Anti-BCMA antibodies, antigen-binding fragments thereof, and their medical uses

Abstract

Anti-BCMA antibodies, antigen-binding fragments thereof, and medical uses thereof are provided. Furthermore, chimeric and humanized antibodies containing the CDR regions of the anti-BCMA antibodies, pharmaceutical compositions containing the anti-BCMA antibodies or antigen-binding fragments thereof, and their uses as anti-cancer agents and for treating autoimmune diseases are also provided. Specifically, humanized anti-BCMA antibodies, their use in the preparation of drugs for treating BCMA-mediated diseases or conditions, and their use in the detection and diagnosis of diseases are provided.

Description

[Technical field]

[0001] The present disclosure relates to anti-BCMA antibodies, antigen-binding fragments thereof, and medical uses. Further, the present disclosure relates to anti-BCMA antibodies and antigen-binding fragments thereof, and pharmaceutical compositions comprising same for use in the diagnosis or treatment of BCMA-mediated diseases or conditions. [Background technology]

[0002] B cells are lymphocytes that play a key role in adaptive humoral immunity and the production of antibodies that specifically recognize antigens. The three subtypes of B cells are naive B cells, immune memory B cells, and plasma cells. In the process of VDJ recombination, two or three fragments of DNA selected from a genomic library are recombined to generate a combinatorial sequence of antibody variable domains. This allows the creation of up to 10 9 Unique B cell lineages of the B cell lineage are generated by further modifying the variable domains encoded by B cells of different lineages, resulting in antibodies specific for unique targets. B cells are involved in many diseases. Malignant transformation of B cells leads to cancers, including several lymphomas such as multiple myeloma and Hodgkin's lymphoma. B cells are also involved in autoimmune diseases such as systemic lupus erythematosus (SLE) and IgA nephropathy. B cell-related cancers and autoimmune diseases can be considered as abnormalities in B cell function. Therefore, a possible strategy to control such diseases is to use antibodies that target pathological B cells.

[0003] BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily and is a non-glycosylated endogenous membrane receptor for the ligands BAFF (B cell activating factor) and APRIL (proliferation-inducing ligand). BCMA and its associated ligands can regulate various functions of humoral immunity, B cell development, and homeostasis. BCMA is detected in the spleen, lymph nodes, thymus, adrenal gland, and liver. It is expressed by human plasmablasts, plasma cells from tonsils, spleen, and bone marrow, as well as tonsillar immune memory B cells and germinal center B cells, and various analyses of B cell lineages have shown that BCMA expression levels increase after maturation. BCMA is highly expressed in B cell lymphomas and multiple myeloma.

[0004] Antibodies against BCMA have been described, for example, in Gras MP. et al., Int Immunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949, and WO2012163805. Antibodies against BCMA and their use to treat lymphoma and multiple myeloma have been described, for example, in WO2002066516 and WO2010104949. WO2013154760 relates to a chimeric antigen receptor comprising a portion which recognizes BCMA and a portion which activates T cells.

[0005] US9273141 provides an internalizable antigen binding protein that specifically binds to BCMA and inhibits the binding of BAFF and / or APRIL to BCMA, as well as a conjugate comprising the antigen binding protein and a cytotoxin.

[0006] The present disclosure provides anti-BCMA antibodies with high affinity, high specificity, low immunogenicity, high biological activity, significant anti-tumor efficacy, and enhanced plasma membrane penetrating properties, as well as cytotoxic conjugates comprising the antibodies and their use in tumor inhibition. Summary of the Invention

[0007] According to some embodiments of the present disclosure, there is provided an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof, comprising: an antibody heavy chain variable region comprising at least one HCDR represented by a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; and An antibody light chain variable region comprising at least one LCDR represented by a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0008] In a preferred embodiment of the disclosure, the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises: HCDR1 represented by SEQ ID NO: 3, HCDR2 represented by SEQ ID NO: 4, and HCDR3 represented by sequence number:5.

[0009] In a preferred embodiment of the disclosure, the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises: LCDR1 represented by SEQ ID NO: 6, LCDR2 represented by SEQ ID NO: 7, and LCDR3 represented by sequence number:8.

[0010] In a preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is HCDR1 represented by SEQ ID NO: 3, HCDR2 represented by SEQ ID NO: 4, and HCDR3 represented by SEQ ID NO:5 Also includes The light chain variable region comprises: LCDR1 represented by SEQ ID NO: 6, LCDR2 represented by SEQ ID NO: 7, and LCDR3 represented by SEQ ID NO: 8 Includes.

[0011] According to some embodiments of the present disclosure, there is provided an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof, the anti-BCMA antibody or antigen-binding fragment thereof comprising at least one HCDR selected from SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or variants thereof.

[0012] According to some embodiments of the present disclosure, there is provided an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof, the anti-BCMA antibody or antigen-binding fragment thereof comprising at least one LDCR selected from SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or variants thereof.

[0013] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises: HCDR1 represented by SEQ ID NO: 3 or a mutant sequence thereof; HCDR2 represented by SEQ ID NO: 4 or a mutant sequence thereof, and HCDR3 represented by SEQ ID NO:5 or a mutant sequence thereof The heavy chain variable region comprises:

[0014] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises: LCDR1 as shown in SEQ ID NO: 6 or a mutant sequence thereof; LCDR2 as set forth in SEQ ID NO: 7 or a mutant sequence thereof; and LCDR3 as shown in SEQ ID NO: 8 or a mutant sequence thereof Includes.

[0015] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is HCDR1 represented by the mutant sequence of SEQ ID NO: 3, HCDR2 represented by the mutant sequence of SEQ ID NO: 4, and HCDR3 represented by the mutant sequence of SEQ ID NO:5 Also includes The light chain variable region comprises: LCDR1 represented by the mutant sequence of SEQ ID NO: 6, LCDR2 represented by the mutant sequence of SEQ ID NO: 7, and LCDR3 represented by the mutant sequence of SEQ ID NO: 8 Includes.

[0016] In a preferred embodiment of the disclosure, there is provided an anti-BCMA antibody or antigen-binding fragment thereof as described above, or a pharma- ceutically acceptable salt thereof, wherein the variant sequence has one to three amino acid mutations in the CDR regions that optimize the activity and stability or reduce immunogenicity of the antibody.

[0017] In a preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof is a murine antibody.

[0018] In a preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof is a chimeric antibody.

[0019] In a preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof is a human antibody.

[0020] In a preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof is a humanized antibody.

[0021] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1 or variant thereof, IgG2 or variant thereof, IgG3 or variant thereof, or IgG4 or variant thereof.

[0022] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, or IgG4.

[0023] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of IgG1 having enhanced ADCC toxicity following amino acid mutation.

[0024] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as set forth in SEQ ID NO:22.

[0025] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain or variant thereof, a lambda chain or variant thereof.

[0026] In a preferred embodiment of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region as set forth in SEQ ID NO:23.

[0027] In further preferred embodiments of the anti-BCMA antibody, or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody, or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof comprises a heavy chain variable region represented by a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0028] In further preferred embodiments of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0029] In further preferred embodiments of the anti-BCMA antibody or antigen-binding fragment thereof according to the present disclosure, or a pharma- ceutically acceptable salt thereof, the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region represented by a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0030] In further preferred embodiments of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

[0031] In further preferred embodiments of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain represented by SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0032] In further preferred embodiments of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0033] In further preferred embodiments of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain represented by SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0034] In further preferred embodiments of the anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0035] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:9 and a light chain variable region set forth in SEQ ID NO:12.

[0036] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from SEQ ID NO:9 or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain variable region is selected from SEQ ID NO:12 or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0037] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:10 and a light chain variable region set forth in SEQ ID NO:13.

[0038] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from SEQ ID NO:10 or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain variable region is selected from SEQ ID NO:13 or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0039] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:11 and a light chain variable region set forth in SEQ ID NO:14.

[0040] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from SEQ ID NO:11 or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain variable region is selected from SEQ ID NO:14 or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0041] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain represented by SEQ ID NO:15 and a light chain represented by SEQ ID NO:18.

[0042] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain is selected from SEQ ID NO:15 or has at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain is selected from SEQ ID NO:18 or has at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0043] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain represented by SEQ ID NO:16 and a light chain represented by SEQ ID NO:19.

[0044] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain is selected from SEQ ID NO:16 or has at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain is selected from SEQ ID NO:19 or has at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0045] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain represented by SEQ ID NO:17 and a light chain represented by SEQ ID NO:20.

[0046] In a more preferred embodiment of the disclosure, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain is selected from SEQ ID NO:17 or has at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain is selected from SEQ ID NO:20 or has at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0047] In yet another embodiment of the present disclosure, there is provided a polynucleotide encoding an anti-BCMA antibody or antigen-binding fragment thereof according to the present disclosure.

[0048] In yet another embodiment of the present disclosure, there is provided an expression vector containing a polynucleotide according to the present disclosure.

[0049] In yet another embodiment of the present disclosure, a host cell is provided that is transfected with or contains an expression vector according to the present disclosure.

[0050] In yet another embodiment of the present disclosure, a host cell transformed with an expression vector according to the present disclosure is provided.

[0051] In a preferred embodiment of the present disclosure, said host cell is a bacterium, preferably E. coli.

[0052] In a preferred embodiment of the present disclosure, the host cell is a yeast, preferably Pichia pastoris.

[0053] In a preferred embodiment of the present disclosure, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.

[0054] In another aspect, the disclosure provides a method for producing an anti-BCMA antibody comprising the steps of: Culturing a host cell according to the present disclosure; isolating the antibody from the culture; and Purifying the antibody.

[0055] In the methods for producing anti-BCMA antibodies provided by the present disclosure, culturing the host cells according to the present disclosure preferably comprises culturing HEK293 cells; isolating the antibody from the culture preferably comprises isolating from the cell culture medium; and purifying the antibody by affinity chromatography, preferably by chromatographic methods.

[0056] In yet another embodiment of the present disclosure, there is provided a pharmaceutical composition comprising said anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof according to the present disclosure, and a pharma- ceutical acceptable excipient, diluent or carrier.

[0057] In yet another embodiment of the present disclosure, there is provided a detection or diagnostic reagent comprising an anti-BCMA antibody or antigen-binding fragment thereof according to the present disclosure, or a pharma- ceutical acceptable salt thereof.

[0058] In yet another embodiment of the present disclosure, there is provided a detection or diagnostic kit containing an anti-BCMA antibody or antigen-binding fragment thereof according to the present disclosure, or a pharma- ceutically acceptable salt thereof, as well as a labeled secondary antibody, buffers and substrates useful for detection or diagnosis.

[0059] In a further embodiment of the present disclosure, there is provided a detection or diagnostic kit containing an anti-BCMA antibody or antigen-binding fragment thereof according to the present disclosure, or a pharma- ceutically acceptable salt thereof, and one or more reagents capable of detecting binding of the BCMA antibody or antigen-binding fragment thereof to BCMA.

[0060] In another aspect, the disclosure provides an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical composition comprising same according to the disclosure, for use in treating or preventing a BCMA-mediated disease or condition.

[0061] In another aspect, the disclosure provides the use of an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical composition according to the disclosure comprising same, in the preparation of a medicament for treating or preventing a BCMA-mediated disease or condition.

[0062] In another aspect, the disclosure provides an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof, or a detection or diagnostic reagent according to the disclosure comprising same, for use in the detection, diagnosis and prognosis of a BCMA-mediated disease or condition.

[0063] In another aspect, the disclosure provides the use of an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof, or a detection or diagnostic reagent according to the disclosure comprising same, in the preparation of a kit for the detection, diagnosis and prognosis of a BCMA-mediated disease or condition.

[0064] In certain embodiments, said BCMA-mediated disease or condition is cancer, preferably a BCMA-expressing cancer, more preferably lymphoma, leukemia, or myeloma, most preferably multiple myeloma.

[0065] In a particular embodiment, said BCMA-mediated disease or condition is an autoimmune disease, preferably lupus erythematosus, IgA nephropathy and rheumatoid arthritis.

[0066] In another aspect, the present disclosure provides an antibody drug conjugate comprising an anti-BCMA antibody or antigen-binding fragment thereof according to the present disclosure conjugated to a cytotoxic agent, preferably wherein the cytotoxic agent is selected from the group consisting of MMAF, SN-38, and exatecan.More preferably, the antibody drug conjugate according to the present disclosure has a structural formula selected from the group consisting of: JPEG0007627258000001.jpg123122 where Ab is an anti-BCMA antibody or antigen-binding fragment thereof according to the present disclosure as described above.

[0067] The present disclosure further provides a use of an antibody drug conjugate according to the present disclosure in the preparation of a medicament for treating a BCMA-mediated disease or condition, the BCMA-mediated disease or condition being selected from the group consisting of cancer and autoimmune disease, the cancer being preferably a BCMA-expressing cancer, more preferably lymphoma, leukemia, or myeloma, most preferably multiple myeloma, the autoimmune disease being preferably selected from the group consisting of lupus erythematosus, IgA nephropathy, and rheumatoid arthritis.

[0068] In another aspect, the disclosure provides a method for treating a BCMA-mediated disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-BCMA antibody or antigen-binding fragment thereof, or a pharma- ceutically acceptable salt thereof according to the disclosure, or a pharmaceutical composition thereof, or an antibody-drug conjugate of the disclosure.

[0069] The anti-BCMA antibody or antigen-binding fragment thereof provided by the present disclosure has high affinity, high specificity, low immunogenicity, high biological activity, and significant antitumor effect. Meanwhile, the antibody of the present disclosure can be rapidly internalized after binding to the BCMA antigen. They are indicated for application in ADC form for therapeutic use or other uses requiring rapid internalization. The antibody of the present disclosure in ADC form can efficiently kill tumor cells expressing BCMMAFMA.

[0070] Detailed Description term To facilitate the understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless expressly defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0071] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0072] The term "antibody" as described in this disclosure refers to an immunoglobulin with a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence order of the heavy chain constant region of the immunoglobulins are different, so their antigenicity is also different. This leads to five types of immunoglobulins, known as immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Ig of the same type can be classified into different subclasses depending on the difference in amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ and λ chains depending on the difference in the constant region. Each of the five types of Ig can have a κ or λ chain.

[0073] In the present disclosure, the antibody light chain according to the present disclosure can further comprise a light chain constant region comprising a human or mouse kappa chain, lambda chain, or a variant thereof.

[0074] In the present disclosure, the antibody heavy chain according to the present disclosure can further comprise a heavy chain constant region comprising human or mouse IgG1, IgG2, IgG3, and IgG4, or variants thereof.

[0075] Approximately 110 amino acid sequences near the N-terminus of the heavy and light chains of an antibody are highly variable 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 regions contain three hypervariable regions (HVRs) and four framework regions (FRs), the sequences of which are relatively conservative. The three hypervariable regions determine the specificity of the antibody and are also known as complementarity determining regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions. The order of arrangement from amino terminus to carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The numbers and positions of CDR amino acid residues in the VL and VH regions of an antibody or antigen-binding fragment according to the present disclosure are in accordance with the known Kabat numbering criteria and Kabat, AbM, or IMGT definition criteria (http: / / bioinf.org.uk / abs / ).

[0076] The term "antigen-presenting cell" or "APC" refers to a cell that presents foreign antigens complexed with MHC on its surface. T cells recognize such complexes using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.

[0077] The term "antigen presentation" refers to the process of capturing antigens by APCs and enabling them to be recognized by T cells, for example as components of MHC-I / MHC-II complexes.

[0078] The term "BCMA" includes any variant or isoform of BCMA that is naturally expressed by a cell. The antibodies of the present disclosure may cross-react with BCMA from a non-human species. Alternatively, the antibodies may be specific for human BCMA and not cross-reactive with other species. BCMA or any variant or isoform thereof may be isolated from cells or tissues that naturally express it or may be produced by recombinant techniques using techniques commonly known in the art and described herein. Preferably, the anti-BCMA antibody targets human BCMA with a normal glycosylation pattern.

[0079] The term "recombinant human antibody" includes human antibodies prepared, expressed, produced, or isolated by recombinant methods, the techniques and methods of which are well known in the art. For example, 1. Antibodies isolated from transgenic and transchromosomal animals (e.g., mice) carrying human immunoglobulin genes, or from hybridomas prepared therefrom; 2. An antibody isolated from a host cell, such as a transfectoma, that has been transformed to express the antibody; 3. Antibodies isolated from a recombinant combinatorial human antibody library; and 4. Antibodies prepared, expressed, engineered, or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences.

[0080] Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also contain subsequent rearrangements and mutations that occur during antibody maturation.

[0081] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human BCMA prepared according to the knowledge and techniques in the art. During preparation, a subject is injected with BCMA antigen, and then fusion cells expressing an antibody with the desired sequence or functional properties are isolated. In a preferred embodiment of the present disclosure, the mouse BCMA antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse kappa chain, lambda chain, or variants thereof, or may further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0082] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").

[0083] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the framework of human antibody variable regions. Humanized antibodies can overcome the drawback of strong immune responses elicited by chimeric antibodies carrying large amounts of mouse protein components. To avoid the reduced activity caused by reduced immunogenicity, human antibody variable regions can be subjected to minimal back mutations to maintain their activity.

[0084] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody. This can reduce the immune response induced by mouse antibodies. To establish a chimeric antibody, the following steps are required: first, establish a hybridoma secreting mouse-specific monoclonal antibody; then, clone the variable region gene from the mouse hybridoma; and then, optionally, clone the constant region gene of a human antibody and link the mouse variable region gene with the human constant region gene to form a chimeric gene, which is inserted into a human expression vector; and finally, express the chimeric antibody molecule in a eukaryotic or prokaryotic industrial system. The human antibody constant region can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof, preferably the heavy chain constant region of human IgG1, IgG2, or IgG4, or the heavy chain constant region of IgG1 with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation.

[0085] The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs that typically contain at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least a portion of the binding specificity of the parent antibody. Generally, the antibody fragment retains at least 10% of the binding activity of the parent when the activity is expressed in molar terms. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody with 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.

[0086] A "Fab fragment" is composed of one light chain, CH1, and the variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0087] The "Fc" region comprises two antibody heavy chain fragments containing the CH1 and CH2 domains, held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0088] A "Fab' fragment" contains a light chain and a portion of a heavy chain including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains of the two Fab' fragments, thereby forming an F(ab')2 molecule.

[0089] An "F(ab')2 fragment" comprises two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0090] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0091] The term "multispecific antibody" is used in its broadest sense and encompasses antibodies with specificity for multiple epitopes. These multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable region VH and a light chain variable region VL, where the VH-VL unit has specificity for multiple epitopes; antibodies with two or more VL and VH regions, where each VH-VL unit binds to a different target or a different epitope on the same target; antibodies with two or more single variable regions, where each single variable region binds to a different target or a different epitope on the same target; full length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked antibody fragments, etc.

[0092] The term "single-chain antibody" refers to a single-chain recombinant protein formed by linking the heavy chain variable domain VH and the light chain variable domain VL of an antibody via a linker peptide. It is the smallest antibody fragment that contains a complete antigen-binding site.

[0093] The term "domain antibody fragment" refers to an immunoglobulin fragment having an immunological function, and includes either a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same antigen or different antigens.

[0094] In this disclosure, the term "binding to BCMA" refers to the ability to interact with human BCMA.

[0095] The term "antigen-binding site" in this disclosure refers to the three-dimensional site recognized by an antibody or antigen-binding fragment of the disclosure.

[0096] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually lost after treatment with denaturing solvents. Epitopes usually contain at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art and include immunoblotting, immunoprecipitation detection analysis, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art and described herein, such as x-ray crystallography, two-dimensional nuclear magnetic resonance, and the like.

[0097] As used in this disclosure, the terms "specifically bind" and "selectively bind" refer to an antibody binding to an epitope on a given antigen. Generally, when recombinant human BCMA is used as the analyte and an antibody is used as the ligand, the antibody binds to an epitope on a given antigen at a binding affinity of about 10 -7 The equilibrium dissociation constant (K D ) and its binding affinity for the predetermined antigen is at least twice its binding affinity for a non-specific antigen other than the predetermined antigen or a closely related antigen (such as BSA). The term "antibody that recognizes an antigen" can be used interchangeably with the term "antibody that specifically binds" herein.

[0098] The term "cross-reactivity" refers to the ability of an antibody of the present disclosure to bind to BCMA from a different species. For example, an antibody of the present disclosure that binds to human BCMA can also bind to BCMA from other species. Cross-reactivity is measured in binding assays (such as SPR or ELISA) by detecting specific reactivity with purified antigen, or binding or functional interaction with cells that physiologically express BCMA. Methods for measuring cross-reactivity include standard binding assays described herein, such as surface plasmon resonance (SPR) analysis or flow cytometry.

[0099] The terms "inhibition" or "blocking" can be used interchangeably and include both partial and complete inhibition / blocking. Inhibition / blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blockage. Inhibition and blocking also include a measurable decrease in the binding affinity of a ligand when contacted with an anti-BCMA antibody compared to a ligand not contacted with an anti-BCMA antibody.

[0100] The term "growth inhibition" (eg, cell growth inhibition) is intended to include a measurable decrease in cell proliferation.

[0101] The terms "induced immune response" and "enhanced immune response" can be used interchangeably and refer to an immune response (i.e., passive or adaptive) stimulated by a particular antigen. The term "inducing", such as inducing CDC or ADCC, refers to stimulating a specific direct cell killing mechanism.

[0102] In the present disclosure, "ADCC", i.e., antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors by recognizing the Fc segment of the antibody. The Fc segment of IgG can be modified to enhance, reduce, or eliminate the ADCC function of the antibody. The modification refers to a mutation in the heavy chain constant region of the antibody.

[0103] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as in Cold Spring Harbor's "Antibodies: A Laboratory Manual," Chapters 5-8, 15. For example, mice can be immunized with human BCMA or a fragment thereof, and the resulting antibodies can be renatured and purified and subjected to amino acid sequence determination by conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to non-human CDR regions. Human FR germline sequences are available from the ImmunoGeneTics (IMGT) Website http: / / imgt.cines.fr, or from Immunoglobulin Facts Book, 2001 ISBN 012441351.

[0104] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody 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 preferred prior art, mammalian expression systems can provide glycosylation of the antibody, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing an antibody that specifically binds to a human antigen. Positive clones are grown in serum-free medium in a bioreactor to produce the antibody. The culture fluid from which the antibody is secreted can be purified and harvested by conventional methods. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers can also be removed by conventional methods, e.g., molecular sieves or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0105] The antibody of the present disclosure refers to a monoclonal antibody. The monoclonal antibody (mAb) of the present disclosure refers to an antibody obtained from a single cloned cell line, examples of which include, but are not limited to, a eukaryotic cell line, a prokaryotic cell line, or a phage cloned cell line. The monoclonal antibody or antigen-binding fragment can be obtained by recombination using, for example, fusion cell technology, recombinant technology, phage display technology, synthetic technology (such as CDR grafting), or other existing technology.

[0106] "Administering", "giving" and "treating" when applied to an animal, human, experimental subject, cell, tissue, organ, or bodily fluid, refers to contacting an exogenous agent, therapeutic agent, diagnostic agent, or composition with the animal, human, experimental subject, cell, tissue, organ, or bodily fluid. "Administering", "giving" and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treating a cell includes contacting a reagent with a cell, and contacting a reagent with a fluid, where the fluid is in contact with the cell. "Administering", "giving" and "treating" also refer to, for example, treating a cell with a reagent, diagnostic agent, binding composition, or another cell, in vitro and ex vivo. "Treating" when applied to a human, animal, or experimental subject, refers to therapeutic or prophylactic measures, research, and diagnostic applications.

[0107] "Treatment" refers to providing an internal or external therapeutic agent, including, for example, any one of the antibodies of the present disclosure, to a patient with one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. In general, the therapeutic agent is provided in an amount effective to alleviate one or more disease symptoms in a subject or population to be treated, induce regression of such symptoms, or inhibit the onset of such symptoms to a clinically measurable extent. The amount of a therapeutic agent effective to alleviate a particular disease symptom (also called a "therapeutically effective amount") may vary depending on various factors, such as the patient's medical condition, age, and weight, as well as 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 testing method commonly used by physicians or other medical professionals to evaluate the severity or progression of the condition. While embodiments of the present disclosure (e.g., methods of treatment or products) may not be effective in alleviating each disease symptom of interest, they should alleviate the disease symptom of interest in a statistically significant number of patients as determined by statistical testing methods known in the art, such as Student's t test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0108] As used throughout this specification and claims, the term "consisting essentially of" or variations thereof means the inclusion of all recited elements or elements, and optionally other elements that are similar or different in nature to the recited elements that do not materially alter the basic or novel characteristics of a given dosage regimen, method, or composition.

[0109] In this disclosure, the term "naturally occurring" as applied to a particular entity refers to the fact that the entity can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally artificially modified in a laboratory is naturally occurring.

[0110] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or condition of a disease state. Effective amount also refers to an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or animal subject may vary depending on factors such as, for example, the condition being treated, the patient's general health, the method, route and dose of drug administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regime that avoids significant side effects or toxic effects.

[0111] "Exogenous" refers to a substance produced outside an organism, cell, or body depending on the context.

[0112] "Endogenous" refers to a substance produced within a cell, organism, or body in response to a context.

[0113] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If the positions of two aligned 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, 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 two sequences divided by the number of positions compared, multiplied by 100%. For example, in optimal sequence alignment, if 6 out of 10 positions of two sequences are matching or homologous, the two sequences are 60% homologous. In general, alignment is performed when two sequences are aligned to obtain the maximum percentage of identity.

[0114] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. Thus, the terms "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of passages. It should be understood that not all progeny are completely identical in DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the original transformed cell are included. When different designations are referenced, they will be clearly understood from the context.

[0115] "Any" or "optionally" means that an event or circumstance described below may occur but does not necessarily occur, and the description includes cases where the event or circumstance occurs or does not occur. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence can be present but does not have to be present.

[0116] A "pharmaceutical composition" is meant to contain one or more antibodies or antigen-binding fragments described herein, as well as other components, such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism, such as to facilitate absorption of the active ingredient to exert its biological activity.

[0117] "Drug loading" (DAR) is represented by y and is the average number of cytotoxic drugs per antibody in the antibody-drug conjugate. The drug loading (DAR) of the antibody-drug conjugates produced from the conjugation reaction can be characterized by conventional means, such as mass spectrometry, HPLC, and ELISA. These means allow the quantitative distribution of antibody-drug conjugates in y values ​​to be measured. Detailed Description of the Invention

[0118] The present disclosure is further described with reference to the following examples, which should not be construed as limiting the scope of the present disclosure. If no specific conditions are described in the examples of the present disclosure, the experimental methods generally follow commonly used conditions such as those in Cold Spring Harbor's "Antibody: A Laboratory Manual" and "Molecular Cloning: A Laboratory Manual", or conditions recommended by manufacturers of raw materials or products. Reagents whose sources are not listed are commonly used reagents purchased from the market.

[0119] Example 1: Antigen Preparation A protein encoding the His-tagged extracellular domain of human BCMA (BCMA-His) was synthesized according to SinoBiologics (catalog number: 10620-H08H). BCMA-His sequence: MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAHHHHHHHHHHH SEQ ID NO: 21

[0120] Example 2: Obtaining mouse hybridomas and antibody sequences A total of 5 Balb / c and 5 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. Immunogen and adjuvant were thoroughly mixed in a 1:1 ratio and emulsified to prepare a stable "water in oil" solution. The injection volume was 25 μg / 200 μL / mouse.

[0121] Table 1 Immunization scheme JPEG0007627258000002.jpg37139

[0122] The serum titer and the ability to bind to cell surface antigens of the sera of mice immunized according to Example 3 were evaluated by indirect ELISA. The initiation of cell fusion was measured based on the detection result of the titer (dilution of 100,000 times or more). Immunized mice with high serum titer, affinity and FACS binding were selected for one final immunization, and then they were sacrificed. The spleen cells were fused with SP2 / 0 myeloma cells and plated to obtain fusion cells. The desired fusion cells were screened by indirect ELISA and established as monoclonal cell lines by limiting dilution. The obtained antibody-positive lines were further screened by indirect ELISA to select fusion cells that bind to the recombinant protein. The fusion cells in the logarithmic growth phase were harvested. RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-initiator set (Novagen, TB326 Rev.B0503). Finally, the sequence of mouse antibody M1 was obtained.

[0123] The heavy and light chain variable region sequences of mouse monoclonal antibody M1 are as follows: M1 HCVR QVQLQQSGAELVRPGASVKLSCKALGYSFSDYEMHWVRQTPVHGLEWIGGIHPGSGGSAYNQKFKGKATLTVDKSSSTAYMELSSLTSEDSAVYYCTRLDYGYSWAWFPYWGQGTLVTVSA SEQ ID NO: 1 M1 LCVR EILLTQSPAIIVTSPGEKVTITCSASSSVIYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPLTFGAGTKLELK SEQ ID NO: 2

[0124] Table 2. CDR sequences of the heavy and light chain variable regions of mouse monoclonal antibody M1 JPEG0007627258000003.jpg56139

[0125] Example 3: Method for detecting in vitro binding activity of an antibody (1) In vitro indirect ELISA binding assay: BCMA His protein (Sino Biological Inc., Cat. No. 10620-H08H) was diluted to a concentration of 1 μg / ml in pH 7.4 PBS and added to a 96-well high affinity ELISA plate at 100 μl / well and incubated overnight (16-20 hours) in a 4°C refrigerator. After washing the plate four times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 3% bovine serum albumin (BSA) blocking solution diluted in PBST was added at 150 μl / well and incubated at room temperature for 1 hour for blocking. After blocking was completed, the blocking solution was discarded and the plate was washed four times with PBST buffer.

[0126] Test antibodies were diluted 10-fold in PBST containing 3% BSA (initial concentration was 1 μM after 10-fold dilution) and added to the microtiter plate at 100 μl / well and incubated at room temperature for 1 h. After incubation, the plate was washed 4 times with PBST. HRP-conjugated goat anti-human secondary antibody (Abcam, Cat. No. ab97225) diluted in PBST containing 3% BSA was added at 100 μl / well and incubated at room temperature for 1 h. After washing the plate 4 times with PBST, TMB chromogenic substrate (Cell Signaling Technology, Cat. No. 7004S) was added at 100 μl / well and incubated at room temperature in the dark for 1 min. The reaction was stopped by adding stop solution (Cell Signaling Technology, Cat. No. 7002S) at 100 μl / well. Absorbance values ​​were read at 450 nm using a microplate reader (BioTek, model Synergy H1). The data was analyzed. The results were analyzed by plotting concentration-signal curves as shown in the following table.

[0127] Table 3. Affinity of mouse antibodies to human BCMA antigen (EC 50 value) JPEG0007627258000004.jpg17139

[0128] (2) In vitro cell binding assay: Cultured cells with high BCMA expression (BCMA-overexpressing HEK293T cells; BCMA-expressing tumor cells, NCI-H929, ATCC deposit number CRL-9068) were harvested. The cell density was adjusted and the cells were plated in a 96-well U-bottom plate at 1 × 10 5 ~2×10 5 Cells / well were seeded. The plate was centrifuged at 1200g for 5 minutes, and the supernatant was removed. 100ul of gradient diluted antibody solution or mouse immune serum was added and incubated at 4°C for 60 minutes. The plate was centrifuged at 1200g for 5 minutes, and the supernatant was removed. The cells were washed twice with PBS. 100ul / well of fluorescently labeled secondary antibody (PE-GAM or PE-GAH) was added and incubated at 4°C for 60 minutes. The plate was centrifuged at 1200g for 5 minutes, and the supernatant was removed. The cells were washed twice with PBS, and then resuspended in PBS. Signals were detected using a flow cytometer, and a concentration curve was plotted for result analysis.

[0129] Table 4. Affinity of mouse antibodies to BCMA-expressing cells (EC 50 value) JPEG0007627258000005.jpg25139

[0130] Example 4: Humanization experiment of mouse antibodies Humanization of mouse anti-human BCMA monoclonal antibodies was carried out according to the methods published in many documents in the art. Briefly, the parent (mouse antibody) constant domain was replaced with a human constant domain. According to the identity between mouse and human antibodies, human germline antibody sequences were selected. In this disclosure, mouse antibody M1 was humanized.

[0131] Based on the typical structures of the VH / VL CDRs of the obtained mouse antibodies, the sequences of the heavy and light chain variable regions were aligned with a human antibody germline database to obtain human germline templates with high identity.

[0132] The CDR region of mouse antibody M1 was grafted into the selected related humanized template. Then, based on the three-dimensional structure of the mouse antibody, the buried residues, the residues that directly interact with the CDR region, and the residues that have a significant effect on the conformation of VL and VH were subjected to back mutation to optimize the chemically unstable amino acid residues in the CDR region. After expression testing and comparison of the number of back mutations, the sequence of the humanized heavy chain variable region HCVR was selected and designed as follows: HCVR1 QVQLVQSGAEVKKPGASVKVSCKASGYSFSDYEMHWVRQAPGQGLEWMGGIHPGSGGSAYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRLDYGYSWAWFPYWGQGTLVTVSA SEQ ID NO: 9 HCVR2 QVQLVQSGAEVKKPGASVKVSCKASGYSFSDYEMHWVRQAPGQGLEWIGGIHPGSGGSAYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCTRLDYGYSWAWFPYWGQGTLVTVSA SEQ ID NO: 10 HCVR3 QVQLVQSGAEVKKPGASVKVSCKASGYSFSDYEMHWVRQAPGQGLEWIGGIHPGSGGSAYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCTRLDYGYSWAWFPYWGQGTLVTVSA SEQ ID NO: 11

[0133] The sequences of the humanized light chain variable region LCVR were selected and designed as follows: LCVR1 EIVLTQSPATLSLSPGERATLSCSASSSVIYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK SEQ ID NO: 12 LCVR2 EIVLTQSPATLSLSPGERATLSCSASSSVIYMNWYQQKPGQSPKIWIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK SEQ ID NO: 13 LCVR3 EILLTQSPATLSLSPGERATLTCSASSSVIYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK SEQ ID NO: 14

[0134] The designed heavy and light chain variable region sequences were linked to IgG1 heavy and light chain constant region sequences, respectively. Exemplary heavy and light chain constant region sequences are as follows, respectively: IgG1 C ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 22 Ig kappa C RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 23

[0135] Examples of heavy and light chain sequences obtained after such ligation are as follows: Ab1 HC QVQLVQSGAEVKKPGASVKVSCKASGYSFSDYEMHWVRQAPGQGLEWMGGIHPGSGGSAYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRLDYGYSWAWFPYWG QGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 15 Ab2 HC QVQLVQSGAEVKKPGASVKVSCKASGYSFSDYEMHWVRQAPGQGLEWIGGIHPGSGGSAYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCTRLDYGYSWAWFPYWG QGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16 Ab3HC QVQLVQSGAEVKKPGASVKVSCKASGYSFSDYEMHWVRQAPGQGLEWIGGIHPGSGGSAYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCTRLDYGYSWAWFPYWG QGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 Ab1 LC EIVLTQSPATLSLSPGERATLSCSASSSVIYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 18 Ab2 LC EIVLTQSPATLSLSPGERATLSCSASSSVIYMNWYQQKPGQSPKIWIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 19 Ab3 LC EILLTQSPATLSLSPGERATLTCSASSSVIYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20

[0136] Table 5. Antibodies and SEQ ID NOs for heavy and light chains and their variable regions JPEG0007627258000006.jpg38137

[0137] According to the amino acid sequences of the light and heavy chains of each of the above humanized antibodies, cDNA fragments were synthesized and inserted into a pcDNA3.1 expression vector (Life Technologies, catalog number V790-20). The expression vector was used with a gene transfer reagent PEI (Polysciences, Inc., catalog number 23966) at a ratio of 1:2 to transfect HEK293 cells (Life Technologies, catalog number 11625019). The cells were then incubated at 4°C for 1 h at 4°C for 2 h at 4°C. 2 The cells were incubated in an incubator for 4-5 days. The cell culture fluid was collected, centrifuged, and filtered. The sample was then loaded onto an antibody purification affinity column. The column was washed with phosphate buffer. The sample was eluted with glycine-HCl buffer (pH 2.7, 0.1M Gly-HCl), neutralized with 1M Tris-HCl pH 9.0, and dialyzed against phosphate buffer to obtain the humanized antibody protein of the present disclosure.

[0138] Example 5: In vitro binding affinity and kinetic assays The affinity (EC 50 ) were measured and the results are shown in Table 6 below. Table 6. Affinity of each humanized antibody for human BCMA antigen (EC 50 ) JPEG0007627258000007.jpg33128

[0139] The affinity (EC 50 ) were measured and the results are shown in Table 7 below. Table 7. Affinity of each humanized antibody for NCI-H929 tumor cells (EC 50 ) JPEG0007627258000008.jpg33126

[0140] Example 6: Antibody Invagination into the Plasma Membrane NCI-H929 (ATCC Accession No. CRL-9068) was used to assess whether the antibodies of the present disclosure could be co-invaginated into cells with human BCMA after binding to BCMA. NCI-H929 cells were digested with trypsin (after washing once with PBS for approximately 2 minutes at 37° C.), harvested, and resuspended in pre-chilled FACS buffer. The cell concentration was 1×10 6The cell concentration was adjusted to 100 μg / mL. 1 mL of cell suspension was added to an EP tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. 1 mL of the prepared antibody to be tested was added to resuspend the cells, and the final concentration of the antibody was 20 μg / mL. The cells were incubated in a shaker at 4° C. for 1 hour, centrifuged (4° C., 1500 rpm×5 minutes), and the supernatant was discarded. The cells were washed twice with FACS buffer, and the supernatant was removed. 100 μL of fluorescent secondary antibody working solution was added to each tube, and the cells were resuspended. The cells were incubated in a shaker at 4° C. for 30 minutes, centrifuged (4° C., 1500 rpm×5 minutes), and the supernatant was discarded. The cells were washed twice with FACS buffer, and the supernatant was removed. 1.0 mL of pre-warmed NCI-H929 cell complete medium was added to each tube, and the cells were resuspended and mixed thoroughly. The cell suspension was dispensed into 4 tubes, 200 μL / tube, for 0 min group, blank group, 30 min group, and 2 h group, respectively. The 0 min group and blank group were left on ice, and the remaining groups were left in a 37°C incubator for 30 min and 2 h, respectively, for plasma membrane invagination. At the corresponding time points, the EP tubes were taken out and left on ice to pre-cool for 5 min. All treatment groups were centrifuged (4°C, 1500 rpm x 5 min), and the supernatant was discarded. The cells were washed once with FACS buffer, and the supernatant was removed. 250 μL of strip buffer was added to the EP tubes of all treatment groups except the 0 min group. The cells were incubated at room temperature for 8 min, centrifuged (4°C, 1500 rpm x 5 min), and the supernatant was discarded. The cells were washed twice with FACS buffer, and the supernatant was removed. All treatment groups were added with 100 μL of fixative and left at 4°C for more than 30 minutes for immunostaining, and detected by flow cytometer DxFlex. Percentage (%) of BCMA antibody plasma membrane invagination = (fluorescence intensity value at each time point - mean fluorescence intensity value of blank group) / mean fluorescence intensity value at zero point - mean fluorescence intensity value of blank group. The results are shown in Table 8 below. Table 8. Plasma Membrane Invagination (EC) of Antibodies into NCI-H929 Tumor Cells 50 ) JPEG0007627258000009.jpg53139

[0141] As shown in the results, the antibodies of the present disclosure have higher plasma membrane invagination efficiency compared to the anti-BCMA antibody J6M0 (described in U.S. Patent No. 9,273,141).

[0142] Example 7: Conjugation of antibodies to MC-MMAF The antibody of the present disclosure has cell affinity activity and plasma membrane engulfment activity, so it is suitable for conjugating with drugs to form antibody-drug conjugates for treating BCMA-mediated diseases. The antibody of the present disclosure was conjugated with MC-MMAF to form antibody-drug conjugates. The conjugation process is shown in the following formula, where Ab represents Ab2 or Ab3 antibody. JPEG0007627258000010.jpg75140

[0143] In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use. The above prepared acetonitrile solution containing S-(3-hydroxypropyl)thioacetate was added to acetic acid / sodium acetate buffer pH=4.3 (10.35 mg / mL, 9.0 mL, 0.97 mol) containing antibody. Then, 1.0 mL of aqueous solution containing sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise and reacted at 25° C. for 2 hours with shaking. After the reaction was completed, the reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS solution, pH 6.5) to obtain a solution containing product 1f. The solution was concentrated to 10 mg / mL and used directly in the next reaction.

[0144] In the second step, 0.35 mL of 2.0 M carboxamide hydrochloride solution was added to the solution of 1f (11.0 mL) and reacted with shaking for 30 min at 25° C. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution, pH 6.5) to obtain a solution containing product 2f (concentration 6.17 mg / mL, 14.7 mL).

[0145] In the third step, compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile, added to the solution of 2f (concentration 6.17 mg / mL, 3.0 mL), and reacted with shaking at 25°C for 4 hours. The reaction solution was then desalted, purified using a Sephadex G25 gel column (elution phase: 0.05M PBS solution, pH 6.5), and filtered under sterile conditions using a filter to obtain product Ab2-MC-MMAF. The average value y of DAR of product Ab2-MC-MMAF measured by HIC-HPLC was 4. The antibody-drug conjugate in PBS buffer (3.7 mg / mL, 4.7 mL) was refrigerated at 4°C. Product Ab3-MC-MMAF was prepared by the method described above. The average value y of DAR of product Ab3-MC-MMAF measured by HIC-HPLC was 4.1. The antibody-drug conjugate in PBS buffer (3.5 mg / mL, 5.0 mL) was refrigerated at 4°C.

[0146] Example 8: Conjugation of antibodies to SN-38 The antibody-conjugated drug was prepared by the following conjugation process: where Ab represents Ab2. JPEG0007627258000011.jpg90139

[0147] In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use. The above prepared acetonitrile solution containing S-(3-hydroxypropyl)thioacetate was added to acetic acid / sodium acetate buffer pH=4.3 (10.35 mg / mL, 9.0 mL, 0.97 mol) containing antibody. Then, 1.0 mL of aqueous solution containing sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise and reacted at 25° C. for 2 hours with shaking. After the reaction was completed, the reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS solution, pH 6.5) to obtain a solution containing product 1h. The solution was concentrated to 10 mg / mL and used directly in the next reaction.

[0148] In the second step, 0.35 mL of 2.0 M carboxamide hydrochloride solution was added to the solution of 1h (11.0 mL) and reacted at 25° C. for 30 minutes with shaking. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution, pH 6.5) to obtain a solution containing product 2h (concentration 6.2 mg / mL, 15.0 mL). The solution was concentrated to about 10 mg / mL and used in the next reaction.

[0149] In the third step, compound MC-SN-38 (1.3 mg, 1.2 mol) was dissolved in 0.3 mL of acetonitrile, added to the 2h solution (concentration 6.2 mg / mL, 3.0 mL), and reacted at 25°C for 4 hours with shaking. Next, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS solution, pH 6.5), and then filtered under sterile conditions using a filter to obtain the product Ab-SN-38 antibody drug conjugate in PBS buffer (3.7 mg / mL, 4.7 mL), which was refrigerated at 4°C. The average value y was measured by ultraviolet light. A cuvette filled with sodium succinate buffer was placed in the reference absorption cell and the absorption cell for sample measurement, respectively, and after subtracting the solvent blank, a cuvette filled with the test solution was placed in the absorption cell for sample measurement. The absorbance at 280 nm and 370 nm was measured.

[0150] Data Processing: A standard curve was prepared and the antibody content Cmab was measured by measuring the absorbance at a wavelength of 280 nm. The small molecule content CDrug was measured by measuring the absorbance at a wavelength of 370 nm. Mean drug loading y = CDrug / Cmab The average DAR y of the antibody-drug conjugate Ab2-SN-38 measured by the above method was 3.9.

[0151] Example 9: Conjugation of antibodies to exatecan JPEG0007627258000012.jpg95143

[0152] In the first step, 2a (2 g, 17.2 mmol) was dissolved in 75 mL of acetonitrile, and potassium carbonate (9.27 g, 67.2 mmol), benzyl bromide (20 mL, 167.2 mmol), and tetrabutylammonium iodide (620 mg, 1.68 mmol) were added successively. The reaction solution was stirred at room temperature for 48 h and filtered through diatomaceous earth. The filter cake was rinsed with ethyl acetate (20 ml). The filtrate was pooled and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to give product 5a (3.2 g, yield: 90.1%).

[0153] In the second step, 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were added to the reaction flask. 6 mL of tetrahydrofuran was added. The reaction mixture was exchanged with argon three times and cooled to 0-5 °C in an ice-water bath. Potassium tert-butoxide (164 mg, 1.46 mmol) was added. The ice bath was removed and the reaction mixture was heated to room temperature and stirred for 40 min. 15 mL of ice water was added and the reaction mixture was extracted with ethyl acetate (40 mL × 2) and chloroform (20 mL × 5). The organic phases were pooled and concentrated. The resulting residue was dissolved in 6 mL of dioxane, 3 mL of water, sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorenylmethyl chloroformate (190 mg, 0.734 mmol) were added and stirred at room temperature for 2 h. 30 mL of water was added and the reaction mixture was extracted with ethyl acetate (20 mL × 3). The organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain product 5b benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundeca-11-carboxylate (73 mg, yield: 19.4%). MS m / z (ESI): 515.0 [M+1].

[0154] In the third step, 5b (30 mg, 0.058 mmol) was dissolved in 6.75 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1). Palladium on carbon (18 mg, 10% content, dry) was added, and the reaction mixture was exchanged with hydrogen three times and reacted at room temperature with stirring for 1 h. The reaction solution was filtered through diatomaceous earth. The filter cake was rinsed with ethyl acetate. The filtrate was concentrated to give the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid (20 mg), which was used directly in the next reaction without purification. MS m / z(ESI):424.9 [M+1].

[0155] In the fourth step, 1b (15 mg, 28.2 μmol) was added to the reaction flask. 1.5 mL of N,N-dimethylformamide was added. The reaction mixture was exchanged with argon three times and cooled to 0-5 °C in an ice-water bath. A drop of triethylamine was added, followed by the addition of crude product 5c (20 mg, 47.1 μmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine (25.4 mg, 86.2 μmol), and the reaction mixture was reacted with stirring in an ice bath for 40 min. 15 mL of water was added, and the reaction mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were pooled. The organic phase was washed with saturated sodium chloride solution (20 mL × 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 5d (9H-fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolozino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (23.7 mg, yield: 78.9%). MS m / z(ESI):842.1 [M+1].

[0156] In the fifth step, 5d (30 mg, 35.7 μmol) was dissolved in 3 mL of dichloromethane. 1.5 mL of diethylamine was added and the reaction mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure. 1.5 mL of toluene was added and the reaction mixture was concentrated under reduced pressure, which was repeated twice. 4.5 mL of n-hexane was added to the residue and homogenized. After settling, the supernatant was poured off and the solid remained. The solid residue was concentrated under reduced pressure and pump dried to give crude product 5e 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolozino[1,2-b]quinolin-1-yl)acetamide (23 mg), which was used directly in the next reaction without purification. MS m / z(ESI):638.0 [M+18].

[0157] In the sixth step, the crude product 5e (20 mg, 32.3 μmol) was dissolved in 1 mL of N,N-dimethylformamide and exchanged with argon three times. The reaction mixture was cooled to 0-5°C in an ice-water bath. 0.5 mL of 4g N,N-dimethylformamide solution (31.8 mg, 67.3 μmol) was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine (27.8 mg, 94.3 μmol), and the mixture was reacted with stirring in an ice bath for 10 minutes. The ice bath was removed, and the reaction mixture was heated to room temperature and reacted with stirring for 1 hour to produce compound 5. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 um 19*250 mm; mobile phase: A-water (10 mmol NH 4 OAc):B-acetonitrile, gradient elution, flow rate: 18 mL / min. The corresponding components were collected and concentrated under reduced pressure to give products 5-A and 5-B (3.6 mg, 2.6 mg). MS m / z(ESI):1074.4 [M+1].

[0158] Single component compound 5-A (with shorter retention time): UPLC analysis: Retention time: 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC18 1.7 um 2.1*50 mm, mobile phase: A-water (5 mmol NH 4 OAc), B-Acetonitrile). JPEG0007627258000013.jpg41137TIFF0007627258000014.tif225

[0159] Single component compound 5-B (with longer retention time): UPLC analysis: Retention time: 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC18 1.7 um 2.1*50 mm, mobile phase: A-water (5 mmol NH 4 OAc), B-Acetonitrile). JPEG0007627258000015.jpg42138

[0160] The preparation method of other intermediates was the same as that of Intermediate 5.

[0161] At 37° C., the prepared aqueous solution containing tris(2-carboxyethyl)phosphine (10 mM, 0.347 mL, 3.47 μmol) was added to an aqueous PBS buffer solution containing antibody Ab2 (pH 6.5, 0.05 M PBS buffer solution; 7.3 ml, 13.8 mg / ml, 0.681 μmol). The reaction mixture was left on a water bath shaker and reacted with shaking at 37° C. for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25° C. in a water bath and diluted to 14.0 ml, and 3.3 ml of the solution was taken out for the next reaction.

[0162] Compound 5-A (3.0 mg, 3.72 μmol) was dissolved in 0.15 mL of DMSO and added to 3.3 ml of the above solution. The reaction mixture was left on a water bath shaker and reacted with shaking at 25° C. for 3 hours. The reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: pH 6.5, 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the exemplary products Ab-exatecan and Ab2-exatecan (1.35 mg / mL, 13 mL) in PBS buffer, which were stored frozen at 4° C.

[0163] The average value y was measured by the ultraviolet method. A cuvette filled with sodium succinate buffer was placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after subtracting the solvent blank, a cuvette filled with the test solution was placed in the sample measurement absorption cell. The absorbance at 280 nm and 370 nm was measured.

[0164] Data Processing: A standard curve was prepared and the antibody content Cmab was measured by measuring the absorbance at a wavelength of 280 nm. The small molecule content CDrug was measured by measuring the absorbance at a wavelength of 370 nm. Mean drug loading y = CDrug / Cmab. The exemplary product Ab2-exatecan had a y of 7.6 as measured by the method described above. A sample of Ab2-exatecan (y=8) was obtained by UV-HPLC purification.

[0165] Example 10: Tumor killing activity of antibody-drug conjugates To further examine the killing effect of the antibody-drug conjugates on tumors formed in vivo, tumors implanted with NCI-H929 cells were formed in mice, and the anti-tumor effect of the antibody-drug conjugates of the present disclosure was evaluated.

[0166] (1) 9×10 6NCI-H929 cells were subcutaneously injected into 8-week-old immunodeficient nude mice (NOD-SCID). Eight days later, intravenous injections of antibody-drug conjugates Ab2-MC-MMAF (Example 7, y=4) and Ab2-exatecan (Example 9, y=8) were started once a week at a dose of 1 mg / kg. Human IgG1 protein was used as a control at a dose of 1 mg / kg. Five mice were used in each of the control and treatment groups. The tumor volumes were measured to calculate the tumor inhibition rate. Tumor inhibition rate = 100% - (tumor volume of treatment group on day 14 - tumor volume of treatment group on day 0) / (tumor volume of control group on day 14 - tumor volume of control group on day 0). The experimental results are shown in Table 9. The antibody-drug conjugates Ab2-MC-MMAF (Example 7, y=4) and Ab2-exatecan (Example 9, y=8) both show tumor inhibitory effects. Table 9. Tumor killing activity of antibody-drug conjugates JPEG0007627258000016.jpg25128

[0167] (2) 9×10 6 NCI-H929 cells were subcutaneously injected into 8-week-old immunodeficient nude mice (NOD-SCID). Eight days later, intravenous injections of antibody-drug conjugates Ab2-MC-MMAF (Example 7, y=4) and Ab3-MC-MMAF (Example 7, y=4.1) were started twice weekly at a dose of 3 mg / kg. Human IgG1 protein was used as a control at a dose of 3 mg / kg. Five mice were used in each of the control and treatment groups. Tumor volumes were measured to calculate the tumor inhibition rate. Tumor inhibition rate TGI=100%-(tumor volume of treatment group on day 14-tumor volume of treatment group on day 0) / (tumor volume of control group on day 14-tumor volume of control group on day 0). The experimental results are shown in Table 10. Both Ab2-MC-MMAF (Example 7, y=4) and Ab3-MC-MMAF (Example 7, y=4.1) showed a killing effect on the tumor. Table 10. Tumor killing activity of antibody-drug conjugates JPEG0007627258000017.jpg25126

Claims

1. 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 an HCDR1 represented by SEQ ID NO:3, an HCDR2 represented by SEQ ID NO:4, and an HCDR3 represented by SEQ ID NO:5; and the light chain variable region comprises an LCDR1 represented by SEQ ID NO:6, an LDCR2 represented by SEQ ID NO:7, and an LCDR3 represented by SEQ ID NO:

8.

2. 2. The anti-BCMA antibody or antigen-binding fragment thereof of claim 1, wherein the anti-BCMA antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, or a humanized antibody.

3. 2. The anti-BCMA antibody or antigen-binding fragment thereof of claim 1, wherein the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

4. The anti-BCMA antibody or antigen-binding fragment thereof described in claim 3, further comprising a heavy chain constant region derived from human IgG1, IgG2, or IgG4.

5. The anti-BCMA antibody or antigen-binding fragment thereof described in claim 3, further comprising an IgG1 heavy chain constant region represented by sequence number:

22.

6. The anti-BCMA antibody or antigen-binding fragment thereof of claim 1, further comprising a light chain constant region derived from a human kappa chain, lambda chain, or a variant thereof.

7. The anti-BCMA antibody or antigen-binding fragment thereof described in claim 6, further comprising a light chain constant region represented by sequence number:

23.

8. 8. The anti-BCMA antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:

11.

9. 8. The anti-BCMA antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO:

14.

10. 9. The anti-BCMA antibody or antigen-binding fragment thereof of claim 8, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:

17.

11. 10. The anti-BCMA antibody or antigen-binding fragment thereof of claim 9, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:

20.

12. the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region represented by SEQ ID NO:9 and a light chain variable region represented by SEQ ID NO:12; or the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region represented by SEQ ID NO:10 and a light chain variable region represented by SEQ ID NO:13; or the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region represented by SEQ ID NO: 11 and a light chain variable region represented by SEQ ID NO: 14; An anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

13. the anti-BCMA antibody comprises a heavy chain represented by SEQ ID NO:15 and a light chain represented by SEQ ID NO:18; or the anti-BCMA antibody comprises a heavy chain represented by SEQ ID NO:16 and a light chain represented by SEQ ID NO:19; or the anti-BCMA antibody comprises a heavy chain represented by SEQ ID NO: 17 and a light chain represented by SEQ ID NO: 20; 13. The anti-BCMA antibody or antigen-binding fragment thereof of claim 12.

14. A polynucleotide encoding the anti-BCMA antibody or antigen-binding fragment thereof of any one of claims 1 to 13.

15. An expression vector containing the polynucleotide of claim 14.

16. A host cell transfected with or containing the expression vector of claim 15.

17. 17. The host cell of claim 16, wherein the host cell is a bacterial, yeast, or mammalian cell.

18. The host cell of claim 17, wherein the host cell is Escherichia coli, Pichia pastoris, a CHO cell, or a HEK293 cell.

19. 1. A method for producing an anti-BCMA antibody, comprising: Cultivating a host cell according to any one of claims 16 to 18; isolating the antibody from the culture; and purifying said antibody.

20. A method for producing the anti-BCMA antibody of claim 19, comprising: the host cell is a HEK293 cell, the culture is a cell culture medium, and The method, wherein the purifying step comprises purifying the antibody by chromatographic methods.

21. 14. A pharmaceutical composition comprising an anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, and a pharma- ceutical acceptable excipient or carrier.

22. 14. A detection or diagnostic kit comprising an anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

23. The detection or diagnostic kit of claim 22, further comprising one or more reagents capable of detecting binding of the BCMA antibody or antigen-binding fragment thereof to BCMA.

24. 14. An antibody drug conjugate comprising the anti-BCMA antibody or antigen-binding fragment thereof of any one of claims 1 to 13, linked to a cytotoxic agent.

25. The antibody-drug conjugate of claim 24, wherein the cytotoxic agent is selected from the group consisting of MMAF, SN-38, and exatecan.

26. 25. Use of an anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or a pharmaceutical composition according to claim 21, or an antibody drug conjugate according to claim 24, in the preparation of a medicament for treating or preventing a BCMA-mediated disease or condition.

27. 14. Use of an anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 in the preparation of a kit for the detection, diagnosis or prognosis of a BCMA-mediated disease or condition.

28. The use of claim 26 or 27, wherein the BCMA-mediated disease or condition is cancer or an autoimmune disease.

29. The use of claim 28, wherein the cancer is a BCMA-expressing cancer and the autoimmune disease is selected from the group consisting of lupus erythematosus, IgA nephropathy, and rheumatoid arthritis.

30. The use of claim 29, wherein the BCMA-expressing cancer is lymphoma, leukemia, or myeloma.

31. The use of claim 29, wherein the BCMA-expressing cancer is multiple myeloma.

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