in the variable region of the light chain of the antigen-binding portion specifically binding to human CD3 is replaced by cysteine. The amino acid sequence is: ADIELTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGCGTKLELKR.

[0023] More preferably, in one embodiment of the present invention, the amino acid sequence of the above-mentioned bispecific heteroantibody is shown in SEQ ID No. 11.

[0024] Another aspect of the present invention is to provide an isolated nucleic acid molecule that encodes the aforementioned bispecific antibody;

[0025] Preferably, in one embodiment of the present invention, the nucleotide sequence of the above-mentioned nucleic acid molecule is as shown in SEQ ID No. 12.

[0026] Another aspect of the present invention is to provide an expression vector comprising the aforementioned nucleic acid molecule.

[0027] Furthermore, the polynucleotide sequence of the present invention can be inserted into any suitable expression vector using appropriate methods, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. Preferably, in one embodiment of the present invention, the expression vector is a eukaryotic expression vector pcDNA. TM 3.4.

[0028] Another aspect of the present invention is to provide a host cell comprising the expression vector described above.

[0029] Another aspect of the present invention is to provide a pharmaceutical composition comprising the above-described bispecific antibody or a nucleic acid molecule isolated as described above, and a pharmaceutically acceptable carrier.

[0030] In addition to the above-mentioned components, the pharmaceutical compositions of the present invention may also contain any pharmaceutically permissible additives, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, etc.

[0031] Similarly, the pharmaceutical compositions of the present invention can also be used in combination with other suitable anticancer agents, such as cytarabine, daunorubicin, demethoxydaunorubicin, mitoxantrone, etc.

[0032] Another aspect of the present invention is to provide an immunoconjugate comprising the aforementioned bispecific antibody, which is linked to a therapeutic agent.

[0033] The aforementioned therapeutic agents can be selected from cytotoxic agents, immunomodulators, therapeutic proteins, biopolymers, or oligonucleotides. Preferably, in embodiments of the present invention, the aforementioned therapeutic agents are cytotoxic agents, including but not limited to anthracyclines, auristatin, camptothecin, cobustatin, dolasstatin, duocarmycin, enediyne, galdmycin, indoline-benzodiazepine dimer, maytansine, puromycin, pyrrole benzodiazepine dimer, taxanes, vinca alkaloids, tubulysin, hemiasterlin, spliceostatin, pladienolide, and their stereoisomers, isosteres, analogs, or derivatives.

[0034] In an embodiment of the present invention, the connection method for linking the therapeutic agent described above can be: bispecific antibody - tag containing acyl donor glutamine - linker - cytotoxic agent.

[0035] Another aspect of the present invention is to provide the use of the above-mentioned bispecific antibody, the above-mentioned isolated nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host cell, the above-mentioned pharmaceutical composition or the above-mentioned immune conjugate in the preparation of a product for treating tumors;

[0036] Preferably, in an embodiment of the present invention, the tumor is a hematologic tumor;

[0037] More preferably, in some embodiments of the present invention, the aforementioned hematologic malignancies are multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, small aneuploid lymphoma, endemic Burkitt's lymphoma, diffuse Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal mononuclear B-cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphadenoma, primary mediastinal B-cell lymphoma, pulmonary B-cell angiogenic lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, or glioblastoma.

[0038] More preferably, in one embodiment of the present invention, the aforementioned hematologic tumor is multiple myeloma.

[0039] Another aspect of the present invention is to provide a method for treating tumors, the method comprising administering an effective dose of the aforementioned bispecific antibody, the aforementioned pharmaceutical composition, or the aforementioned immunoconjugate to a patient suffering from a tumor.

[0040] Preferably, in an embodiment of the present invention, the tumor is a hematologic tumor;

[0041] More preferably, in some embodiments of the present invention, the aforementioned hematologic malignancies are multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, small aneuploid lymphoma, endemic Burkitt's lymphoma, diffuse Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal mononuclear B-cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphadenoma, primary mediastinal B-cell lymphoma, pulmonary B-cell angiogenic lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, or glioblastoma.

[0042] More preferably, in one embodiment of the present invention, the aforementioned hematologic tumor is multiple myeloma.

[0043] The beneficial effects of this invention are as follows:

[0044] The bispecific antibody targeting human BCMA and CD3 provided by this invention has high affinity for both BCMA and CD3 antigens, with an affinity constant Kd value of 1.963 x 10⁻⁶ at its BCMA binding terminus. -9 The affinity constant at the M;CD3 binding end is 1.961 x 10⁻⁶. -9M. exhibits high specificity, specifically binding to the BCMA-positive cell line H929 and the CD3-positive cell line Jurkat, without cross-reactivity with the BCMA- and CD3-negative cell lines K562 and HL-60. This invention can mediate antigen-specific activation, proliferation, and specific killing of target cells in PBMCs by simultaneously targeting human BCMA and CD3. In a mouse subcutaneous myeloma model, it has a significant inhibitory effect on tumor growth. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the construction of the tetravalent bispecific antibody Tandab (CD3 / BCMA) in an embodiment of the present invention, wherein VHBCMA and VLBCMA represent the heavy chain variable region and light chain variable region sequences of the anti-human BCMA antibody, respectively; VH3 and VL3 represent the heavy chain variable region and light chain variable region sequences of the anti-human CD3 antibody HIT3a, respectively; and L1 and L2 are linker peptide sequences.

[0046] Figure 2 This is a diagram showing the expression, purification, and identification of the tetravalent bispecific antibody Tandab (CD3 / BCMA) in an embodiment of the present invention. Lane 1 is the unpurified supernatant, lanes 2 and 3 are the eluent, lane 4 is the elution buffer, lanes 5, 6, 7, and 8 are the bands after reduction of lanes 1, 2, 3, and 4, respectively, and lane M is the protein marker.

[0047] Figure 3 The figure shows the results of FACS detection of the binding of the tetravalent bispecific antibody Tandab (CD3 / BCMA) to human BCMA-positive cell lines H929, 3T3-BCMA and human CD3-positive cell line Jurkat in this embodiment of the invention.

[0048] Figure 4 This is a diagram showing the cross-reactivity results of the tetravalent bispecific antibody Tandab (CD3 / BCMA) with BCMA and CD3 double-negative cell lines K562 and HL60 detected by FACS in an embodiment of the present invention.

[0049] Figure 5 The figure shows the results of FACS detection of the affinity constants of the tetravalent bispecific antibody Tandab (CD3 / BCMA) with the human BCMA-positive cell line H929 and the human CD3-positive cell line Jurkat in this embodiment of the invention.

[0050] Figure 6 This is a graph showing the results of FACS detection in this embodiment of the invention regarding the competition between the tetravalent bispecific antibody Tandab (CD3 / BCMA) and commercial antibodies against BCMA and CD3 binding to BCMA protein on the surface of H929 cells and CD3 protein on the surface of Jurkat cells.

[0051] Figure 7 This is a diagram showing the results of FACS detection in this embodiment of the invention of upregulation of expression of the tetravalent bispecific antibody Tandab (CD3 / BCMA) mediated by the activation markers CD25 and CD69 on the surface of PBMCs.

[0052] Figure 8 This is a diagram showing the results of ElISA detection of cytokine release from PBMCs mediated by the tetravalent bispecific antibody Tandab (CD3 / BCMA) in an embodiment of the present invention.

[0053] Figure 9 This is a diagram showing the dose-dependent results of the tetravalent bispecific antibody Tandab (CD3 / BCMA) mediating the killing of myeloma cells NCI-H929 by PBMCs in an embodiment of the present invention.

[0054] Figure 10 This is a diagram showing the results of FACS detection of PBMC proliferation mediated by the tetravalent bispecific antibody Tandab (CD3 / BCMA) in an embodiment of the present invention.

[0055] Figure 11 This is a time-sharing diagram of the treatment of the subcutaneous myeloma model NCI-H929 with the quadrivalent bispecific antibody Tandab (CD3 / BCMA) in an embodiment of the present invention.

[0056] Figure 12 This is a graph showing the comparison of tumor volume in a subcutaneous myeloma model treated with the tetravalent bispecific antibody Tandab (CD3 / BCMA) in an embodiment of the present invention.

[0057] Sequence Description

[0058] SEQ ID No. 1 is the amino acid sequence of the CDR1 region of the light chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0059] SEQ ID No. 2 is the amino acid sequence of the CDR2 region of the light chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0060] SEQ ID No. 3 is the amino acid sequence of the CDR3 region of the light chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0061] SEQ ID No. 4 is the amino acid sequence of the CDR1 region of the heavy chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0062] SEQ ID No. 5 is the amino acid sequence of the CDR2 region of the heavy chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0063] SEQ ID No. 6 is the amino acid sequence of the CDR3 region of the heavy chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0064] SEQ ID No. 7 is the amino acid sequence of the light chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0065] SEQ ID No. 8 is the amino acid sequence of the heavy chain variable region of the antigen-binding moiety that specifically binds to human BCMA in this invention;

[0066] SEQ ID No. 9 is the amino acid sequence of the light chain variable region of the antigen-binding moiety that specifically binds to human CD3 in this invention;

[0067] SEQ ID No. 10 is the amino acid sequence of the heavy chain variable region of the antigen-binding moiety that specifically binds to human CD3 in this invention;

[0068] SEQ ID No. 11 is the amino acid sequence of the tetravalent bispecific antibody in this invention;

[0069] SEQ ID No. 12 is the nucleotide sequence of the tetravalent bispecific antibody in this invention. Detailed Implementation

[0070] This invention discloses a bispecific antibody that simultaneously targets human BCMA and human CD3. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve this antibody. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately change and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0071] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components.

[0072] The following is an explanation of some of the terms used in this invention.

[0073] As used in this invention, the term "antigen-binding moiety" refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding fragment." See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab′, F(ab′)2, Fd, Fv, etc.

[0074] The term "bispecific" or "double-specific" as used in this invention refers to a hybrid antibody having two different antigen-binding sites. The two antigen-binding sites of a bispecific antibody bind to two different epitopes, which may be located on the same or different protein targets.

[0075] The term "bivalent antibody" as used in this invention refers to an antibody that contains two antigen-binding sites per molecule (e.g., IgG). In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific. Similarly, a "quadrivalent antibody" contains four antigen-binding sites per molecule.

[0076] The term "specifically binding" as used in this invention has a well-known definition in the art, for example, meaning that a molecule exhibits "specific binding" or "preferential binding" if it reacts or correlates with a particular molecule or substance more frequently, more rapidly, for a longer duration, and / or with a greater affinity compared to alternative cells or substances. An antibody is said to "specifically bind" or "preferentially bind" to a target if it binds with greater affinity, faster, and / or for a longer duration compared to other substances it binds to. For example, an antibody that specifically or preferentially binds to a BCMA epitope or CD3 epitope is an antibody that binds to that epitope with greater affinity, faster, and / or for a longer duration compared to other BCMA epitopes, non-BCMA epitopes, CD3 epitopes, or non-CD3 epitopes. By reading this definition, it should also be understood that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) specific binding. Generally speaking, but not necessarily, mentioning combination indicates priority in combination.

[0077] As used herein, the term “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together tightly by the FRs, and also by those from other chains, which contribute to the formation of the antigen-binding site of the antibody. At least two techniques are used to determine the CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273: 927-948). As used herein, a CDR may refer to a CDR defined by either method or by a combination of both methods.

[0078] As used in this invention, the term "humanized" refers to the form of a non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) comprising a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the complementarity-determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody) with the desired specificity, affinity, and capacity, such as mouse, rat, or rabbit. In some cases, residues from the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the receptor antibody or the introduced CDR or framework sequence, but included to further refine and optimize antibody performance. Generally, humanized antibodies contain substantially all, typically two, variable domains, wherein all or substantially all CDR regions correspond to those regions of non-human immunoglobulins, and all or substantially all FR regions are those regions of the common sequence of human immunoglobulins. Humanized antibodies preferably also contain at least a portion of immunoglobulin constant regions or domains (Fc), typically constant regions of human immunoglobulins. Antibodies having Fc regions modified as described in WO 99 / 58572 are preferred. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) modified from the original antibody, also referred to as one or more CDRs “derived” from one or more CDRs of the original antibody.

[0079] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0080] Example 1: Construction of the tetravalent bispecific antibody Tandab (CD3 / BCMA)

[0081] First, using the monoclonal antibody 69G8 targeting BCMA and the monoclonal antibody HIT3a targeting CD3, obtained through hybridoma technology in the laboratory, as templates, primers were designed to extract the variable region gene of the heavy and light chains. The bispecific antibody gene fragment was then synthesized using gene synthesis methods and ligated into the eukaryotic expression vector pcDNA. TM 3.4. Recombinant plasmid pcDNA was obtained. TM 3.4-Tandab (CD3 / BCMA). See the diagram for the construction. Figure 1 The recombinant plasmid was transformed into E. coli DH5α competent cells using the heat shock method. The cells were plated, and positive single clones were picked for sequencing comparison. The cells with correct sequencing results were selected for amplification and preservation.

[0082] The aforementioned monoclonal antibody 69G8 targeting BCMA and monoclonal antibody HIT3a targeting CD3 are both stored at the Institute of Hematology, Chinese Academy of Medical Sciences.

[0083] Similarly, since the nucleotide sequences of each gene fragment of the bispecific antibody are known, the above gene fragments can also be obtained using a simpler chemical synthesis method, and then sequentially ligated into a eukaryotic expression vector to construct a recombinant plasmid.

[0084] Example 2: Expression, purification, and identification of the tetravalent bispecific antibody Tandab (CD3 / BCMA)

[0085] First, the recombinant plasmid pcDNA TM 3,4-Tandab (CD3 / BCMA) was transfected into ExpiCHO-STM cells using the ExpiFectamine CHO Transfection Kit (Thermo). After 14 days of culture at maximum titer, the cell culture medium was collected, filtered through a 0.45 μm filter, and purified by nickel affinity chromatography to obtain a large quantity of the target protein. Western blotting was then performed to determine its molecular weight and to preliminarily validate the target protein. Results are shown below. Figure 2 The results showed that Tandab (CD3 / BCMA) was successfully expressed.

[0086] Experimental Example 1: FACS Detection of the tetravalent bispecific antibody Tandab (CD3 / BCMA) with BCMA+ cell line H929 and CD3 + Binding experiment of the Jurkat cell line

[0087] The 10 nM bispecific antibody obtained in Example 2 was used with 1 × 10 6 H929 and Jurkat cells were incubated at room temperature for 30 minutes, followed by two washes with PBS. Cells were resuspended in 100 μL, and 0.2 μL of APC-labeled anti-His secondary antibody was added. Cells were incubated at room temperature in the dark for 30 minutes, followed by two washes with PBS. An isotype control tube was also included. Cells were resuspended in 200 μL of PBS buffer and analyzed by FACS. Results are shown below. Figure 3 The results showed that the bispecific antibody Tandab (CD3 / BCMA) can effectively bind to H929 and Jurkat.

[0088] Experiment Example 2: Cross-reactivity experiment of tetravalent bispecific antibody Tandab (CD3 / BCMA) with BCMA and CD3 double-negative cells

[0089] Using the 10 nM bispecific antibody Tandab (CD3 / BCMA) obtained in Example 2, respectively with 1x106 K562 and HL-60 cells (BCMACD3 double-negative) were incubated. Cells were incubated at room temperature for 30 min, washed twice with PBS, and then resuspended in 100 μL of the solution. 0.2 μL of APC-labeled anti-His secondary antibody was added, and the cells were incubated at room temperature in the dark for 30 min, washed twice with PBS. A secondary antibody control tube was also prepared. Cells were resuspended in 200 μL of PBS buffer and analyzed by FACS. Results are shown below. Figure 4 The results showed that the bispecific antibody Tandab (CD3 / BCMA) did not cross-react with either K562 or HL-60 cells.

[0090] Experiment Example 3: Detection of BCMA-binding and CD3-binding affinity constants of the tetravalent bispecific antibody Tandab (CD3 / BCMA)

[0091] The bispecific antibody Tandab (CD3 / BCMA) obtained in Example 2 was used at final concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.2 nM, 1.6 nM, 0.8 nM, 0.4 nM, 0.2 nM, 0.1 nM, 0.05 nM, and 0.025 nM, respectively, in combination with 1×10⁻⁶ ppm. 6 Cells were incubated with H929 or Jurkat. Incubation was performed at room temperature for 30 min, followed by two washes with PBS. Cells were resuspended in 100 μL, and 0.2 μL of APC-labeled anti-his secondary antibody was added. Cells were incubated at room temperature in the dark for 30 min, followed by two washes with PBS. An isotype control tube was also included. Cells were resuspended in 200 μL of PBS buffer, and fluorescence intensity was measured using FACS. Mean values ​​were calculated. The Kd value of the bispecific antibody was calculated using GraphPad Prism7. Results are shown below. Figure 5 The results showed that the affinity constants (Kd) of the bispecific antibody Tandab (CD3 / BCMA) with BCMA and CD3 on the cell membrane surface were 1.963 x 10⁻⁶ and 1.963 x 10⁻⁶, respectively. -9 M and 1.961x10 -9 M.

[0092] Experiment Example 4: FACS assay to detect the competition between the tetravalent bispecific antibody Tandab (CD3 / BCMA) and the commercial BCMA antibody 19F2 and the commercial CD3 antibody HIT3a.

[0093] Using the 10nMTandab (CD3 / BCMA) and 10nMAPC conjugated anti-human BCMA commercial antibody 19F2 (brand: Biolegend, catalog number: 357506) obtained in Example 2, simultaneously with 1×10 6H929 cells were incubated at room temperature in the dark for 30 min. After centrifugation at 1200 rpm for 6 min, the supernatant was discarded, and the cells were washed with PBS. This process was repeated three times. The cells were then resuspended in 200 μl of PBS, and fluorescence intensity was measured using FACS. The results of incubation of H929 cells with 10 nM commercial antibody 19F2 alone served as a pre-competition control. See [details omitted]. Figure 6 The left figure shows that Tandab (CD3 / BCMA) can competitively bind to the BCMA protein on the surface of target cells with the commercial antibody 19F2.

[0094] HIT3a (brand: Biolegend, catalog number: 300312), a commercially available anti-human CD3 antibody conjugated with 10nMTandab (CD3 / BCMA) and 10nMAPC, was administered simultaneously with 1×10 6 Jurkat cells were incubated at room temperature in the dark for 30 min. After centrifugation at 1200 rpm for 6 min, the supernatant was discarded, and cells were washed with PBS. This process was repeated three times. Cells were resuspended in 200 μl of PBS, and fluorescence intensity was measured using FACS. The results of incubation of Jurkat cells with 10 nM commercial antibody HIT3a alone served as a pre-competition control. See [details omitted]. Figure 6 The right figure shows that Tandab (CD3 / BCMA) can competitively bind to the CD3 protein on the surface of target cells with the commercial antibody HIT3a.

[0095] Experiment Example 5: Assay on Upregulation of PBMC Activation Marker Expression and Cytokine Release Mediated by the Quadrivalent Bispecific Antibody Tandab (CD3 / BCMA)

[0096] PBMCs were used as effector cells, NCI-H929 cells as target cells, and K562 cells as negative controls. At an effector-to-target ratio of 10:1, the bispecific antibodies obtained in Example 2 (from 10 nM to 0.001 nM) were serially diluted. After incubation at 37°C with 5% CO2 for 24 h, cells were collected by centrifugation at 1800 rpm for 8 min. Each tube was then incubated with 1 μl of FITC-labeled anti-human CD8 monoclonal antibody, 1 μl of PE-CY7-labeled anti-human CD4 monoclonal antibody, 1 μl of APC-labeled anti-human CD69 monoclonal antibody, and 1 μl of PE-labeled anti-human CD25 monoclonal antibody. The cells were incubated at room temperature for 30 min, washed twice with PBS, and resuspended in 200 μl of PBS. The expression levels of CD25 and CD69 in the CD4 and CD8 subsets were detected by FACS. The culture supernatant was collected, and ELISA was used to quantify IL-2, TNF-α, and IFN-γ released from PBMCs into the supernatant. Results are shown below. Figure 7 and Figure 8 The results showed that Tandab (CD3 / BCMA)-mediated activation marker expression and cytokine release in PBMCs were concentration-dependent, and the activation mediated by H929 as the target cell was significantly higher than that mediated by K562.

[0097] Experiment Example 6: Detection of the killing effect of Tandab (CD3 / BCMA)-mediated PBMCs on H929 and K562 by LDH lactate dehydrogenase cytotoxicity assay.

[0098] PBMCs were used as effector cells, NCI-H929 cells as target cells, and K562 cells as negative control cells. At an effector-to-target ratio of 10:1, Tandab (CD3 / BCMA) obtained in Example 2 was serially diluted (from 10 nM to 0.001 nM), incubated at 37°C with 5% CO2 for 24 h, and then the cytotoxic effect of Tandab (CD3 / BCMA) on target cells in PBMCs was detected under different concentrations. The following groups were set up: spontaneous release group of target cells (target cells + culture medium); spontaneous release group of effector cells (effector cells + culture medium); background blank control group (cell-free culture medium); volume correction control group (LDH lysis buffer + culture medium); and maximum release group of target cells (target cells + culture medium). One hour before the end of the culture time, 10% volume of LDH cell lysis buffer was added to each group and the mixture was repeatedly pipetted and mixed. Three parallel wells were set up for each group. After incubation at 37°C in a 5% CO2 incubator for a period of time, the cells were centrifuged at 2500g for 3 min. 50 μl of culture supernatant was aspirated from each well into a new 96-well plate. 50 μl of LDH working solution was added to each well and mixed. The cells were incubated at room temperature in the dark for 30 min, and the OD490 was measured. The absorbance of each group should be subtracted from the absorbance of the background blank control group (where the absorbance of the target cell maximum release group is subtracted from the absorbance of the volume correction group). The killing rate is calculated as follows: (Experimental group - effector cell spontaneous release group - target cell spontaneous release group OD value) / (target cell maximum release group - target cell spontaneous release group OD value) × 100%. Results are shown below. Figure 9 The results showed that the killing effect of Tandab (CD3 / BCMA) on myeloma cells was concentration-dependent, and the cytotoxicity mediated was significantly better than that of the K562 control group.

[0099] Experiment Example 7: PBMC Proliferation Assay Mediated by the Tetravalent Bispecific Antibody Tandab (CD3 / BCMA)

[0100] PBMCs were stained with CFSE-Far Red dye, and the specific steps were as follows: The density of PBMCs was adjusted to 1x10⁶ / ml with PBS, 1 μl of CFSE dye was added per ml of PBMCs, and the mixture was incubated at 37°C in the dark for 20 min. Then, 5 times the volume of the original staining solution was added to the culture medium and incubated for 5 min to terminate the reaction. The mixture was centrifuged at 1000 rpm for 6 min and resuspended in complete culture medium.

[0101] PBMCs were used as effector cells, NCI-H929 cells as target cells, and K562 cells as negative control cells. At an effector-to-target ratio of 10:1, Tandab (CD3 / BCMA) obtained in Example 2 was serially diluted (from 10 nM to 0.001 nM), and incubated at 37°C with 5% CO2 for 24 h. Flow cytometry was then used to detect the proliferation of PBMCs mediated by Tandab (CD3 / BCMA) at different concentrations. Results are shown below. Figure 10 The results showed that Tandab (CD3 / BCMA) mediated the proliferation of PBMCs in a dose-dependent manner, and the proliferation mediated by H929 as the target cell was significantly higher than that mediated by K562.

[0102] Experiment Example 8: Antitumor effect of tetravalent bispecific antibody Tandab (CD3 / BCMA) on a subcutaneous myeloma model

[0103] A xenograft tumor model was established using the H929 multiple myeloma cell line. NOD / SCID female mice, 6-8 weeks old and weighing 16-18g, were subcutaneously inoculated with 5 x 10⁵ cells per cell line at the dorsal aspect of the right hind limb root. 6 / 0.2ml cells. The tumor diameter in the xenograft model was measured using calipers to dynamically observe the antitumor effect of the drug. Tumor diameter was measured every two days. The tumor volume was calculated using the formula: V = 1 / 2 × a × b 2 a and b are the long and short diameters of the tumor, respectively. The mouse weight is recorded each time the tumor volume is measured. The tumor grows to 50-100 mm in approximately 5-7 days. 3 The mixture was divided into five groups: ①PBS; ②PBS + 1x10 7 PBMC; ③1mg / kgTandab(CD3 / BCMA)+1x10 7 PBMC; ④0.5mg / kgTandab(CD3 / BCMA)+1x10 7 PBMC; ⑤0.25mg / kgTandab(CD3 / BCMA)+1x10 7 PBMC; treatment begins on day 8, twice a week, for two weeks. See the treatment flowchart below. Figure 11 Mice were sacrificed 22 days after tumor inoculation. Tumor volume at the treatment endpoint was compared. Figure 12 The results showed that, compared with the PBS control group and the PBMC control group, 1 mg / kg and 0.5 mg / kg doses of Tandab (CD3 / BCMA) significantly delayed tumor growth.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Chinese Academy of Medical Sciences Blood Diseases Hospital (Institute of Hematology, Chinese Academy of Medical Sciences) <120> A bispecific antibody that simultaneously targets human BCMA and human CD3 <130> none <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 15 <212> PRT <213> Artificial <400> 1 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial <400> 2 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial <400> 3 Gln His Ser Arg Asp Leu Pro Tyr Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial <400> 4 Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Glu 1 5 10 <210> 5 <211> 17 <212> PRT <213> Artificial <400> 5 Val Ile Thr Pro Gly Arg Gly Asp Thr Lys Tyr Asn Ala Lys Phe Ala 1 5 10 15 Gly <210> 6 <211> 8 <212> PRT <213> Artificial <400> 6 Gly Thr Thr Ala Trp Phe Pro Tyr 1 5 <210> 7 <211> 113 <212> PRT <213> Artificial <400> 7 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Arg Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala <210> 8 <211> 117 <212> PRT <213> Artificial <400> 8 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Pro Glu Trp Ile 35 40 45 Gly Val Ile Thr Pro Gly Arg Gly Asp Thr Lys Tyr Asn Ala Lys Phe 50 55 60 Ala Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Thr Thr Ala Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 9 <211> 108 <212> PRT <213> Artificial <400> 9 Ala Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro 1 5 10 15 Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr 20 25 30 Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile 35 40 45 Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 10 <211> 119 <212> PRT <213> Artificial <400> 10 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Thr Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 11 <211> 509 <212> PRT <213> Artificial <400> 11 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 50 55 60 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys 115 120 125 Arg Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu 130 135 140 Val Val Arg Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly 145 150 155 160 Tyr Ala Phe Thr Asn Tyr Leu Ile Glu Trp Val Lys Gln Arg Pro Gly 165 170 175 Gln Gly Pro Glu Trp Ile Gly Val Ile Thr Pro Gly Arg Gly Asp Thr 180 185 190 Lys Tyr Asn Ala Lys Phe Ala Gly Lys Ala Thr Leu Thr Ala Asp Lys 195 200 205 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Asp Asp 210 215 220 Ser Ala Val Tyr Phe Cys Ala Arg Gly Thr Thr Ala Trp Phe Pro Tyr 225 230 235 240 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser 245 250 255 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln 260 265 270 Ser Pro Ala Ser Leu Thr Val Ser Leu Gly Gln Arg Ala Thr Ile Ser 275 280 285 Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 290 295 300 Trp Tyr Gln Gln Lys Pro Arg Gln Pro Pro Lys Leu Leu Ile Tyr Leu 305 310 315 320 Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 325 330 335 Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp 340 345 350 Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro Tyr Thr Phe 355 360 365 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Gly Ser 370 375 380 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 385 390 395 400 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 405 410 415 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile 420 425 430 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 435 440 445 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 450 455 460 Met Glu Leu Thr Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 465 470 475 480 Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly 485 490 495 Thr Thr Val Thr Val Create Create His His His His His 500 505 <210> 12 <211> 1527 <212> DNA <213> Artificial <400> 12 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgctgaca tcgagctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120 gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgaactggta ccagcagaag 180 tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct 240 gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cggcatggag 300 gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccatt cacgttcggc 360 tgtgggacca agctggagct gaaacggggt ggcggagggt cgcaggtcca gctgcagcag 420 tctggagctg aggtggtaag gcctgggact tcagtgaagg tgtcctgcaa ggcttctgga 480 tacgccttca ctaattactt gatagagtgg gtaaagcaga ggcctggaca gggccctgag 540 tggattggag tgattactcc tggacgtggt gatactaaat acaatgcgaa attcgcgggc 600 aaggcaacac tgactgcaga caaatcctcc agcactgcct acatgcagct cagcagcctg 660 acatttgatg actctgcggt ttattctgt gcaagaggga ctacggcctg gtttccttac 720 tggggccaag ggactctggt cactgtctcc gcaggaggcg gcggtagcgg cggaggggt 780 tcaggaggtg ggggcagtga cattgtgctg acacagtctc ctgcttcctt aactgtatct 840 ctggggcaga gggccaccat ctcatgcagg gccagcaaaa gtgtcagtac atctggctat 900 agttatatgc actggtacca acagaaacca agaacagccac ccaaactcct catctatctt 960 gcatccaacc tagaatctgg ggtccctgcc aggttcagtg gcagtgggtc tggcacagac 1020 ttcaccctca acatccatcc tgtggaggag gaggatgctg caacctatta ctgtcagcac 1080 agtagggacc ttccgtacac gttcggaggg gggaccaagc tggaaataaa acgggctggt 1140 ggcggagggt cgcaggtgca gctgcagcag tctggggctg aactggcaag acctggggcc 1200 tcagtaaaga tgtcctgcaa ggcttctggc tacaccttta ctaggtacac gatgcactgg 1260 gtaaaacaga ggcctggaca gtgcctggaa tggattggat acattaatcc tagccgtggt tatactaatt acaatcaga gttcaaggac aaggccacat tgactacaga caaatcctcc agcacagcct atatggagct cactaggctg acatctgagg actctgcagt ctattactgt gcaagatatt acgatgatca ttacagcctt gactactggg gccaaggcac cacggtcacc gtctcctcac atcatcacca tcaccat 1527

Claims

1. A bispecific antibody that simultaneously targets human BCMA and human CD3, characterized in that, It consists of the following parts: a) Specifically binds to the antigen-binding portion of human BCMA; b) Specifically binds to the antigen-binding portion of human CD3; and c) Connectors between the antigen-binding portions; The antigen-binding portion that specifically binds to human BCMA includes the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID No. 1-3, and the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID No. 4-6. The antigen-binding moiety that specifically binds to human CD3 includes the amino acid sequence of the light chain variable region as shown in SEQ ID No. 9 and the amino acid sequence of the heavy chain variable region as shown in SEQ ID No. 10; The bispecific antibody is a tetravalent bispecific antibody. The primary structure of the tetravalent bispecific antibody is arranged from the N-terminus to the C-terminus in the following order: light chain variable region sequence of the antigen-binding moiety specifically binding to human CD3 - heavy chain variable region sequence of the antigen-binding moiety specifically binding to human BCMA - light chain variable region sequence of the antigen-binding moiety specifically binding to human BCMA - heavy chain variable region sequence of the antigen-binding moiety specifically binding to human CD3; the 100th serine residue of the light chain variable region of the antigen-binding moiety specifically binding to human CD3 is replaced by a cysteine ​​residue.

2. The bispecific antibody according to claim 1, characterized in that, The antigen-binding portion that specifically binds to human BCMA comprises the amino acid sequence of the light chain variable region as shown in SEQ ID No. 7 and the amino acid sequence of the heavy chain variable region as shown in SEQ ID No.

8.

3. The bispecific antibody according to claim 1 or 2, characterized in that, The bispecific antibody is a humanized antibody.

4. The bispecific antibody according to claim 1 or 2, characterized in that, The connector is (GGGGS) n , where n is an integer and 1≤n≤3.

5. The bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the bispecific antibody is shown in SEQ ID No.

11.

6. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific antibody as described in any one of claims 1 to 5.

7. The isolated nucleic acid molecule according to claim 6, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.

12.

8. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 6.

9. A host cell, characterized in that, The host cell contains the expression vector as described in claim 8.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a bispecific antibody as described in any one of claims 1 to 5 or an isolated nucleic acid molecule as described in claims 6 and 7, and a pharmaceutically acceptable carrier.

11. Use of the bispecific antibody as described in any one of claims 1 to 5, the isolated nucleic acid molecule as described in claims 6 and 7, the expression vector as described in claim 8, the host cell as described in claim 9, or the pharmaceutical composition as described in claim 10 in the preparation of a product for treating multiple myeloma.

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