Specific antigen-binding molecule, its preparation method and medical use

Abstract

Provided are anti-BCMA antibodies, bispecific antigen-binding molecules that bind BCMA and CD3, and pharmaceutical compositions thereof, as well as their use in the preparation of anti-cancer drugs and drugs for the treatment of autoimmune diseases.

Description

[0001] This application claims the priority of the patent application No. 202010307360.1 filed on April 17, 2020 and the patent application No. 202110119870.0 filed on January 28, 2021. Technical Field

[0002] The present invention relates to the field of biomedicine. Specifically, the present invention relates to anti-BCMA antibodies, antigen-binding fragments thereof, bispecific antigen-binding molecules that bind BCMA and CD3, methods for preparing the same, and their medical uses. Background Art

[0003] B cells are lymphocytes that play an important role in humoral immunity and the production of antibodies that specifically recognize antigens. The three subtypes of B cells are naive B cells, memory B cells, and plasma cells.

[0004] In the process of VDJ recombination, DNA recombines to generate a combinatorial array of antibody variable domains, which are further diversified by variable domains encoded by different lineages of B cells, resulting in up to 10 9 unique B cell lineages that produce antibodies with specificities.

[0005] Multiple diseases involve B cells. Malignant transformation of B cells leads to cancers, including lymphomas (such as multiple myeloma and Hodgkin's lymphoma). Autoimmune diseases also involve B cells, including systemic lupus erythematosus (SLE) and IgA nephropathy. Cancers and autoimmune diseases involving B cells are considered to be due to abnormal B cell function. Therefore, a possible strategy for controlling such diseases is to use antibodies that target pathological B cells.

[0006] BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily and is a non-glycosylated integral membrane receptor for the ligands BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand). BCMA and its corresponding ligands can regulate humoral immunity, B cell development, and homeostasis. BCMA is detected in the spleen, lymph nodes, thymus, adrenal glands, and liver, and is also expressed in tonsil memory B cells and germinal center B cells. Analysis of multiple B cell lines has shown that the expression level of BCMA increases after maturation. BCMA is highly expressed in B cell lymphoma and multiple myeloma.

[0007] Current therapies targeting BCMA mainly fall into three categories: chimeric antigen receptor T cell therapy (CAR-T), bispecific antibodies (BsAb), and antibody-drug conjugates (ADC). Among them, GSK's ADC drug Blenrep was approved by the US FDA in August 2020, becoming the first approved immune-related drug for anti-BCMA. However, its treatment has adverse reactions such as vision impairment and late-stage failure, making the bispecific antibody strategy another popular research and development direction for this target. Among them, Amgen's bispecific antibody AMG-420 has priority to enter the late clinical stage. This antibody uses a BiTE design with good penetrability, but has a small molecular weight and a short half-life, reducing patient compliance. SUMMARY OF THE INVENTION

[0008] According to some embodiments of the present invention, there is provided an anti-BCMA antibody or an antigen-binding fragment thereof, which comprises:

[0009] A heavy chain variable region of the antibody, wherein the heavy chain variable region of the antibody comprises at least 1 HCDR selected from the following sequences:

[0010] SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 9; and

[0011] A light chain variable region of the antibody, wherein the light chain variable region of the antibody comprises at least 1 LCDR selected from the following sequences: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10.

[0012] In a specific embodiment of the present invention, the heavy chain variable region of the anti-BCMA antibody or an antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 or SEQ ID NO: 9, and HCDR3 shown in SEQ ID NO: 5.

[0013] In a specific embodiment of the present invention, the light chain variable region of the anti-BCMA antibody or an antigen-binding fragment thereof comprises: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 or SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8.

[0014] In a specific embodiment of the present invention, there is provided an anti-BCMA antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:

[0015] i)

[0016] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and

[0017] The light chain variable region comprises: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8,

[0018] or ii)

[0019] The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9, and HCDR3 shown in SEQ ID NO: 5; and

[0020] The light chain variable region comprises: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8,

[0021] or iii)

[0022] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9, and HCDR3 shown in SEQ ID NO: 5;

[0023] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8,

[0024] or iv)

[0025] The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and

[0026] The light chain variable region comprises: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8.

[0027] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment is a murine antibody or its antigen-binding fragment.

[0028] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment is a chimeric antibody or its antigen-binding fragment.

[0029] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment.

[0030] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0031] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0032] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof.

[0033] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the heavy chain constant region of human IgG1.

[0034] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises a variant of the heavy chain constant region of human IgG1.

[0035] In a specific embodiment of the present invention, the variant of the human IgG1 heavy chain constant region has reduced ADCC toxicity compared to the wild-type IgG1 heavy chain constant region.

[0036] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the heavy chain constant region as shown in SEQ ID NO: 31, or the variant of the heavy chain constant region as shown in SEQ ID NO: 42, or the variant of the heavy chain constant region as shown in SEQ ID NO: 33, or the variant of the heavy chain constant region as shown in SEQ ID NO: 34.

[0037] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the heavy chain constant region as shown in SEQ ID NO: 31 or the variant of the heavy chain constant region as shown in SEQ ID NO: 42.

[0038] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the variant of the heavy chain constant region as shown in SEQ ID NO: 42.

[0039] In some embodiments of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the light chain constant region derived from human antibody κ chain, λ chain or a variant thereof.

[0040] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region derived from the human antibody κ chain.

[0041] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises the light chain constant region shown in SEQ ID NO: 32.

[0042] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or,

[0043] The anti-BCMA antibody or its antigen-binding fragment comprises a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0044] In a specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment contains a heavy chain as shown in the following sequence, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequence: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 43; and / or,

[0045] The anti-BCMA antibody or its antigen-binding fragment contains a light chain as shown in the following sequence, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequence: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.

[0046] In a more specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises:

[0047] The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14; or,

[0048] The heavy chain variable region shown in SEQ ID NO: 12 and the light chain variable region shown in SEQ ID NO: 15; or,

[0049] The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 16; or,

[0050] The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 16.

[0051] In a more specific embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises:

[0052] The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20; or,

[0053] The heavy chain shown in SEQ ID NO: 18 and the light chain shown in SEQ ID NO: 21; or,

[0054] The heavy chain shown in SEQ ID NO: 19 and the light chain shown in SEQ ID NO: 22; or,

[0055] The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 22; or,

[0056] The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 20.

[0057] According to some embodiments of the present invention, there is provided an anti-CD3 antibody or its antigen-binding fragment, which comprises a heavy chain variable region and a light chain variable region, wherein:

[0058] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;

[0059] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.

[0060] According to some embodiments of the present invention, there is provided an anti-CD3 antibody or its antigen-binding fragment, which comprises the heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25.

[0061] According to some embodiments of the present invention, there is provided an antigen-binding fragment of an anti-CD3 antibody, which is scFv. In a specific embodiment, scFv is as shown in SEQ ID NO: 41.

[0062] According to some embodiments of the present invention, there is provided a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, comprising:

[0063] a first binding region that specifically binds BCMA; and

[0064] a second binding region that specifically binds CD3.

[0065] In some specific embodiments, the first binding region is selected from the anti-BCMA antibodies or antigen-binding fragments thereof as described above.

[0066] In some specific embodiments, the second binding region is selected from anti-CD3 antibodies or antigen-binding fragments thereof.

[0067] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8.

[0068] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 11 and a light chain variable region shown in SEQ ID NO: 14.

[0069] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0070] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1.

[0071] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a variant of the heavy chain constant region of human IgG1, and the variant of the heavy chain constant region of human IgG1 has reduced ADCC toxicity compared with the heavy chain constant region of human IgG1.

[0072] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO: 31.

[0073] In a preferred embodiment of the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain constant region variant as shown in SEQ ID NO: 42.

[0074] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region of a human antibody κ chain, λ chain or a variant thereof, preferably the light chain constant region of a human antibody κ chain, and most preferably the light chain constant region as shown in SEQ ID NO: 32.

[0075] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 17 or SEQ ID NO: 43 and a light chain as shown in SEQ ID NO: 20.

[0076] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 43 and a light chain as shown in SEQ ID NO: 20.

[0077] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0078] The heavy chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof comprises HCDR1 as shown in SEQ ID NO: 35, HCDR2 as shown in SEQ ID NO: 36, and HCDR3 as shown in SEQ ID NO: 37;

[0079] The light chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof comprises LCDR1 as shown in SEQ ID NO: 38, LCDR2 as shown in SEQ ID NO: 39, and LCDR3 as shown in SEQ ID NO: 40.

[0080] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-CD3 antibody or antigen-binding fragment thereof contains a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO: 25.

[0081] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the second binding region comprises a heavy chain variable region and a light chain variable region linked by a peptide linker.

[0082] The peptide linker linking the heavy chain variable region and the light chain variable region of the second binding region is selected from (GGGGS) n , where n is an integer selected from 1-5, and preferably, n is 3.

[0083] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the second binding region comprises the sequence shown in SEQ ID NO: 41.

[0084] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the N-terminus of the heavy chain variable region of the second binding region is linked to the C-terminus of the light chain constant region of the first binding region.

[0085] In some embodiments, the N-terminus of the heavy chain variable region of the second binding region is linked to the C-terminus of the light chain constant region of the first binding region by a peptide linker selected from (GGGGS) n , where n is an integer selected from 1-5, and preferably, n is 3.

[0086] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, comprising a first binding region that specifically binds BCMA and a second binding region that specifically binds CD3, wherein:

[0087] The first binding region comprises a heavy chain variable region and a light chain variable region,

[0088] The heavy chain variable region of the first binding region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5;

[0089] The light chain variable region of the first binding region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8, and

[0090] The second binding region comprises a heavy chain variable region and a light chain variable region,

[0091] The heavy chain variable region of the second binding region comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;

[0092] The light chain variable region of the second binding domain comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.

[0093] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein:

[0094] The first binding domain comprises a heavy chain variable region shown in SEQ ID NO: 11 and a light chain variable region shown in SEQ ID NO: 14, and

[0095] The second binding domain comprises a heavy chain variable region shown in SEQ ID NO: 24 and a light chain variable region shown in SEQ ID NO: 25.

[0096] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein:

[0097] The first binding domain comprises a heavy chain as shown in SEQ ID NO: 17 or SEQ ID NO: 43 and a light chain as shown in SEQ ID NO: 20, and

[0098] The second binding domain comprises a heavy chain variable region shown in SEQ ID NO: 24 and a light chain variable region shown in SEQ ID NO: 25.

[0099] In a preferred embodiment of the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first binding domain comprises a heavy chain as shown in SEQ ID NO: 43 and a light chain as shown in SEQ ID NO: 20, and the second binding domain comprises a heavy chain variable region shown in SEQ ID NO: 24 and a light chain variable region shown in SEQ ID NO: 25.

[0100] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the heavy chain variable region and the light chain variable region of the second binding domain are connected by a peptide linker, and the peptide linker connecting the heavy chain variable region and the light chain variable region of the second binding domain is selected from (GGGGS) n , n is an integer selected from 1-5 (1, 2, 3, 4, 5), and preferably, n is 3.

[0101] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the N-terminus of the heavy chain variable region of the second binding domain is connected to the C-terminus of the light chain constant region of the first binding domain; preferably, the N-terminus of the heavy chain variable region of the second binding domain is connected to the C-terminus of the light chain constant region of the first binding domain through a peptide linker.

[0102] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first binding region comprises at least one Fab fragment of an anti-BCMA antibody, and the second binding region comprises at least one scFv fragment of an anti-CD3 antibody.

[0103] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first binding region comprises two Fab fragments of an anti-BCMA antibody, and the second binding region comprises two scFv fragments of an anti-CD3 antibody.

[0104] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first binding region is selected from the above-mentioned anti-BCMA antibodies comprising a light chain and a heavy chain, and the second binding region comprises two scFv fragments of the above-mentioned anti-CD3 antibodies or their antigen-binding fragments.

[0105] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the N-terminus of the scFv fragment is linked to the Fab fragment or to the C-terminus of the light chain constant region of the anti-BCMA antibody; preferably, the N-terminus of the scFv fragment is linked to the Fab fragment or to the C-terminus of the light chain constant region of the anti-BCMA antibody through a peptide linker.

[0106] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first binding region is selected from anti-BCMA antibodies comprising a light chain and a heavy chain, and the second binding region comprises two scFv fragments of an anti-CD3 antibody.

[0107] For a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the N-terminus of the scFv fragment is linked to the Fab fragment or the C-terminus of the light chain constant region of the anti-BCMA antibody through a peptide linker, and the peptide linker is selected from (GGGGS) n , where n is an integer from 1 to 5, preferably n is 3.

[0108] In a specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention has a four-chain structure and comprises 2 identical first chains and 2 identical second chains, wherein:

[0109] The first chain, from the N-terminus to the C-terminus, comprises the heavy chain variable region of the first binding region and the heavy chain constant region of the first binding region;

[0110] The second chain, from the N-terminus to the C-terminus, comprises the light chain variable region of the first binding region, the light chain constant region of the first binding region and the first peptide linker, as well as the heavy chain variable region of the second binding region, the second peptide linker and the light chain variable region of the second binding region.

[0111] The bispecific antibody or antigen-binding fragment thereof according to the present invention, wherein the heavy-chain variable region of the second binding region and the light-chain variable region of the second binding region in the second chain are selected from the heavy-chain variable region and the light-chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof according to the present invention, and the second peptide linker is selected from (GGGGS) n , n is an integer selected from 1 to 5, and preferably, n is 3.

[0112] The bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first binding region comprises a Fab fragment of an anti-BCMA antibody, and the second binding region comprises a scFv fragment of an anti-CD3 antibody.

[0113] According to a specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention comprises:

[0114] A first polypeptide, comprising a heavy-chain variable region of the first binding region and a heavy-chain constant region of the first binding region from the N-terminus to the C-terminus;

[0115] A second polypeptide, comprising a light-chain variable region of the first binding region and a light-chain constant region of the first binding region from the N-terminus to the C-terminus;

[0116] A third polypeptide, comprising a heavy-chain variable region of the second binding region, a first peptide linker, a light-chain variable region of the second binding region, a second peptide linker, and a heavy-chain constant region of the first binding region from the N-terminus to the C-terminus.

[0117] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the second peptide linker in the third polypeptide is selected from (GGGGS)n, n is an integer selected from 1 to 5, and preferably, n is 2.

[0118] In some embodiments, when the peptide linker (i.e., the first peptide linker) connects the heavy-chain variable region of the second binding region and the light-chain variable region of the second binding region, n is 3.

[0119] In some embodiments, when the peptide linker (i.e., the second peptide linker) connects the light-chain variable region of the second binding region and the heavy-chain constant region, n is 2.

[0120] For the bispecific antibody or antigen-binding fragment thereof according to the present invention, the heavy-chain variable region of the first binding region of the first polypeptide is selected from the heavy-chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof according to the present invention.

[0121] For the bispecific antibody or antigen-binding fragment thereof according to the present invention, the light-chain variable region of the first binding region of the second polypeptide is selected from the light-chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof according to the present invention.

[0122] The bispecific antibody or antigen-binding fragment thereof according to the present invention, wherein the heavy chain variable region of the second binding region of the third polypeptide and the light chain variable region of the second binding region are respectively selected from the heavy chain variable region and the light chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof according to the present invention; the first peptide linker is selected from (GGGGS)n, and n is an integer selected from 1 to 5.

[0123] For the bispecific antibody or antigen-binding fragment thereof according to the present invention, the heavy chain constant region in the first polypeptide comprises a heavy chain constant region derived from human IgG, and the heavy chain constant region in the third polypeptide comprises a heavy chain constant region domain derived from human IgG. Preferably, the heavy chain constant region domain of IgG comprises (from the N-terminus to the C-terminus) a hinge region, CH2, and CH3. Among them, human IgG is selected from IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably IgG1.

[0124] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the heavy chain constant regions of the first polypeptide and the third polypeptide comprise one or more cysteine residues in the hinge region to form disulfide bonds.

[0125] For the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the heavy chain constant regions of the first polypeptide and the third polypeptide further comprise one or more cysteine residues outside the hinge region to form disulfide bonds.

[0126] In some embodiments, the cysteine residues are located at sites or combinations selected from the following: 349, 354 or equivalent sites.

[0127] For the numbering of antibody amino acids, there are multiple different numbering rules. It is possible to compare the numbering rules to determine equivalent residue positions. The Kabat numbering rule was developed based on the positions of the same hypervariable regions. The Chothia numbering rule (Al-Lazikani, 1997) is the same as Kabat's scheme, but corrects the first VH CDR. IMGT (Lefranc, 2003) and AHo (Honegger and Plückthun, 2001) define the variable domains of antibodies and T cell receptors (TCRs), so the equivalent residue positions of the two can be compared.

[0128] The Eu numbering rule was established based on human IgG1 (named Eu) purified by Gerald M. Edelman et al. Gerald M. Edelman et al. determined the amino acid sequence of IgG1 and numbered it. In the prior art, many tools can be used to determine or align the amino acid numbering of antibodies, such as but not limited to ANARCI, abYsis.

[0129] In the context of the present application, the amino acid positions in the heavy chain constant region follow the EU numbering rule, and positions 349 or 354 refer to the amino acid positions under the EU numbering rule. It will be obvious to those skilled in the art that cysteine residues at equivalent positions are also expected to perform the same function (i.e., promote the formation of disulfide bonds between the first polypeptide and the third polypeptide). For example, the numbering rules for determining antibody amino acid positions also include: Kabat, Chothia, IMGT, and those skilled in the art can determine the corresponding equivalent positions in different numbering rules. For another example, those skilled in the art can determine such equivalent positions through alignment analysis of amino acid sequences.

[0130] In a specific embodiment, the heavy chain constant regions of the first polypeptide and the third polypeptide respectively contain 349C and 354C.

[0131] In a specific embodiment, for the bispecific antibody or its antigen-binding fragment that binds BCMA and CD3 according to the present invention, the heavy chain constant region of the first polypeptide contains 354C, and the heavy chain constant region of the third polypeptide contains 349C.

[0132] In other embodiments, for the bispecific antibody or its antigen-binding fragment that binds BCMA and CD3 according to the present invention, the heavy chain constant regions of the first polypeptide and the third polypeptide respectively contain a knob and a hole domain, and the interaction between the hole and the knob domains promotes the heterodimerization of the first polypeptide and the third polypeptide.

[0133] In a specific embodiment, the heavy chain constant region contains a knob domain, preferably containing a tryptophan residue at position 366 or an equivalent position.

[0134] In a specific embodiment, the heavy chain constant region contains a hole domain, preferably containing an amino acid residue selected from: S at position 366 or an equivalent position, A at position 368 or an equivalent position, V at position 407 or an equivalent position. As described above, those skilled in the art can determine such equivalent positions.

[0135] In a specific embodiment, for the bispecific antibody or its antigen-binding fragment that binds BCMA and CD3 according to the present invention, the heavy chain constant region of the first polypeptide contains a knob domain, and the heavy chain constant region of the third polypeptide contains a hole domain.

[0136] The mutations of the heavy chain constant region are numbered according to the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0137] A bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first polypeptide heavy chain constant region comprises the sequence shown in SEQ ID NO: 33, and the third polypeptide heavy chain constant region comprises the sequence shown in SEQ ID NO: 34.

[0138] In a preferred embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention contains the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27.

[0139] According to a preferred embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention has a four-chain structure and comprises 2 (preferably identical) first chains and 2 (preferably identical) second chains, wherein the first chain comprises the sequence shown in SEQ ID NO: 26; and the second chain comprises the sequence shown in SEQ ID NO: 27.

[0140] In a more specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention contains the sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 20.

[0141] According to a specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention is a trispecific antibody, wherein the first polypeptide contains the sequence shown in SEQ ID NO: 28, the second polypeptide contains the sequence shown in SEQ ID NO: 20, and the third polypeptide contains the sequence shown in SEQ ID NO: 29.

[0142] The present invention also provides a polynucleotide encoding the anti-BCMA antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, of the present invention.

[0143] The present invention also provides an expression vector containing the polynucleotide of the present invention.

[0144] The present invention also provides a host cell into which or that contains the expression vector of the present invention.

[0145] In a preferred embodiment of the present invention, the host cell is a bacterium, preferably Escherichia coli (E. coli).

[0146] In a preferred embodiment of the present invention, the host cell is a yeast, preferably Pichia pastoris (P. pastoris).

[0147] In a preferred embodiment of the present invention, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.

[0148] On the other hand, the present invention provides a method for producing an anti-BCMA antibody or an antigen-binding fragment thereof, or a bispecific antibody that binds BCMA and CD3 or an antigen-binding fragment thereof, comprising the steps of:

[0149] culturing the host cell as described above;

[0150] isolating the antibody from the culture; and

[0151] purifying the antibody.

[0152] The present invention also provides a pharmaceutical composition comprising:

[0153] the anti-BCMA antibody or an antigen-binding fragment thereof or the bispecific antibody that binds BCMA and CD3 or an antigen-binding fragment thereof as described in the present invention; and

[0154] a pharmaceutically acceptable excipient, diluent or carrier.

[0155] On the other hand, the present invention provides a use, comprising the anti-BCMA antibody or an antigen-binding fragment thereof, the bispecific antibody that binds BCMA and CD3 or an antigen-binding fragment thereof as described in the present invention, or the pharmaceutical composition as described above for treating or preventing BCMA-mediated diseases or disorders.

[0156] In a preferred embodiment of the present invention, the disease or disorder is cancer; preferably cancer expressing BCMA; more preferably lymphoma and myeloma.

[0157] In a preferred embodiment of the present invention, the disease or disorder is an autoimmune disease; preferably lupus erythematosus, IgA nephropathy and rheumatoid arthritis.

[0158] The anti-BCMA antibody or an antigen-binding fragment thereof provided by the present invention can specifically bind to the BCMA antigen and cells expressing BCMA, and has strong targeting.

[0159] The bispecific antibody provided by the present invention can maintain the affinity for each antigen (or epitope), has obvious tumor suppression effects in vitro and in vivo. While having better affinity activity than AMG-420, the in vivo drug effect is prolonged and the compliance is better. Each chain of the bispecific antibody of the present invention can be correctly paired, is easy to express, has a simple preparation process, is stable in nature, has good drug-forming properties, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0160] Figure 1 : Schematic model of a bispecific antibody, A: the first chain; B: the second chain.

[0161] Figure 2:Schematic model of bispecific antibody, C: first polypeptide; D: second polypeptide; E: third polypeptide. Detailed implementation mode

[0162] Term

[0163] To facilitate the understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0164] The three-letter and single-letter codes of amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).

[0165] The term "antibody" as used in the present invention refers to an immunoglobulin, which is a four-peptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or called isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain according to the difference in the constant region. Each of the five classes of Ig can have κ chain or λ chain.

[0166] In the present invention, the antibody light chain described in the present invention may further comprise a light chain constant region, and the light chain constant region comprises human or murine κ, λ chain or its variant.

[0167] In the present invention, the antibody heavy chain described in the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or its variant.

[0168] The sequences of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly and are the variable regions (V regions); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions (C regions). The variable region includes 3 hypervariable regions (HVRs) and 4 relatively conserved framework regions (FRs). The 3 hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each variable region of the light chain (VL) and the variable region of the heavy chain (VH) consists of 3 CDR regions and 4 FR regions, and the order arranged from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment described in the present invention conform to the known Kabat numbering rules and the Kabat or ABM definition rules in terms of quantity and position.

[0169] The term "BCMA" includes any variant or isotype of BCMA expressed by cells. The antibodies or fragments thereof of the present invention can cross-react with BCMA obtained from non-human species. As an alternative, the antibody can also be specific for human BCMA and may not cross-react with BCMA of other species. BCMA or any of its variants or isotypes can be isolated from cells or tissues that naturally express them, or can be produced by recombinant techniques using techniques common in the art and those described herein. As an example, the anti-BCMA antibody or its fragment targets human BCMA with glycosylation modification.

[0170] "Specifically binds to BCMA" means that the antibody or fragment thereof of the present invention recognizes and binds to BCMA or its epitope.

[0171] The term "CD3" includes any variant or isotype of CD3 expressed by cells. The antibodies or fragments thereof of the present invention can cross-react with CD3 obtained from non-human species. As an alternative, the antibody can also be specific for human CD3 and may not cross-react with CD3 of other species. CD3 or any of its variants or isotypes can be isolated from cells or tissues that naturally express them, or can be produced by recombinant techniques using techniques common in the art and those described herein.

[0172] "Specifically binds to CD3" means that the antibody or fragment thereof of the present invention recognizes and binds to CD3 or its epitope.

[0173] The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, and the techniques and methods involved are well-known in the art, such as:

[0174] 1. Antibodies isolated from transgenic or transchromosomal animals (e.g., mice) carrying human immunoglobulin genes or from hybridomas prepared therefrom;

[0175] 2. Antibodies isolated from host cells (such as transfected tumors) transformed to express antibodies;

[0176] 3. Antibodies isolated from recombinant combinatorial human antibody libraries; and

[0177] 4. Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences, etc.

[0178] Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.

[0179] The term "murine antibody" in the present invention refers to monoclonal antibodies against human BCMA or its epitopes prepared according to knowledge and skills in the art. When preparing, a test subject is injected with BCMA antigen or its fragment, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a preferred embodiment of the present invention, the murine BCMA antibody or its antigen-binding fragment may further comprise a light chain constant region of murine κ, λ chain or its variant, and / or further comprise a heavy chain constant region of murine IgG1, IgG2, IgG3 or IgG4 or its variant.

[0180] The term "human antibody" includes antibodies having variable and constant regions with human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutations). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as mice) have been grafted onto a human framework sequence (i.e., "humanized antibodies").

[0181] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences of a non-human species into the framework of the variable region of a human antibody. Humanized antibodies can overcome the drawback of chimeric antibodies in inducing immune response reactions due to carrying a large amount of heterologous protein components. To avoid a decrease in activity while reducing immunogenicity, back mutations can be made to the variable region of the human antibody to maintain activity.

[0182] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, which can reduce the immune response induced by heterologous antibodies. As an example, to establish a chimeric antibody, a hybridoma secreting a murine-derived specific monoclonal antibody is first established, then the variable region gene is cloned from the murine hybridoma cells, and then the constant region gene of a human antibody is cloned as needed. The murine variable region gene and the human constant region gene are ligated into a chimeric gene and then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or their variants, preferably including the heavy chain constant regions of human IgG1, IgG2 or IgG4, or the IgG1 heavy chain constant region with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

[0183] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region of the parental antibody (e.g., one or more CDRs). Antibody fragments retain at least some of the binding specificities of the parental antibody. Generally, when activities are expressed on a molar basis, antibody fragments retain at least 10% of the parental binding activity. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody for the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.

[0184] A "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0185] The "Fc" region is two heavy chain fragments containing the CH2 and CH3 domains. The two heavy chain fragments are held together by one or more disulfide bonds.

[0186] A "Fab’ fragment" contains a light chain and a portion of a heavy chain (which includes the VH domain, CH1 domain, and the region between the CH1 and CH2 domains); an interchain disulfide bond can be formed between two Fab’ fragments to form an F(ab’)2 molecule.

[0187] The "Fv region" contains the variable regions of the heavy and light chains, but lacks the constant regions.

[0188] The terms "single-chain antibody", "single-chain Fv" or "scFv" refer to single-chain recombinant proteins formed by linking the variable region VH of the heavy chain and the variable region VL of the light chain of an antibody by a linker peptide, and are the smallest antibody fragments with complete antigen-binding sites. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 23) amino acid sequences or variants thereof, for example using 1-4 (including 1, 2, 3 or 4) repeated variants (Holliger et al. (1993), Proc Natl Acad Sci USA. 90: 6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur J Immuno. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J Mol Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol Immunother. 50: 51-59.

[0189] The term "domain antibody fragment" refers to an immunoglobulin fragment containing only the variable region of the heavy chain or the light chain and having immunological function. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.

[0190] The term "binding to BCMA" in the present invention means being able to interact with human BCMA.

[0191] The term "antigen-binding site" in the present invention refers to the three-dimensional spatial site recognized by the antibody or antigen-binding fragment of the present invention.

[0192] The term "epitope" refers to the site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent amino acids, or by non - adjacent amino acids juxtaposed through the tertiary folding of a protein. Epitopes formed by adjacent amino acids generally remain after exposure to denaturing solvents, while epitopes formed by tertiary folding generally are lost after treatment with denaturing solvents. Epitopes generally include at least 3 - 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well - known in the art and include immunoblotting and immunoprecipitation assays, etc. Methods for determining the spatial conformation of epitopes include techniques in the art and techniques described herein, such as X - ray crystallography and two - dimensional nuclear magnetic resonance, etc.

[0193] The terms "specifically binds" and "selectively binds" as used in the present invention refer to an antibody binding to an epitope on a predetermined antigen. Generally, when using human BCMA as an analyte and an antibody as a ligand, and measuring by surface plasmon resonance (SPR) technology in an instrument, the antibody binds to the predetermined antigen with an equilibrium dissociation constant (K -7 ) of about less than 10 D M or even smaller, and its affinity for binding to the predetermined antigen is at least twice its affinity for binding to a non - specific antigen (such as BSA, etc.) other than the predetermined antigen or closely related antigens. The term "antibody that recognizes an antigen" can be used interchangeably with the term "antibody that specifically binds" herein.

[0194] A "bispecific" antibody or "bifunctional" antibody is a hybrid antibody having two different epitope - binding sites. The two epitopes can be from the same antigen, or from different antigens. As an example, the two epitopes are from BCMA and CD3 respectively. Bispecific antibodies can be produced by well - known methods, including but not limited to hybridoma fusion or linking Fab′ fragments. See, for example, the methods disclosed in Songsivilai et al., Clin. Exp. Immunol., 79:315 - 321 (1990); Kostelny et al., J. Immunol., 148:1547 - 1553 (1992).

[0195] The term "cross - reactive" refers to the ability of the antibodies of the present invention to bind to BCMA from different species. For example, an antibody of the present invention that binds to human BCMA can also bind to BCMA of another species. Cross - reactivity is measured by detecting specific reactivity with a purified antigen, or binding or functional interaction with cells physiologically expressing BCMA in a binding assay (such as SPR and ELISA). Methods for determining cross - reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0196] The terms "inhibit" or "block" are used interchangeably and cover both partial and complete inhibition / blockade. Inhibition / blockade of a ligand preferably reduces or alters the normal level or type of activity that occurs upon ligand binding in the absence of inhibition or blockade. Inhibition and blockade are also intended to include any measurable decrease in ligand binding affinity upon contact with an anti-BCMA antibody compared to a ligand that has not been contacted with an anti-BCMA antibody.

[0197] The term "inhibit growth" (e.g., as it pertains to cells) is intended to include any measurable decrease in cell growth.

[0198] As used herein, the term "EC 50 " or "half maximal effective concentration" refers to the concentration of a molecule (e.g., an antibody or an antigen-binding fragment thereof in this application) that is able to induce a 50% maximal response after a specific exposure time. Methods for determining EC 50 are well known in the art. For example, a concentration-effect fitting curve can be plotted using software such as GraphPad Prism to calculate EC 50 .

[0199] As used in the present invention, "ADCC", namely antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc portion of the antibody. The ADCC effector function of an antibody can be enhanced, reduced, or eliminated by modification of the Fc portion on IgG. The said modification refers to mutation in the constant region of the heavy chain of the antibody.

[0200] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as in the Antibody Engineering Laboratory Manual of Cold Spring Harbor, Chapters 5-8 and 15. For example, mice can be immunized with human BCMA or a fragment thereof, and the resulting antibodies can be renatured, purified, and their amino acid sequences can be determined using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) database or from the Journal of Immunology, 2001 ISBN012441351.

[0201] The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. The cDNA sequences of the corresponding antibodies can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. The positive clones are expanded in a serum-free medium in a bioreactor to produce antibodies. The culture broth secreting the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0202] The antibodies of the present invention refer to monoclonal antibodies. The monoclonal antibodies (mAbs) described in the present invention refer to antibodies obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines. Monoclonal antibodies or antigen-binding fragments can be recombinantly obtained by, for example, hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other prior art.

[0203] "Administer", "give", and "treat", when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administer", "give", and "treat" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes the contact of a reagent with the cells, as well as the contact of the reagent with a fluid, where the fluid contacts the cells. "Administer", "give", and "treat" also mean treatment of cells in vitro and ex vivo by a reagent, diagnostic, binding composition, or by another cell. "Treat", when applied to human, veterinary medicine, or research subjects, refers to therapeutic treatment, preventive or prophylactic measures, research, and diagnostic applications.

[0204] "Treatment" means administering to a patient a therapeutic agent, either internally or externally, such as any antibody of the present invention, to a patient having one or more disease symptoms, wherein the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the treated patient or population in an amount effective to relieve one or more disease symptoms, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to relieve any specific disease symptom (also referred to as "therapeutically effective amount") may vary depending on various factors, such as the disease state, age and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical test method commonly used by a doctor or other professional healthcare provider to evaluate the severity or progression of the symptom. Although the embodiments of the present invention (e.g., treatment methods or products) may not be effective in relieving the target disease symptoms in each patient, according to any statistical test methods known in the art such as Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test, it should relieve the target disease symptoms in a statistically significant number of patients.

[0205] "Effective amount" includes an amount sufficient to ameliorate or prevent the symptoms of a medical condition. An effective amount also means an amount sufficient to permit or facilitate a diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the overall health of the patient, the method of administration, route and dosage, and the severity of side effects. The effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0206] As used herein, the term "lupus erythematosus" refers to a chronic autoimmune disease in which the immune system becomes overactive and attacks normal tissues. This attack triggers inflammation. Lupus erythematosus is an "organ-nonspecific" type of autoimmune disease.

[0207] As used herein, the term "IgA nephropathy" is characterized by the deposition of IgA on glomerular mesangial cells. IgA nephropathy belongs to proliferative glomerulonephritis caused by an immune response against glomerular mesangial cells.

[0208] As used herein, the term "rheumatoid arthritis" refers to an autoimmune disease in which the immune system abnormally attacks the joints or body parts of the self and causes inflammation, manifested by symptoms such as pain and swelling in joints such as hands, feet, wrists, and knees, and may also cause abnormal systemic chronic inflammatory diseases in organs such as muscles, skin, lungs, and eyes.

[0209] As used herein, the term "lymphoma" refers to a malignant tumor originating from the lymphohematopoietic system. The classification of lymphoma is carried out according to the criteria of the 2016 WHO Lymphoma Classification.

[0210] As used herein, the term "myeloma" (also known as plasmacytoma) is a malignant tumor originating from plasma cells in the bone marrow. The classification of myeloma is carried out according to the criteria of the WHO (2013) Classification of Bone Tumors.

[0211] "Exogenous" refers to a substance produced outside a living organism, cell, or human body according to the context.

[0212] "Endogenous" refers to a substance produced inside a living organism, cell, or human body according to the context.

[0213] "Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when two sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% identical. Generally, comparison is carried out when the maximum percentage of identity is obtained by aligning the two sequences.

[0214] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformant" and "transformed cell" include the primary test cells and the cultures derived therefrom, regardless of the number of passages. It should also be understood that due to deliberate or inadvertent mutations, all progeny may not be precisely identical in terms of DNA content. This includes mutant progeny having the same function or biological activity as that screened in the original transformed cells. When it is intended to refer to different situations, it is clear from the context.

[0215] "Optionally" or "optionally" means that the subsequently described event or circumstance may but does not have to occur, and this description includes the cases where the event or circumstance occurs or does not occur. For example, "optionally comprising 1 - 3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may but do not have to be present.

[0216] "Pharmaceutical composition" means a composition containing one or more antibodies or antigen - binding fragments thereof described herein, as well as other components such as a physiological / pharmaceutically acceptable carrier and excipient. The purpose of the pharmaceutical composition is to facilitate the administration to an organism, promote the absorption of the active ingredient, and thus exert its biological activity. Detailed implementation manners

[0218] The following embodiments are used to further describe the present invention, but these embodiments do not limit the scope of the present invention. For the experimental methods without specific conditions noted in the embodiments of the present invention, they are usually carried out under conventional conditions, such as the antibody technology experimental manual of Cold Spring Harbor, the molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. The reagents without specific sources noted are conventional reagents purchased from the market.

[0219] Example 1: Antigen preparation

[0220] The human BCMA (BCMA-His) protein encoded with a His tag was synthesized by SinoBiologics (Cat No.: 10620-H08H).

[0221] BCMA-His sequence:

[0222]

[0223] Example 2: Obtaining murine hybridomas and antibody sequences

[0224] Five Balb / c mice and five A / J mice, female, 10 weeks old, were immunized with the human antigen BCMA-His. Sigma complete Freund's adjuvant (CFA) and Sigma incomplete Freund's adjuvant (IFA) were used. The immunogen and the immunoadjuvant were fully mixed and emulsified at a ratio of 1:1 to form a stable "water-in-oil" liquid; the injection dose was 25 μg / 200 μL / mouse.

[0225] Table 1. Immunization protocol

[0226] Day 1 First immunization, complete Freund's adjuvant. Day 21 Second immunization, incomplete Freund's adjuvant. Day 35 Third immunization, incomplete Freund's adjuvant. Day 42 Blood collection and serum titer detection (blood samples collected after the third immunization) Day 49 Fourth immunization, incomplete Freund's adjuvant. Day 56 Blood collection and serum titer detection (blood samples collected after the fourth immunization)

[0227] The serum of the immunized mice was evaluated for serum titer and the ability to bind cell surface antigens using the indirect ELISA method described in Example 3. Cell fusion was carried out according to the control titer detection situation (greater than 100,000-fold dilution). Immunized mice with strong serum titer, affinity, and FACS binding were selected for a final immunization and then sacrificed. After fusing splenocytes and SP2 / 0 myeloma cells, hybridomas were obtained; target hybridomas were screened by indirect ELISA, and the cell lines were established as monoclonal cell lines by the limiting dilution method. The obtained positive antibody strains were further screened using indirect ELISA to select the hybridomas that bind to the recombinant protein. Logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TMReverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using the mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced, and finally the sequence of the murine antibody M1 was obtained.

[0228] The variable region sequences of the heavy and light chains of murine monoclonal antibody M1 are as follows:

[0229]

[0230] Table 2. CDR sequences of the variable regions of the heavy and light chains of murine monoclonal antibody M1

[0231] Name Sequence Number HCDR1 GYTFTNYVMH SEQ ID NO: 3 HCDR2 YIIPYNDGTKYNEKFKGKA SEQ ID NO: 4 HCDR3 TRLIFDGYYFDY SEQ ID NO: 5 LCDR1 RASKSVSTSGFSYMH SEQ ID NO: 6 LCDR2 SLASNLES SEQ ID NO: 7 LCDR3 QHSRELPWT SEQ ID NO: 8

[0232] Example 3: Method for detecting the in vitro binding activity of an antibody

[0233] 1. In vitro indirect ELISA binding experiment:

[0234] The BCMA His protein (Sino Biological Inc., cat#10428-H08H) was diluted to a concentration of 1 μg / ml with PBS at pH 7.4 and added to a 96-well high-affinity ELISA plate at a volume of 100 μl / well, and incubated overnight (16 - 20 hours) in a refrigerator at 4°C. After washing the plate 4 times with PBST (PBS at pH 7.4 containing 0.05% Tween-20), 150 μl / well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST was added and incubated at room temperature for 1 hour for blocking. After the blocking was completed, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

[0235] The antibody to be tested was diluted with PBST containing 3% BSA; starting from 1 μM, it was diluted in a 10-fold gradient to obtain 10 concentration gradients; 100 μl / well was added to the ELISA plate and incubated at room temperature for 1 hour. After the incubation was completed, the plate was washed 4 times with PBST, 100 μl / well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with PBST containing 3% BSA was added, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, 100 μl / well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S) was added and incubated at room temperature in the dark for 1 minute, and then 100 μl / well of stop solution (Cell Signaling Technology, cat#7002S) was added to terminate the reaction; the absorbance was read at 450 nm using an ELISA reader (BioTek, model Synergy H1) to analyze the data. A concentration-signal value curve was made to analyze the results. As shown in the following table:

[0236] Table 3. Affinity of murine antibodies for human BCMA antigen (EC 50 value)

[0237] Mouse antibody <![CDATA[Binding EC to human BCMA His antigen 50 (nM)]]> M1 0.111

[0238] 2. In vitro cell binding assay:

[0239] Collect cells highly expressing BCMA (HEK-293T cells overexpressing BCMA and tumor cells expressing BCMA, NCI-H929 myeloma cell line); after adjusting the cell density, seed the cells in a 96-well U-bottom plate at 1×10 5 to 2×10 5 cells per well. Centrifuge at 1200 g for 5 min, discard the supernatant; add 100 μl of diluted antibody solution or mouse immune serum, incubate at 4 °C for 60 min; centrifuge at 1200 g for 5 min, discard the supernatant; wash the cells twice with PBS, then add 100 μl of fluorescently labeled secondary antibody (PE-GAM or PE-GAH) per well, incubate at 4 °C for 60 min. Centrifuge at 1200 g for 5 min, discard the supernatant. After washing the cells twice with PBS, resuspend the cells in PBS; use a flow cytometer to detect the signal and generate a concentration curve to analyze the results.

[0240] Table 4. Affinity of murine antibodies for cells expressing BCMA (EC 50 value)

[0241]

[0242] Example 4: Humanization of murine antibodies

[0243] Humanize the murine anti-human BCMA monoclonal antibody by the method disclosed in many documents in the art. Briefly, replace the constant domain of the parental (murine antibody) with the human constant domain, and select the human antibody sequence according to the homology between the murine antibody and the human antibody. The murine antibody M1 of the present invention is humanized.

[0244] Based on the typical structure of the VH / VL CDR of the obtained murine antibody, compare the variable region sequences of the heavy and light chains with the human antibody germline database to obtain a human germline template with high homology.

[0245] Transfer the CDR regions of the murine antibody M1 to the selected humanized template. Then, based on the three-dimensional structure of the murine antibody, perform back mutations on the buried residues, residues that directly interact with the CDR regions, and residues that have an important impact on the conformation of VL and VH, and optimize the chemically unstable amino acid residues in the CDR regions. The sequences are as follows:

[0246] Table 5. Mutations in the CDR2 region

[0247] Name Sequence Number HCDR1 GYTFTNYVMH SEQ ID NO: 3 HCDR2 YIIPYNDGTKYNEKFKGRV SEQ ID NO: 9 HCDR3 TRLIFDGYYFDY SEQ ID NO: 5 LCDR1 RASKSVSTSGFSYMH SEQ ID NO: 6 LCDR2 SLASNLES SEQ ID NO: 7 LCDR3 QHSRELPWT SEQ ID NO: 8

[0248] Table 6. Mutations in the CDR2 region

[0249] Name Sequence Number HCDR1 GYTFTNYVMH SEQ ID NO: 3 HCDR2 YIIPYNDGTKYNEKFKGrKA SEQ ID NO: 4 HCDR3 TRLIFDGYYFDY SEQ ID NO: 5 LCDR1 RASKSVSTSGFSYMH SEQ ID NO: 6 LCDR2 YLASNLES SEQ ID NO: 10 LCDR3 QHSRELPWT SEQ ID NO: 8

[0250] Through the comparison of expression tests and the number of revertant mutations, the sequence of the humanized heavy chain variable region HCVR was selected, and the sequence is as follows:

[0251]

[0252]

[0253] The sequence of the humanized light chain variable region LCVR was selected, and the sequence is as follows:

[0254]

[0255] The designed heavy chain and light chain variable region sequences were respectively linked to the heavy chain constant region and light chain constant region sequences of human antibodies. Exemplarily, they were linked to the human IgG1 heavy chain constant region (SEQ ID NO: 31), the human IgG1 heavy chain constant region variant (SEQ ID NO: 42), and the human antibody κ chain constant region sequence (SEQ ID NO: 32). Exemplarily, the heavy chain and light chain sequences are as follows:

[0256]

[0257]

[0258]

[0259] Exemplary constant region sequences are shown as follows:

[0260] Human IgG1 heavy chain constant region

[0261] Human IgG1 heavy chain constant region variant

[0262]

[0263] Human antibody κ chain constant region

[0264]

[0265] Table 7. Sequence numbers of heavy chain, light chain, and variable regions

[0266]

[0267] cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of the above-mentioned humanized antibodies and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2 and incubated in a CO2 incubator for 4-5 days. The cell culture medium was collected, centrifuged and filtered, and then loaded onto an antibody purification affinity column. After washing the column with phosphate buffer, eluting with glycine hydrochloride buffer (pH 2.7 0.1M Gly-HCl), neutralizing with 1M Tris-HCl pH 9.0, and dialyzing with phosphate buffer, the humanized antibody of the present invention was obtained.

[0268] Example 5: In vitro binding affinity and kinetic experiments

[0269] Using the in vitro indirect ELISA binding experiment of Example 3, the affinity (EC 50 ) of each humanized antibody for the human BCMA antigen was determined as shown in the following table:

[0270] Table 8. Affinity (EC 50 ) of humanized antibodies for the human BCMA antigen

[0271]

[0272] Using the in vitro cell binding experiment of Example 3, the affinity (EC 50 ) of each humanized antibody for NCI-H929 tumor cells was determined as shown in the following table:

[0273] Table 9. Affinity (EC 50 ) of humanized antibodies for NCI-H929 tumor cells

[0274]

[0275] Example 6: Construction of bispecific antibodies

[0276] Antigen-binding fragment specifically binding to CD3: The CD3HCVR and CD3LCVR shown in the following sequences were ligated into a single-chain fragment through a linker sequence; the linker sequence is a linker well-known in the art, and an exemplary linker can be (GGGGS) n , where n is selected from 1, 2, 3, 4 or 5.

[0277]

[0278] The CDR sequences of the anti-CD3 antibody or its antigen-binding fragment are as follows:

[0279] Table 10. CDR Sequences

[0280] Name Sequence Number HCDR1 KYAMN SEQ ID NO: 35 HCDR2 RIRSKYNNYATYYADSVKD SEQ ID NO: 36 HCDR3 HGNFGNEYISYWAY SEQ ID NO: 37 LCDR1 GSSTGAVTSGNYPN SEQ ID NO: 38 LCDR2 GTKFLAP SEQ ID NO: 39 LCDR3 VLWYSNRWV SEQ ID NO: 40

[0281] The antigen-binding fragment that specifically binds to BCMA (the first binding region) and the antigen-binding fragment that specifically binds to CD3 (the second binding region) are linked by different methods to obtain a bispecific antibody containing the following sequences:

[0282] The Ab4 antibody is constructed as follows (in the order from the N-terminus to the C-terminus) (the structure is as shown in Figure 1 ):

[0283] The first chain, the heavy chain variable region and the heavy chain constant region of the first binding region;

[0284] The second chain, the light chain variable region, the light chain constant region and the peptide linker of the first binding region, and the heavy chain variable region, the peptide linker and the light chain variable region of the second binding region.

[0285] The Ab5 antibody is constructed as follows (in the order from the N-terminus to the C-terminus) (the structure is as shown in Figure 2 ):

[0286] The first polypeptide, containing the heavy chain variable region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;

[0287] The second polypeptide, containing the light chain variable region and the light chain constant region of the first binding region from the N-terminus to the C-terminus;

[0288] The third polypeptide, containing the heavy chain variable region, the peptide linker, the light chain variable region, the peptide linker and the heavy chain constant region of the second binding region from the N-terminus to the C-terminus.

[0289] In the above constructs, the peptide linker is a peptide linker for linking antigen-binding domains, and exemplary linker sequences can be (GGGGS)n, where n is selected from 1, 2, 3, 4 or 5.

[0290] In the above constructs, the heavy chain constant region can be selected from human antibody heavy chain constant regions (such as the human IgG1 heavy chain constant region) or their variants (such as variants with reduced ADCC activity). The heavy chain constant region can be a constant region (or constant region domain) with the same sequence, or a constant region (or constant region domain) containing the Knob and Hole spatial structures. It can also further introduce disulfide bonds on the basis of the sequence containing the Knob and Hole constant region (or constant region domain) to promote dimer formation.

[0291] The heavy chain constant region sequences contained in the exemplary bispecific antibodies of the present invention are as follows:

[0292]

[0293] The sequence of the constructed bispecific antibody is as follows:

[0294]

[0295]

[0296]

[0297] Table 11. Sequence numbers of the antibody and its heavy chain, light chain, and variable region

[0298]

[0299] According to the above amino acid sequence of the bispecific antibody, cDNA fragments were synthesized and inserted into the pcDNA3.1 expression vector (Life Technologies Cat.No.V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat.No.23966) were transfected into HEK293 cells (Life Technologies Cat.No.11625019) at a ratio of 1:2 and incubated in a CO2 incubator for 4 - 5 days. The cell culture medium was collected, centrifuged and filtered, and then loaded onto an antibody purification affinity column. After washing the column with phosphate buffer, eluting with glycine hydrochloride buffer (pH 2.7 0.1M Gly-HCl), neutralizing with 1M Tris-HCl pH 9.0, and dialyzing with phosphate buffer, the bispecific antibody of the present invention was obtained. The concentrations and SEC purities of the prepared bispecific antibodies are as follows:

[0300] Table 12. Concentrations and SEC purities of the bispecific antibodies

[0301] Bispecific antibody Concentration (mg / ml) SEC purity Ab4 0.506 100％ Ab5 2.33 90.12％

[0302] Example 7: In vitro binding affinity experiment

[0303] Using the in vitro indirect ELISA binding experiment of Example 3, the affinity (EC 50 ) of the bispecific antibody for the human BCMA antigen was measured, as shown in Table 13 below:

[0304] Table 13. Affinity (EC 50 ) of the bispecific antibody for the human BCMA antigen

[0305]

[0306] Dilute the CD3D / CD3E heterodimer protein (Acrobiosystems, cat#CDD-H82W0) to a concentration of 1 μg / ml with PBS at pH 7.4, and add it to a 96-well high-affinity ELISA plate at a volume of 100 μl / well. Incubate overnight (16 - 20 hours) in a refrigerator at 4°C. After washing the plate 4 times with PBST (PBS at pH 7.4 containing 0.05% Tween-20), add 150 μl / well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate at room temperature for 1 hour for blocking. After the blocking is completed, discard the blocking solution and wash the plate 4 times with PBST buffer. Dilute the antibody to be tested with PBST containing 3% BSA; start from 1 μM and dilute it in a 10-fold gradient to obtain 10 concentration gradients; add 100 μl / well to the ELISA plate and incubate at room temperature for 1 hour. After the incubation is completed, wash the plate 4 times with PBST, add 100 μl / well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with PBST containing 3% BSA, and incubate at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 μl / well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate in the dark at room temperature for 1 minute, add 100 μl / well of stop solution (Cell Signaling Technology, cat#7002S) to terminate the reaction, and read the absorbance at 450 nm with an ELISA reader (BioTek, model Synergy H1) to analyze the data. Make a concentration-signal value curve to analyze the results, as shown in the following table:

[0307] Table 14. Affinity of bispecific antibody for human CD3 (EC 50 )

[0308]

[0309] Example 8: Tumor cell killing effect mediated by bispecific antibody

[0310] The BCMA-binding part of the bispecific antibody binds to tumor cells expressing BCMA, and the CD3-binding part binds to the TCR receptor on the surface of effector T cells. An immunological synapse is formed under the guidance of the bispecific antibody, and the T cells lyse and kill the tumor cells through a series of cytokines such as granzyme and perforin.

[0311] Rapidly thaw the cryopreserved PBMCs in a 37°C water bath, transfer the cell suspension to complete cell culture medium (90% RPMI-1640 containing 10% FBS, 100 U / 100 μg / mL penicillin / streptomycin, 2 mM glutamine), resuspend the cells, centrifuge at 2000 rpm / min for 5 min at room temperature, and discard the supernatant; add fresh complete medium to resuspend the cell pellet; count the cells and adjust the cell density to 1.5x10 5 / mL, and use the EasySep Human T Cell Iso Kit (Stemcell, cat#: 17951) to isolate the total human T cell population; add 33.3 μl of the T cell suspension at 5x10 3 cells / well to a clear-bottom 96-well U-bottom plate (Corning, Cat#: 3799). Suspend NCI-H929-LUC (Cobioer product number CBP30061L) in complete cell culture medium, count the cells; adjust the cell density to 1.5x10 6 / mL. Add 33.3 μl of the NCI-H929-LUC suspension at 5x10 4 / well to the experimental plate containing the total human T cell population. Dilute the bispecific antibody 5-fold from the highest concentration of 3000 nM with complete medium to obtain 9 concentration gradients. Add 33.3 μl of each concentration of the antibody suspension to the experimental plate, mix the cells well, and co-incubate in a 37°C, 5% CO2 incubator for 48 hours. Then, equilibrate the experimental plate at room temperature for 10 minutes; at the same time, add 100 μl / well of the aliquoted ONE-Glo TM Luciferase Assay (Promega, Cat#: E6120) detection reagent to the experimental plate and incubate for 5 minutes. Transfer the cell lysate to the experimental plate (Corning, Cat#: 3610) (180 μl / well); read the signal value using a microplate reader (Bio-Tek, Synergy H1).

[0312] Calculate the killing efficiency according to the following formula:

[0313] Cell killing efficiency % = (1 - signal value of the experimental group / signal value of the control group) × 100%.

[0314] Use GraphPad Prism software to calculate the EC 50 value from the killing efficiency values, as shown in Table 15 below:

[0315] Table 15. Killing of NCI-H929-LUC cells by bispecific antibody-mediated T cells (EC 50 )

[0316]

[0317] Example 9: Tumor killing mediated by bispecific antibody

[0318] NCG mice are immunodeficient mice lacking T, B, and NK immune cells. Female NCG mice aged 8 - 9 weeks and weighing 18 - 22 g were selected. Through intraperitoneal injection, 4x10 6 human PBMC cells were injected into each mouse. On the 3rd day, 1x10 7 NCI-H929 cells were injected subcutaneously into the back of the mice. When the tumor volume grew to about 170 mm 3 ³, the mice were divided into 3 groups with 8 mice in each group. The mice were given IgG1 control antibody or bispecific antibody by intraperitoneal injection at a dose of 20 μg / kg body weight every 3 days. After 14 days, the tumor volume of each mouse was measured and the tumor growth inhibition rate TGI was calculated as shown in the following table.

[0319] Tumor growth inhibition rate TGI = 100% - (tumor volume of the drug-administered group on the 14th day - tumor volume of the drug-administered group on the 0th day) / (tumor volume of the control group on the 14th day - tumor volume of the control group on the 0th day) × 100%.

[0320] Table 16. Inhibition of tumor growth by bispecific antibody

[0321] Dosing group Tumor growth inhibition rate TGI Ab4 90％ Ab5 20％

[0322] Female NCG mice aged 8 - 9 weeks and weighing 18 - 22 g were selected. Through intraperitoneal injection, 5x10 6 human PBMC cells were injected into each mouse, and at the same time, 1x10 7 RPMI-8226 cells were injected subcutaneously into the back of the mice. When the tumor volume grew to about 95 mm³, the mice were divided into 3 groups with 8 mice in each group. The mice were given IgG1 control antibody or bispecific antibody by intraperitoneal injection at a dose of 20 μg / kg body weight every 3 days. After 21 days, the tumor volume of each mouse was measured and the tumor growth inhibition rate TGI was calculated as shown in the following table.

[0323] Tumor growth inhibition rate TGI = 100% - (tumor volume of the drug-administered group on the 21st day - tumor volume of the drug-administered group on the 0th day) / (tumor volume of the control group on the 21st day - tumor volume of the control group on the 0th day) × 100%.

[0324] Table 17. Inhibition of tumor growth by bispecific antibody

[0325] Dosing group Tumor growth inhibition rate TGI Ab4 46％ Ab5 22％

[0326] Example 10: Affinity test of BCMA / CD3 bispecific antibody at the cellular level

[0327] The purpose of this example is to evaluate the differences in the affinity level of the BCMA-binding portion of the candidate BCMA / CD3 bispecific antibody for the BCMA antigen on the surface of tumor cells.

[0328] Centrifuge the cell suspension of NCI-H929 (ATCC, CRL-9068 TM ) in the logarithmic growth phase at 300 g for 5 min; add 5 ml of 2% FBS buffer to resuspend the cells, and adjust the cell density to 1x10 6 cells / ml. Aliquot 100 μL / well into a 96-well V-bottom plate, centrifuge at 300 g × 5 min at 4°C, discard the supernatant, add 100 μL / well of the candidate antibody solution diluted in 10 concentration gradients (from 2500 nM to 0.064 nM, 5-fold dilution gradient), and incubate at 4°C for 1 h; centrifuge at 300 g × 5 min at 4°C, wash twice, add 100 μL / well of goat anti-human IgG Fc, FITC-labeled secondary antibody (abcam, cat: 97224) solution diluted at a ratio of 5 μL / 10 6 cells, and incubate at 4°C for 1 h; centrifuge at 300 g × 5 min at 4°C, wash twice, add 70 μl of 2% FBS solution to resuspend the cells, and detect the mean fluorescence intensity (MFI) in the PE channel using a flow cytometer (Bio-Rad, ZE5), and analyze the EC 50 concentration of the antibody binding to the cells.

[0329] Table 18. Affinity of Bispecific Antibody for Cell Surface Antigen

[0330]

[0331] Example 11: In Vitro Tumor Cell Killing Mediated by BCMA / CD3 Bispecific Antibody

[0332] Preparation of effector cell human total T cell population: Rapidly thaw the cryopreserved PBMC in a 37°C water bath, transfer the cell suspension to complete cell medium (90% RPMI-1640 containing 10% FBS, 100 U / 100 μg / mL penicillin / streptomycin, 2 mM L-glutamine), resuspend the cells, and centrifuge at 400 g / min at room temperature for 5 min; discard the supernatant, add fresh complete medium; resuspend the cell pellet, count, and adjust the cell density to 1x10 7 / mL; use the EasySep Human T Cell Iso Kit (Stemcell, cat#: 17951) to isolate the human total T cell population according to the operating instructions, count the cells, and adjust the cell density to 1.2x10 6 / mL.

[0333] Prepare a suspension of target cells, human RPMI-8226 (ATCC, cat: CCL-155), and adjust the cell density to 1.2x10 5 / mL; Add 50 μl of each of the two cell suspensions to each well of a fully transparent 96-well U-bottom plate (Corning, Cat#: 3799). Dilute the BCMA / CD3 bispecific antibody 10-fold with complete medium to obtain 9 concentration gradients (starting from the highest concentration of 300 nM); complete medium serves as the negative control; Add 50 μl / well of the antibody suspension, and mix the cells. Incubate for 48 h in a 37 °C, 5% CO2 incubator, then equilibrate the experimental plate at room temperature for 10 min; Add Cell viability detection reagent (Promega, Cat#: G7573), shake and lyse in the dark at 350 rpm / min for 5 min; Transfer the cell lysate to a white-bottom transparent experimental plate (Corning, Cat#: 3610) at 180 μL / well, centrifuge at 210 g / min for 1 min, and read the signal value using a microplate reader (Bio-Tek, Synergy H1).

[0334] Calculate the killing efficiency according to the following formula:

[0335] Cell killing efficiency % = (1 - signal value of experimental group / signal value of control group) × 100

[0336] Using GraphPad Prism software, for the dose-effect inhibition formula:

[0337] log(inhibitor) vs effect - variable slope (four parameters)

[0338] (log(inhibitor) vs. response - Variable slope (four parameters)) for curve fitting to obtain the IC 50 value. The results are shown in the following table.

[0339] Table 19. Killing of RPMI-8226 cells by T cells mediated by BCMA / CD3 bispecific antibody

[0340] Sequence Listing <110> Shanghai Hansoh Pharmaceutical Co., Ltd. Jiangsu Hansoh Pharmaceutical Group Co., Ltd. <120> Specific antigen-binding molecule, its preparation method and medical use <130> 721033CPCT <150> 202010307360.1 <151> 2020-04-17 <150> 202110119870.0 <151> 2021-01-28 <160> 43 <170> SIPOSequenceListing 1.0 <210> 1 <211> 119 <212> PRT <213> Mus musculus <400> 1 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 2 <211> 111 <212> PRT <213> Mus musculus <400> 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Val 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 Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Ser 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 Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 10 <212> PRT <213> Mus musculus <400> 3 Gly Tyr Thr Phe Thr Asn Tyr Val Met His 1 5 10 <210> 4 <211> 19 <212> PRT <213> Mus musculus <400> 4 Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly Lys Ala <210> 5 <211> 12 <212> PRT <213> Mus musculus <400> 5 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr 1 5 10 <210> 6 <211> 15 <212> PRT <213> Mus musculus <400> 6 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Phe Ser Tyr Met His 1 5 10 15 <210> 7 <211> 8 <212> PRT <213> Mus musculus <400> 7 Ser Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Mus musculus <400> 8 Gln His Ser Arg Glu Leu Pro Trp Thr 1 5 <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR2 <400> 9 Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly Arg Val <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR2 <400> 10 Tyr Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 11 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCVR <400> 11 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 12 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCVR <400> 12 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 13 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCVR <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 14 <211> 111 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCVR <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 15 <211> 111 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCVR <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Ser 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 Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 16 <211> 111 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCVR <400> 16 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Ser 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 Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 17 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HC <400> 17 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 18 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HC <400> 18 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 19 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HC <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 20 <211> 218 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LC or Ab5-2 <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 21 <211> 218 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LC <400> 21 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Ser 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 Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 22 <211> 218 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LC <400> 22 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Ser 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 Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Linker <400> 23 Gly Gly Gly Gly Ser 1 5 <210> 24 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCVR <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Glu Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 25 <211> 109 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCVR <400> 25 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 26 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ab4-1 <400> 26 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 27 <211> 482 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ab4-2 <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 225 230 235 240 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala 245 250 255 Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Met Asn Trp Val Arg Gln 260 265 270 Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr 275 280 285 Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr 290 295 300 Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn 305 310 315 320 Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn 325 330 335 Phe Gly Asn Glu Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr 340 345 350 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 355 360 365 Gly Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr 370 375 380 Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly 385 390 395 400 Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly 405 410 415 Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly 420 425 430 Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu 435 440 445 Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val 450 455 460 Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 465 470 475 480 Val Leu <210> 28 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ab5-1 <400> 28 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 29 <211> 486 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ab5-3 <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Glu Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185 190 Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 260 265 270 Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 275 280 285 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 290 295 300 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 305 310 315 320 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 325 330 335 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 340 345 350 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 355 360 365 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 370 375 380 Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 385 390 395 400 Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile 405 410 415 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 420 425 430 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys 435 440 445 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 450 455 460 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 465 470 475 480 Ser Leu Ser Pro Gly Lys 485 <210> 30 <211> 65 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> BCMA-His Antigen <400> 30 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala His His His His His His His His His His 50 55 60 His 65 <210> 31 <211> 330 <212> PRT <213> Homo sapiens <400> 31 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 32 <211> 107 <212> PRT <213> Homo sapiens <400> 32 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 33 <211> 330 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> CH-Knob <400> 33 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 34 <211> 227 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> CH-Hole <400> 34 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR <400> 35 Lys Tyr Ala Met Asn 1 5 <210> 36 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR <400> 36 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 37 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR <400> 37 His Gly Asn Phe Gly Asn Glu Tyr Ile Ser Tyr Trp Ala Tyr 1 5 10 <210> 38 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR <400> 38 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 1 5 10 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR <400> 39 Gly Thr Lys Phe Leu Ala Pro 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR <400> 40 Val Leu Trp Tyr Ser Asn Arg Trp Val 1 5 <210> 41 <211> 249 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> scFv <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Glu Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185 190 Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 42 <211> 330 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <223> Heavy chain constant region variant <400> 42 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 43 <211> 449 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HC <400> 43 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Ile Phe Asp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys

Claims

1. A bispecific antibody or an antigen-binding fragment thereof, comprising: a first binding region that specifically binds to BCMA, and a second binding region that specifically binds to CD3; the first binding region is an anti-BCMA antibody or an antigen-binding fragment thereof, and the second binding region is an anti-CD3 antibody or an antigen-binding fragment thereof; wherein, the anti-BCMA antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; the light-chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8; or, the heavy-chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9, and HCDR3 shown in SEQ ID NO: 5; the light-chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8; or, the heavy-chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; the light-chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8; the anti-CD3 antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37; the light-chain variable region comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO:

40.

2. The bispecific antibody or an antigen-binding fragment thereof according to claim 1, wherein the first binding region comprises: a heavy-chain variable region shown in SEQ ID NO: 11 and a light-chain variable region shown in SEQ ID NO: 14; or, a heavy-chain variable region shown in SEQ ID NO: 12 and a light-chain variable region shown in SEQ ID NO: 15; or, a heavy-chain variable region shown in SEQ ID NO: 11 and a light-chain variable region shown in SEQ ID NO: 16; or, a heavy-chain variable region shown in SEQ ID NO: 13 and a light-chain variable region shown in SEQ ID NO:

16.

3. The bispecific antibody or antigen-binding fragment thereof according to claim 2, wherein the first binding region further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

4. The bispecific antibody or antigen-binding fragment thereof according to claim 3, wherein the variant of the heavy chain constant region of human IgG1 has reduced ADCC toxicity compared to the heavy chain constant region of human IgG1.

5. The bispecific antibody or antigen-binding fragment thereof according to claim 2, wherein the first binding region further comprises a heavy chain constant region as shown in SEQ ID NO: 31, or a variant of the heavy chain constant region as shown in SEQ ID NO: 42, or a variant of the heavy chain constant region as shown in SEQ ID NO: 33, or a variant of the heavy chain constant region as shown in SEQ ID NO:

34.

6. The bispecific antibody or antigen-binding fragment thereof according to claim 4, wherein the first binding region further comprises a light chain constant region of human antibody kappa chain or lambda chain or a variant thereof.

7. The bispecific antibody or antigen-binding fragment thereof according to claim 2, wherein the first binding region further comprises a light chain constant region as shown in SEQ ID NO:

32.

8. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the first binding region comprises: a heavy chain as shown in SEQ ID NO: 17 and a light chain as shown in SEQ ID NO: 20; or, a heavy chain as shown in SEQ ID NO: 18 and a light chain as shown in SEQ ID NO: 21; or, a heavy chain as shown in SEQ ID NO: 19 and a light chain as shown in SEQ ID NO: 22; or, a heavy chain as shown in SEQ ID NO: 43 and a light chain as shown in SEQ ID NO: 22; or, a heavy chain as shown in SEQ ID NO: 43 and a light chain as shown in SEQ ID NO:

20.

9. The bispecific antibody or antigen-binding fragment thereof according to claim 2, wherein the second binding region comprises a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO:

25.

10. The bispecific antibody or antigen-binding fragment thereof according to claim 9, wherein the heavy chain variable region and the light chain variable region of the second binding region are linked by a peptide linker.

11. The bispecific antibody or antigen-binding fragment thereof according to claim 10, wherein the second binding region comprises a sequence as shown in SEQ ID NO:

41.

12. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-11, which comprises two first chains and two second chains, wherein: The first chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the first binding region and a heavy chain constant region of the first binding region; The second chain comprises, from the N-terminus to the C-terminus, a light chain variable region of the first binding region, a light chain constant region of the first binding region and a first peptide linker, and a heavy chain variable region of the second binding region, a second peptide linker and a light chain variable region of the second binding region; The first peptide linker and the second peptide linker are the same or different.

13. The bispecific antibody or antigen-binding fragment thereof according to claim 12, wherein the peptide linker is selected from (GGGGS)n, and n is selected from 1, 2, 3, 4 or 5.

14. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising: a first polypeptide comprising, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of a first binding region; a second polypeptide comprising, from N-terminus to C-terminus, a light chain variable region of the first binding region and a light chain constant region of the first binding region; a third polypeptide comprising, from N-terminus to C-terminus, the heavy chain variable region of the second binding region, the first peptide linker, the light chain variable region of the second binding region, the second peptide linker, and the heavy chain constant region; The first peptide linker and the second peptide linker are the same or different.

15. The bispecific antibody or antigen-binding fragment thereof according to claim 14, wherein the peptide linker is selected from (GGGGS)n, and n is selected from 1, 2, 3, 4 or 5.

16. The bispecific antibody or antigen-binding fragment thereof of claim 15, wherein the heavy chain constant region of the first polypeptide comprises a heavy chain constant region of human IgG, the heavy chain constant region of the third polypeptide comprises a heavy chain constant region domain of human IgG, and the heavy chain constant region domain of human IgG comprises, from N-terminus to C-terminus: a hinge region, CH2, and CH3.

17. The bispecific antibody or antigen-binding fragment thereof of claim 16, wherein the first polypeptide and the third polypeptide further comprise one or more cysteine residues outside the hinge region, wherein the cysteine residues are located at a position or combination selected from the group consisting of: 349, 354. The bispecific antibody or antigen-binding fragment thereof according to claim 17 , wherein the first polypeptide and the third polypeptide form one or more disulfide bridges through the cysteine residues.

19. The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 18, wherein the heavy chain constant regions of the first polypeptide and the third polypeptide respectively comprise a knob domain and a hole domain, the knob domain and the hole domain forming a heterodimer, and the knob domain comprises a tryptophan residue at the following position: 366, and the hole domain comprises any one or a combination of the following: a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407.

20. The bispecific antibody or antigen-binding fragment thereof according to claim 14, wherein the heavy chain constant region of the first polypeptide comprises the sequence shown in SEQ ID NO: 33, and the heavy chain constant region of the third polypeptide comprises the sequence shown in SEQ ID NO:

34.

21. The bispecific antibody or antigen-binding fragment thereof according to claim 1, comprising two polypeptides represented by SEQ ID NO: 26 and two polypeptides represented by SEQ ID NO:

27.

22. The bispecific antibody or antigen-binding fragment thereof according to claim 1, comprising the sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO:

20.

23. An anti-BCMA antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8, or, the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9, and HCDR3 shown in SEQ ID NO: 5; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8, or, the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO:

8.

24. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, which is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

25. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 24, which further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

26. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 25, wherein the variant of the heavy chain constant region of human IgG1 has reduced ADCC toxicity compared to the wild-type IgG1 heavy chain constant region.

27. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, further comprising a heavy chain constant region shown in SEQ ID NO: 31, or a variant of the heavy chain constant region shown in SEQ ID NO:

42.

28. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 26, which further comprises a light chain constant region of human antibody kappa chain, lambda chain or a variant thereof.

29. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 27, further comprising a light chain constant region shown in SEQ ID NO:

32.

30. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, which comprises: a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or, a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

31. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, comprising: a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 75% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or, a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 75% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

32. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, comprising: a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 80% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or, a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 80% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

33. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, comprising: a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 85% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or, a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 85% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

34. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, comprising: a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 90% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or, a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 90% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

35. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, comprising: a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 95% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or, a light chain variable region selected from those shown in the following sequences, or a light chain variable region having at least 95% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

36. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, comprising: a heavy chain variable region selected from those shown in the following sequences, or a heavy chain variable region having at least 99% identity compared to the following sequences: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and / or, a light chain variable region selected from those shown in the following sequences, or a light chain variable region having at least 99% identity compared to the following sequences: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

37. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, containing: a heavy chain selected from those shown in the following sequences, or a heavy chain having at least 80% identity compared to the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 43; and / or, a light chain selected from those shown in the following sequences, or a light chain having at least 80% identity compared to the following sequences: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO:

22.

38. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, containing: a heavy chain selected from those shown in the following sequences, or a heavy chain having at least 85% identity compared to the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 43; and / or, a light chain selected from those shown in the following sequences, or a light chain having at least 85% identity compared to the following sequences: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO:

22.

39. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, containing: a heavy chain selected from those shown in the following sequences, or a heavy chain having at least 90% identity compared to the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 43; and / or, a light chain selected from those shown in the following sequences, or a light chain having at least 90% identity compared to the following sequences: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO:

22.

40. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, containing: A heavy chain selected from the sequences shown below or a heavy chain having at least 95% identity compared to the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 43; and / or, a light chain selected from the sequences shown below, or a light chain having at least 95% identity compared to the following sequences: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO:

22.

41. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 23, comprising: A heavy chain selected from the sequences shown below or a heavy chain having at least 99% identity compared to the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 43; and / or, a light chain selected from the sequences shown below, or a light chain having at least 99% identity compared to the following sequences: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO:

22.

42. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 30, comprising: The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14; or, The heavy chain variable region shown in SEQ ID NO: 12 and the light chain variable region shown in SEQ ID NO: 15; or, The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 16; or, The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO:

16.

43. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 37, comprising: The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20; or, The heavy chain shown in SEQ ID NO: 18 and the light chain shown in SEQ ID NO: 21; or, The heavy chain shown in SEQ ID NO: 19 and the light chain shown in SEQ ID NO: 22; or, The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 22; or, The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO:

20.

44. A polynucleotide encoding: The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-22, or the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 23-43.

45. An expression vector containing the polynucleotide according to claim 44.

46. A host cell transfected with or containing: The expression vector according to claim 45, The host cell is selected from bacteria, yeast or mammalian cells.

47. The host cell according to claim 46, wherein the host cell is selected from Escherichia coli, Pichia pastoris or CHO cells or HEK293 cells.

48. A method for producing an anti-BCMA antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, comprising the steps of: culturing the host cell according to any one of claims 46-47; isolating the antibody or its antigen-binding fragment from the culture; and purifying the antibody or its antigen-binding fragment.

49. A pharmaceutical composition comprising: the bispecific antibody or its antigen-binding fragment according to any one of claims 1-22, or the anti-BCMA antibody or its antigen-binding fragment according to any one of claims 23-43; and a diluent or carrier.

50. A pharmaceutical composition comprising: the bispecific antibody or its antigen-binding fragment according to any one of claims 1-22, or the anti-BCMA antibody or its antigen-binding fragment according to any one of claims 23-43; and a pharmaceutically acceptable excipient.

51. Use of the bispecific antibody or its antigen-binding fragment according to any one of claims 1-22, or the anti-BCMA antibody or its antigen-binding fragment according to any one of claims 23-43, or the pharmaceutical composition according to claim 49, in the preparation of a medicament for treating or preventing myeloma.

Images