Anti-BCMA / anti-4-1BB bispecific antibody and its uses

Abstract

Provided are anti-BCMA (B-cell maturation antigen) / anti-4-1BB bispecific antibodies or antigen-binding fragments thereof, and uses thereof. The bispecific antibodies or antigen-binding fragments thereof can have high binding affinity for both BCMA protein and 4-1BB protein, and can be effectively used to prevent or treat diseases (e.g., cancer) associated with BCMA, 4-1BB, or both.

Description

[Technical Field]

[0001] The present invention relates to an anti-BCMA / anti-4-1BB bispecific antibody that can effectively block the interaction between BCMA and its ligand and activate 4-1BB signaling, as well as a method for producing the same and uses thereof. The bispecific antibody can have high binding affinity for both BCMA protein and 4-1BB protein. [Background technology]

[0002] B cell maturation antigen (BCMA) is a protein of approximately 20 kDa and belongs to the tumor necrosis factor receptor (TNFR) superfamily. BCMA is also known to be a ligand for BAFF (B-cell activating factor belonging to the tumor necrosis factor family) and APRIL (A proliferation-inducing ligand). In pathological conditions, BCMA is expressed in neoplastic plasma cells of patients with multiple myeloma (MM), and the survival rate of multiple myeloma patients with higher BCMA expression is lower (Moreaux et al., Eur J Haematol 2009 83: 119-129). Multiple myeloma is a neoplastic disease caused by monoclonal proliferation of plasma cells. The development of drugs such as thalidomide, bortezomib, and lenalidomide, as well as the development of treatment methods, has led to an increase in the initial treatment rate. However, survival rates for patients with multiple myeloma have not improved significantly. 4-1BB is a member of the TNFRSF (TNF-receptor superfamily) and a costimulatory molecule expressed after activation of immune cells, including innate and adaptive immune cells. 4-1BB plays an important role in regulating the activity of various immune cells. Agonist 4-1BB antibodies enhance immune cell proliferation, survival, cytokine secretion, and the cytolytic activity of CD8 T cells. Numerous other studies have shown that activation of 4-1BB enhances immune responses and eliminates tumors in mice. This suggests that 4-1BB is a promising target molecule in cancer immunology. Despite their antitumor efficacy, anti-4-1BB antibodies have induced severe liver toxicity in clinical applications. Multispecific antibodies that target two or more antigens have been developed in various types and forms and are expected to be novel therapeutic antibodies with superior therapeutic effects compared to monoclonal antibodies. Therefore, there is a need for the development of multispecific antibodies that are effective in the treatment of cancers such as multiple myeloma. Summary of the Invention [Problem to be solved by the invention]

[0003] An anti-BCMA (B-cell maturation antigen) / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is provided. Pharmaceutical compositions for the prevention or treatment of diseases involving BCMA, 4-1BB, or both are provided. Methods are provided for preventing or treating diseases involving BCMA, 4-1BB, or both in an individual. Provided are anti-BCMA / anti-4-1BB bispecific antibodies or antigen-binding fragments thereof for use in the prevention or treatment of diseases involving BCMA, 4-1BB, or both. [Means for solving the problem]

[0004] Reference will now be made in detail to the exemplary embodiments illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout the specification. Accordingly, the exemplary embodiments of the present invention may have different configurations and should not be construed as limited to the description set forth herein. Accordingly, the exemplary embodiments may be described solely for the purpose of illustrating the presently described aspects, with reference to the drawings. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. When preceding a list of elements, phrases such as "at least one of" modify the entire list of elements and not individual elements of the list. According to one embodiment of the present disclosure, the anti-BCMA (B-cell maturation antigen) / anti-4-1BB bispecific antibody, or antigen-binding fragment thereof, comprises an anti-BCMA antibody, or antigen-binding fragment thereof, and an anti-4-1BB antibody, or antigen-binding fragment thereof. The term "antibody" is used interchangeably with the term "immunoglobulin (Ig)." A whole antibody is a structure containing two full-length light chains and two full-length heavy chains linked by disulfide bonds. The antibody may be, for example, IgA, IgD, IgE, IgG, or IgM. The antibody may be a monoclonal or polyclonal antibody. The antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that is also a portion of the polypeptide that contains the antigen-binding site, for example, an scFv, (scFv)2, Fv, Fab, Fab', FvF(ab')2, or a combination thereof. There are five types of heavy chains, designated γ, δ, α, μ, and ε. The type of heavy chain defines the class of the antibody. Heavy chain type α and heavy chain type γ each consist of approximately 450 amino acids, while μ and ε each consist of approximately 550 amino acids. Each heavy chain has two regions: a variable region and a constant region. There are two types of light chains, designated λ and κ. Each light chain consists of approximately 211 to 217 amino acids. Each human antibody contains only one type of light chain. Each light chain contains two consecutive domains, including one constant region and one variable region. The variable region refers to the region of the antibody that binds to the antigen. The antibodies or antigen-binding fragments thereof can be modified. For example, the antibodies or antigen-binding fragments thereof can be modified by conjugation, glycosylation, tag attachment, or a combination thereof. The antibodies can also be conjugated to other drugs, such as anticancer drugs. For example, the antibodies or antigen-binding fragments thereof can be conjugated to horseradish peroxidase (HRP), alkaline phosphatase, haptens, biotin, streptavidin, fluorescent substances, radioactive substances, quantum dots, polyethylene glycol (PEG), histidine tags, or a combination thereof. The fluorescent material may be ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 568, ALEXA FLUOR® 680, ALEXA FLUOR® 750, ALEXA FLUOR® 790 or ALEXA FLUOR® 680. TM It's also 350. Anti-BCMA antibody or antigen-binding fragment thereof

[0005] The anti-BCMA antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 10 to 21, a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 22 to 32 and 54 to 64, or both the heavy chain variable region and the light chain variable region. The anti-4-1BB antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 81 to 91, a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 92 to 97, or both the heavy chain variable region and the light chain variable region. The heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof also comprises a CDR (complementarity-determining region)-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 17, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 21. The term "complementarity-determining region (CDER)" refers to the portion of an antibody variable region that confers binding specificity to the antibody or antigen-binding fragment thereof for an antigen. The light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof also comprises CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 25 and SEQ ID NOs: 54 to 62; CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 28; and CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 32, 63, and SEQ ID NO: 64. The anti-BCMA antibody or antigen-binding fragment thereof is also selected from the group consisting of: (1) an antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 10, CDR-H2 having the amino acid sequence of SEQ ID NO: 14, and CDR-H3 having the amino acid sequence of SEQ ID NO: 18; (2) an antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 11, CDR-H2 having the amino acid sequence of SEQ ID NO: 15, and CDR-H3 having the amino acid sequence of SEQ ID NO: 19; (3) an antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 12, CDR-H2 having the amino acid sequence of SEQ ID NO: 16, and CDR-H3 having the amino acid sequence of SEQ ID NO: 20; and (4) An antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 13, CDR-H2 having the amino acid sequence of SEQ ID NO: 17, and CDR-H3 having the amino acid sequence of SEQ ID NO: 21. The anti-BCMA antibody or antigen-binding fragment thereof is also selected from the group consisting of: (1) an antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 22, CDR-L2 having the amino acid sequence of SEQ ID NO: 26, and CDR-L3 having the amino acid sequence of SEQ ID NO: 29; (2) an antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 23, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 30; (3) an antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 24, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 31; (4) an antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 25, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 32; (5) an antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 31; (6) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 55, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 31; (7) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 56, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 31; (8) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 57, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 31; (9) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 58, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 31; (10) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 59, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 32; (11) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 60, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 32; (12) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 25, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 63; (13) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 25, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64; (14) An antibody or antigen-binding fragment 3 comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 61, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 63; (15) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 61, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64; (16) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 63; and (17) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 28, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64. The anti-BCMA antibody or antigen-binding fragment thereof also comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5. The anti-BCMA antibody or antigen-binding fragment thereof also comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 9 and SEQ ID NOs: 41 to 53. The BCMA may be a BCMA polypeptide or a fragment thereof. BCMA is also referred to as TNFRSF17 (tumor necrosis factor receptor superfamily member 17), BCM, CD269, TNFRSF13A, or TNF receptor superfamily member 17. The BCMA polypeptide may comprise the amino acid sequence of GenBank Accession No. NP_001183 (human) or the amino acid sequence of GenBank Accession No. NP_035738 (mouse). The BCMA polypeptide may comprise the amino acid sequence encoded by the polynucleotide of GenBank Accession No. NM_001192 (human) or the polynucleotide of GenBank Accession No. NM_011608 (mouse). The fragment may be a polypeptide comprising a portion of the amino acid sequence of the BCMA polypeptide. The anti-BCMA antibody or antigen-binding fragment thereof has affinity for a BCMA polypeptide or fragment thereof and is thereby capable of specifically binding to BCMA. The anti-BCMA antibody or antigen-binding fragment thereof can inhibit binding between the BCMA protein and a substance that specifically binds to the BCMA protein (also referred to as a ligand), such as B-cell activating factor belonging to the tumor necrosis factor family (BAFF), A proliferation-inducing ligand (APRIL), or a combination thereof. Anti-4-1BB antibody or antigen-binding fragment thereof

[0006] The heavy chain variable region of the anti-4-1BB antibody or antigen-binding fragment thereof also includes CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 81 to 83; CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 86; and CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 87 to 91. The light chain variable region of the anti-4-1BB antibody or antigen-binding fragment thereof also comprises CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 92 and 93; CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 94 and 95; and CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 and 97. The anti-4-1BB antibody or antigen-binding fragment thereof is also selected from the group consisting of: (1) an antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 81, CDR-H2 having the amino acid sequence of SEQ ID NO: 84, and CDR-H3 having the amino acid sequence of SEQ ID NO: 87; (2) an antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 81, CDR-H2 having the amino acid sequence of SEQ ID NO: 84, and CDR-H3 having the amino acid sequence of SEQ ID NO: 88; (3) an antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 81, CDR-H2 having the amino acid sequence of SEQ ID NO: 84, and CDR-H3 having the amino acid sequence of SEQ ID NO: 89; (4) an antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 82, CDR-H2 having the amino acid sequence of SEQ ID NO: 85, and CDR-H3 having the amino acid sequence of SEQ ID NO: 90; and (5) An antibody or antigen-binding fragment comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 83, CDR-H2 having the amino acid sequence of SEQ ID NO: 86, and CDR-H3 having the amino acid sequence of SEQ ID NO: 91. The anti-4-1BB antibody or antigen-binding fragment thereof is also selected from the group consisting of: (1) an antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 92, CDR-L2 having the amino acid sequence of SEQ ID NO: 94, and CDR-L3 having the amino acid sequence of SEQ ID NO: 96; and (2) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 93, CDR-L2 having the amino acid sequence of SEQ ID NO: 95, and CDR-L3 having the amino acid sequence of SEQ ID NO: 97. The anti-4-1BB antibody or antigen-binding fragment thereof is also selected from the group consisting of: (1) an antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 92, CDR-L2 having the amino acid sequence of SEQ ID NO: 94, and CDR-L3 having the amino acid sequence of SEQ ID NO: 96; and (2) An antibody or antigen-binding fragment comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 93, CDR-L2 having the amino acid sequence of SEQ ID NO: 95, and CDR-L3 having the amino acid sequence of SEQ ID NO: 97. The anti-4-1BB antibody or antigen-binding fragment thereof also comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-80. 4-1BB can be a 4-1BB polypeptide or a fragment thereof. 4-1BB is also referred to as CD137, CDw137, ILA, TNFRSF9 (tumor necrosis factor receptor superfamily member 9), or TNF receptor superfamily member 9. The 4-1BB polypeptide can include the amino acid sequence of GenBank Accession No. NP_001552 (human), or the amino acid sequence of GenBank Accession No. NP_001070976, NP_001070977, or NP_035742 (mouse). The 4-1BB polypeptide can also include the amino acid sequence encoded by the polynucleotide of GenBank Accession No. NM_001561 (human), or GenBank Accession No. NM_001077508, NM_001077509, or NM_011612 (mouse). The fragment is also a polypeptide containing a part of the amino acid sequence of the 4-1BB polypeptide. The anti-4-1BB antibody or antigen-binding fragment thereof has affinity for the 4-1BB polypeptide or a fragment thereof and can specifically bind to 4-1BB. The anti-4-1BB antibody or antigen-binding fragment thereof can enhance immune responses and / or treat tumors (cancers) in mammals. The anti-4-1BB antibody or antigen-binding fragment thereof is characterized by being localized and / or activated only in the tumor microenvironment (TME) and / or enhancing immune responses and / or exhibiting significantly reduced hepatotoxicity compared to existing anti-4-1BB antibodies while maintaining efficacy in tumor treatment. Anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof

[0007] In the above-mentioned anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof, the anti-BCMA antibody or antigen-binding fragment thereof, and the anti-4-1BB antibody or antigen-binding fragment thereof are each independently a chimeric antibody, a humanized antibody, or a human antibody. In the bispecific antibody, one of the BCMA targeting moiety and the 4-1BB targeting moiety can be a full-length antibody, and the other can be an antigen-binding fragment (e.g., scFv) comprising the heavy chain CDR, the light chain CDR, or a combination thereof. The full-length antibody targeting one of the BCMA protein and the 4-1BB protein and the antigen-binding fragment targeting the other protein can be chemically linked (e.g., covalently linked) directly or via a peptide linker. The antigen-binding fragment (e.g., scFv) can be linked to the N-terminus of the full-length antibody (e.g., the N-terminus of the light chain or heavy chain of the full-length antibody), the C-terminus of the full-length antibody (e.g., the C-terminus of the heavy chain (or Fc domain or CH3 domain) of the full-length antibody), or both, directly or via a peptide linker (see Figure 1). The anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment may further comprise one or more peptide linkers. The term "peptide linker" may comprise 1 to 100, particularly 2 to 50, amino acids, including any type of amino acid, without limitation. The peptide linker may include a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:98 and SEQ ID NO:99. In one embodiment, the bispecific antibody comprises a full-length anti-BCMA antibody, an antigen-binding fragment (e.g., scFv) of an anti-4-1BB antibody, and a peptide linker therebetween. In another embodiment, the bispecific antibody comprises a full-length anti-4-1BB antibody, an antigen-binding fragment (e.g., scFv) of an anti-BCMA antibody, and a peptide linker therebetween. In the above-mentioned anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof, the antibody may be IgA, IgD, IgE, IgG, or IgM. The antibody may be a monoclonal antibody or a polyclonal antibody. The antigen-binding fragment may be an scFv, (scFv)2, Fv, Fab, Fab', F(ab')2, or a combination thereof. The above-mentioned antibody or antigen-binding fragment thereof may be modified by conjugation or binding, glycosylation, tagging, or a combination thereof. In one embodiment, the scFv contained in the bispecific antibody comprises a heavy chain variable region and a light chain variable region in any order, for example, from N- to C-terminal, a heavy chain variable region and a light chain variable region, optionally with a peptide linker therebetween, or alternatively, a light chain variable region and a heavy chain variable region, optionally with a peptide linker therebetween, from N- to C-terminal. The aforementioned anti-BCMA / anti-4-1BB bispecific antibodies or antigen-binding fragments may be any of IgG-scFv, triomab, knobs-into-holes (kih) IgG with a common light chain, crossmab, ortho-Fab IgG, DVD-Ig (dual variable domain immunoglobulin), 2 in 1-IgG, scFv2-Fc, bi-nanobody, BiTE (bispecific T cell engager), tandAb, DART (dual affinity retargeting) antibody, DART-Fc, scFv-HSA (human serum albumin). The antibody may be in the form of an albumin (scFv), DNL (dock-and-lock)-Fab3, minibody, scFv-Fc, scFv-zipper, scFv, Fab, Fab2 (bispecific), Fab3 (trispecific), scFab, Bis-scFv (bispecific), sdAb (VH / VHH), tetrabody, triabody, diabody, camel Ig, IgNAR, IgG, bispecific construct comprising a knob in hole (KIH), bispecific construct including duobody, tetravalent multispecific antibody, tetravalent construct, tetravalent DVD (dual variable domain) construct, tetravalent IgGScv construct, tetravalent Mbatryn construct, or composite antibody, or a combination thereof. The anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment described above can activate 4-1BB signaling depending on BCMA expressed on the cell surface.

[0008] According to another aspect of the present disclosure, a pharmaceutical composition for the prevention or treatment of a disease associated with BCMA, 4-1BB, or both, comprises an anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to one of the preceding embodiments, and a pharmaceutically acceptable carrier. The BCMA, 4-1BB, anti-BCMA / anti-4-1BB bispecific antibody, and antigen-binding fragments described above are the same as those described above. The aforementioned diseases associated with BCMA, 4-1BB, or both may also be cancers. The cancers may be solid or non-solid cancers. Solid cancers refer to the development of cancerous tumors in solid organs such as the liver, lung, breast, or skin, while non-solid cancers are cancers that affect the blood and are also called hematological cancers. For example, the cancer may be selected from the group consisting of breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, uterine cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymic tumor, mesothelial tumor, esophageal cancer, bile duct cancer, testicular cancer, germinal cancer, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma. The term "prevention" refers to any act of administering the pharmaceutical composition to suppress or delay the onset of a disease associated with BCMA, 4-1BB, or both. The term "treatment" refers to any act of administering the pharmaceutical composition to alleviate the symptoms of a disease associated with BCMA, 4-1BB, or both. The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The term "carrier" may also be interpreted as meaning an excipient, diluent, or adjuvant. The carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, saline, buffers such as phosphate-buffered saline (PBS), methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, glycine, histidine, serine, polysorbate, and mineral oil. The pharmaceutical composition may also include a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof. The pharmaceutical composition can be prepared in any dosage form by a conventional method, for example, in an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral dosage form (e.g., injection). The pharmaceutical composition can also be prepared in a dosage form for systemic delivery or a dosage form for local delivery. The pharmaceutical composition may further comprise an anti-cancer drug, such as cetuximab, panitumumab, erlotinib, gefitinib, trastuzumab, T-DM1, pertuzumab, lapatinib, paclitaxel, tamoxifen, cisplatin, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, 5-fluorouracil (5FU), gemcitabine, or a combination thereof. The pharmaceutical composition may comprise a single composition or separate compositions. For example, the anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof of the pharmaceutical composition may be a composition in a parenteral dosage form, and the anti-cancer agent may be a composition in an oral dosage form. The pharmaceutical composition may also contain an effective amount of the antibody or antigen-binding fragment thereof, the anti-cancer agent, or a combination thereof. As used herein, the term "effective amount" refers to an amount sufficient to prevent or treat a disease associated with BCMA, 4-1BB, or both, when administered to an individual in need of such prevention or treatment. The effective amount can be appropriately selected by those skilled in the art depending on the cell or individual selected. For example, the effective amount may be determined based on factors such as the severity of the disease, the patient's age, weight, health, and sex, the patient's sensitivity to the drug, the duration of administration, the route of administration, the excretion rate, the duration of treatment, other factors including drugs used in combination with or simultaneously with the pharmaceutical composition, and other factors known in the medical field. The effective amount may also be about 0.5 μg to about 2 g of the pharmaceutical composition. The dosage of the pharmaceutical composition may be, for example, about 0.001 mg / kg to about 100 mg / kg for an adult, and the frequency of administration may be, for example, once a day, multiple times a day, once a week to once every four weeks, or once to 12 times a year. According to yet another aspect of the present disclosure, a method for preventing or treating a disease associated with BCMA, 4-1BB, or both, in an individual comprises administering to said individual an anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to one of the preceding embodiments. The BCMA, 4-1BB, anti-BCMA / anti-4-1BB bispecific antibody, antigen-binding fragment, prevention, and treatment are the same as those described above. The individual may be a mammal, such as a human, monkey, cow, horse, pig, dog, sheep, goat, or cat, and may be suffering from or susceptible to a disease involving BCMA, 4-1BB, or both, and the disease may be cancer. The method may further comprise administering to the individual in need thereof an anti-cancer agent, which may be administered simultaneously, separately, or sequentially with the anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to the exemplary embodiments described above. For example, the anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof, anti-cancer agent, or combination thereof can be administered directly to an individual by any method, such as oral, intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The antibody or antigen-binding fragment thereof, anti-cancer agent, or combination thereof can also be administered systemically or locally. The antibody or antigen-binding fragment thereof, anti-cancer agent, or combination thereof can be administered alone or together with a pharmaceutically active compound. The dose of the anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof, anti-cancer agent, or combination thereof varies depending on the patient's condition, weight, disease severity, drug dosage form, administration route, and administration duration, but can be appropriately selected by those skilled in the art. For example, the dose of the antibody or antigen-binding fragment thereof, anti-cancer agent, or combination thereof for an adult is about 0.001 mg / kg to about 100 mg / kg. The frequency of administration can be, for example, once daily, multiple times daily, once per week to once per four weeks, or once to 12 times per year. According to yet another aspect of the present disclosure, there is provided an anti-BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to one of the foregoing embodiments for use in the prevention or treatment of a disease involving BCMA, 4-1BB, or both. The aforementioned BCMA, 4-1BB, anti-BCMA / anti-4-1BB bispecific antibody, antigen-binding fragment, prevention, treatment, and diseases associated with BCMA, 4-1BB, or both are the same as those described above. [Effects of the Invention]

[0009] As described above, one or more exemplary embodiments provide anti-BCMA / anti-4-1BB bispecific antibodies or antigen-binding fragments thereof, and uses thereof. The anti-BCMA / anti-4-1BB bispecific antibodies or antigen-binding fragments thereof can be effectively used to prevent or treat diseases associated with BCMA, 4-1BB, or both. [Brief explanation of the drawings]

[0010] These and / or other aspects will become apparent and more readily understood from the following description of illustrative embodiments when considered in conjunction with the accompanying drawings: [Figure 1] FIG. 1 is a schematic diagram of a bispecific antibody according to one embodiment of the present disclosure. [Figure 2A]1 is a graph showing the results of a target protein binding test using DACE. [Figure 2B] 1 is a graph showing the results of a target protein binding test using DACE. [Figure 2C] 1 is a graph showing the results of a target protein binding test using DACE. [Figure 2D] 1 is a graph showing a cell surface binding test using a FACS system. [Figure 2E] 1 is a graph showing a cell surface binding test using a FACS system. [Figure 2F] 1 is a graph showing a cell surface binding test using a FACS system. [Figure 3A] 1 is a graph showing the results of a 4-1BB reporter bioassay. [Figure 3B] 1 is a graph showing the results of a 4-1BB reporter bioassay. [Figure 3C] 1 is a graph showing the results of a 4-1BB reporter bioassay. [Figure 3D] 1 is a graph showing the results of a 4-1BB reporter bioassay. [Figure 4A] 1 is a graph showing the results of PBMC-based research. [Figure 4B] 1 is a graph showing the results of PBMC-based research. [Figure 4C] 1 is a graph showing the results of PBMC-based research. [Figure 5A] 1 is a graph showing the results of a target protein binding test using DACE (5D5WT mutant and 5D5 mutant). [Figure 5B] 1 is a graph showing the results of a target protein binding test using DACE (5A6WT mutant and 5A6 mutant). [Figure 5C] 1 is a graph showing the results of a target protein binding test using DACE (WT and mutants). [Figure 5D]10 is a graph showing a cell surface binding test using a FACS system (WT and mutants). [Figure 6A] 1 is a graph showing the results of an in vitro 4-1BB activation test. [Figure 6B] 1 is a graph showing the results of an in vitro 4-1BB activation test. [Figure 6C] 1 is a graph showing the results of an in vitro 4-1BB activation test. [Figure 7] 1 is a graph showing the results of a monkey cross-reactivity test using DACE. [Figure 8A] 1 is a graph showing tumor growth inhibition by BCMAx4-1BB bispecific antibody in H929-bearing humanized NOG mice. [Figure 8B] 1 is a graph showing tumor growth inhibition by BCMAx4-1BB bispecific antibody in H929-bearing humanized NOG mice. [Figure 8C] 1 is a graph showing tumor growth inhibition by BCMAx4-1BB bispecific antibody in H929-bearing humanized NOG mice. [Figure 9A] An image showing tumor infiltrating lymphocytes (TILs) is shown. [Figure 9B] 1 is a graph showing the results of an analysis of tumor-infiltrating lymphocytes. [Figure 10A] 1 is a graph showing individual results (n=3) of the pharmacokinetics of anti-BCMA / anti-4-1BB bispecific antibodies in cynomolgus monkeys. [Figure 10B] 1 is a graph showing summing results of the pharmacokinetics of anti-BCMA / anti-4-1BB bispecific antibodies in cynomolgus monkeys. [Figure 11] Graph showing tumor growth profile after administration of anti-BCMA / anti-4-1BB bispecific antibody (*P<0.05; **P<0.01). DETAILED DESCRIPTION OF THE INVENTION

[0011] One or more embodiments of the present disclosure will be described in detail with reference to the following examples, however, such examples are for illustrative purposes only and are not intended to limit the scope of one or more embodiments of the present disclosure. Example 1. Production of anti-BCMA monoclonal antibodies

[0012] 1-1. Antigen production Antigens for anti-BCMA antibody production were prepared as follows: Polypeptides containing amino acid residues 5 to 54, 1 to 54, and 4 to 48, respectively, from the N-terminus of the amino acid sequence of human BCMA (GenBank Accession No. NP_001183.2, SEQ ID NO: 1) were used as antigens. Specifically, we produced an antigen comprising amino acid residues 5 to 54 of human BCMA (GENSCRIPT®, Z02731) ("human BCMA-His(5-54)"); an antigen comprising amino acid residues 1 to 51 of human BCMA fused to the Fc region of human IgG1 (self-produced and expressed in CHO cells) ("human BCMA-Fc(1-51)"); an antigen comprising amino acid residues 1 to 54 of human BCMA fused to an Fc region and a His tag at the C-terminus (10620-H03H, Sino Biological Inc.) ("human BCMA-Fc / His(1-54)"); and an antigen comprising amino acid residues 4 to 48 of human BCMA fused to the Fc region (self-produced and expressed in HEK293 cells) ("human BCMA-Fc(4-48)"). Human BCMA-Fc(4-48) was prepared as follows. A polynucleotide encoding amino acid residues 4 to 48 of human BCMA was cloned into pAB1-Fc, an animal cell expression vector containing a CMV promoter. The cloned vector was transformed into HEK293E cells, and human BCMA-Fc(4-48) was purified using protein A affinity chromatography. Human BCMA-Fc(1-51) was prepared in the same manner as described above. 1-2. Library phage production and phage display panning Human-derived single-chain variable fragment (ScFv) library cells capable of binding to diverse antigens (Mol. Cells OT, pp. 225-235, February 28, 2009) were produced. The produced phage library was infected with helper phage, followed by phage packaging induction. The culture product was then centrifuged at 4,500 rpm for 15 minutes at 4°C. 4% (w / v) PEG 6000 (Fluka, 81253) and 3% (w / v) NaCl (Sigma, S7653) were added to the supernatant, dissolved thoroughly, and incubated on ice for 1 hour. The resulting product was centrifuged at 8,000 rpm for 20 minutes at 4°C. The pellet was suspended in PBS and further centrifuged at 12,000 rpm for 10 minutes at 4°C to obtain the supernatant containing the library phage. The resulting library phages were stored at 4°C until use. To screen for antibodies reactive with human BCMA or cross-reactive with both human and monkey BCMA, panning was performed three times using the following method. Five micrograms of the antigen prepared in Example 1-1 was added to an immunotube (Maxisorp 444202), which was then incubated at 4°C for 16 hours to coat the surface of the tube with protein. The supernatant was removed, and bovine serum albumin (BSA) was added to block nonspecific binding. 10 prepared according to Example 1-2 12CFU of the phage library was mixed with 1.5% (w / v) BSA, and the resulting mixture was added to a target protein-coated immunoassay tube and incubated at 37°C for 1 hour to allow BCMA-specific phages to bind to the target protein. After washing several times with PBS-T (phosphate-buffered saline containing 0.05% (v / v) Tween 20), BCMA-bound phages were recovered using 100 mM triethylamine. The recovered phages were neutralized with 1 M Tris buffer (pH 7.4) and infected with K12 ER2738 E. coli, followed by further phage recovery. This cycle of target binding, elution, neutralization, infection, and recovery was repeated four times. As the panning rounds progressed, the number of washes using PBS-T was increased to amplify and enrich antigen-specific phages. 1-3. Single clone phage antibody screening A monoclonal phage antibody screening procedure was performed to select monoclonal antibodies that specifically bind to BCMA from the phage pool. Specifically, the phage pool obtained in Example 1-2 was serially diluted and cultured on solid medium containing LB-tetracycline / carbenicillin to obtain single colonies. Each colony was cultured in a 96-deep-well plate until the OD600 reached 0.5-0.7. 20 MOI of helper phage was added to each well and incubated at 37°C for 1 hour. Kanamycin was then added to each well and incubated overnight at 30°C. The next day, the culture was centrifuged, the supernatant was collected, and ELISA was performed to select BCMA-specific phages. Each well of the ELISA plate was coated with 100 ng of recombinant BCMA and incubated with PBS-B (PBS containing 3% BSA) to prevent nonspecific binding. The plate was then washed with PBS. The prepared single clone phage was added to each well and incubated at 37°C for 1 hour, and the plate was washed three times with PBS-T. To detect bound phages, horseradish peroxidase (HRP)-conjugated anti-HA (hemagglutinin) antibody was added to each well. After washing with PBS-T, tetramethylbenzidine (TMB, Sigma, T0440) was added. Clones with an absorbance of 0.5 or greater at 450 nm and 5-fold greater than the absorbance of the control group containing anti-HAHRP were selected. Four antibody clones (B58, 5B5, 5D5, and 5A6) that specifically bound to human BCMA were selected. The amino acid sequences of the heavy chain variable regions (SEQ ID NOS: 2 to 5) and the light chain variable regions (SEQ ID NOS: 6 to 9) were analyzed from the nucleotide sequences encoding the selected antibodies, and the complementarity-determining regions (CDRs) were determined according to the Kabat definition. The determined heavy and light chain CDR amino acid sequences (N to C) are shown in Tables 1 and 2, respectively. [Table 1] [Table 2] The nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region are shown in Table 3 below. [Table 3] 1-4. Production of anti-BCMA IgG antibodies from selected anti-BCMA phages Polynucleotides containing nucleotide sequences encoding the antibodies selected in Examples 1-3 were synthesized. The prepared polynucleotides were cloned into animal cell culture vectors (heavy chain expression vector: pAB1-HC, and light chain expression vector: pAB1-LC). A total of eight vectors containing polynucleotides encoding the heavy and light chains of four antibody clones (B58, 5A6, 5D5, and 5B5) were prepared. Each of the vectors prepared for pAB1-HC contained an IgG1-type sequence. CHO-S cells were cultured in CD-CHO (Gibco, 10743) medium, and the prepared vector was introduced into the CHO-S cells using polyethyleneimine (PEI). The transduced CHO-S cells were cultured in CD-CHO medium at 37°C with shaking (110 rpm) in 8% CO for approximately 7 days. Culture supernatants were collected and passed through a MabSelect SuRe column (GE Healthcare, 5 ml) equilibrated with equilibration buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl) to allow the expressed antibodies to bind to the column. The antibodies were eluted with a solution of 50 mM sodium citrate (pH 3.4) and 100 mM NaCl and then neutralized with 1 M Tris-HCl (pH 9.0) to a final pH of 7.2. The buffer was then exchanged with PBS (pH 7.4) and the anti-BCMA IgG antibodies, B58, 5A6, 5D5, and 5B5, were stored at 4°C until use. 1-5. Preparation of 5A6 and 5D5 Mutants To improve the productivity of the selected 5A6 and 5D5 antibodies, one or two amino acid residues in the light chain CDR of the antibody were mutated to produce mutant antibodies with the nucleotide sequences shown in Table 3. The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the 5D5 mutant antibody and the 5A6 mutant antibody are shown in Tables 4 and 5, respectively. In Tables 4 and 5, the underlined and bolded amino acid residues represent the mutated moieties (WT: wild type, LM: light chain mutation). The light chain variable regions of the 5D5 mutant antibody and the 5A6 mutant antibody have the amino acid sequences of SEQ ID NOs: 41 to 45 and 46 to 53, respectively. [Table 4] [Table 5] Example 2. Production of anti-4-1BB monoclonal antibody

[0013] Screening of fully human monoclonal antibodies against 2-1.4-1BB (phage library immunotube panning) For panning of the library against the target molecule, 4-1BB-coated immunotubes were used, and a total of four rounds of panning were performed. Bacterial colonies from the third panning output were cultured in SB medium containing carbenicillin in 96-deep-well plates until turbid, at which point 10 11 pfu of VCSM13 helper phage was added to each well. After incubation at 37°C for 1 hour with gentle shaking (80 rpm), 70 μg / mL kanamycin was added, and the cells were grown overnight at 30°C with shaking at 200 rpm. The next day, the plates were centrifuged, and the phage-containing supernatant was added to a 4-1BB antigen-coated ELISA plate blocked with 3% (w / v) BSA in PBST. After incubation at room temperature for 1 hour, the plate was washed three times with PBST, and anti-M13 antibody was added. The plate was incubated for 1 hour, washed three times with PBST, and the binding activity was measured using tetramethylbenzidine (TMB). 4-1BB-specific antibodies were amplified for plasmid DNA sequencing. The heavy and light chain variable regions (VH and VL) were analyzed to identify unique sequences and determine sequence diversity. Three antibody clones (41B01, 41B02, and AB41) that specifically bind to human 4-1BB were selected. From the selected antibodies, 41B01 M4 mutant, 41B01 M11 mutant, 41B01 M12 mutant, 41B01 M13 mutant, and 41B02 M1 mutant were produced as described in Examples 1-5. The amino acid sequences of the antibody heavy chain variable regions (SEQ ID NOS: 65 to 72) and light chain variable regions (SEQ ID NOS: 73 to 80) were analyzed from the nucleotide sequences encoding the selected antibodies, and CDRs were determined according to the Kabat definition. The determined heavy and light chain CDR amino acid sequences (N to C) are shown in Tables 6 and 7, respectively. [Table 6] [Table 7] 2-2. Antigen-binding ability of anti-4-1BB antibodies to human 4-1BB (1) Antigen binding measured by ELISA To evaluate antigen-binding activity, antibody candidates were subjected to ELISA testing. Briefly, microtiter plates were coated overnight at 4°C with 100 μl / well of 0.1 μg / mL human 4-1BB-Fc protein in PBS and blocked with 100 μl / well of 5% (w / v) BSA. Starting at 10 μg / mL, five-fold serial dilutions of humanized antibody 41B01 and humanized antibody 41B02 were added to each well and incubated at RT for 1 to 2 hours. The plates were washed with PBS / Tween and then incubated with chlorinated anti-human IgG antibody conjugated with HRP (horseradish peroxidase) at RT for 1 hour. After washing, the plates were developed using TMB substrate and analyzed spectrophotometrically at OD 450-630 nm. (2) Cell binding measured by FACS To evaluate antigen-binding properties, antibody candidates were analyzed for their binding to mammalian-expressed 4-1BB by FACS. Briefly, 4-1BB-Jurkat cells were incubated with antibodies (41B01 and 41B02). After washing with FACS buffer (1% (w / v) BSA in PBS), FITC-anti-human IgG antibody was added to each well and incubated for 1 hour at 4°C. The MFI of FITC was assessed using a FACS caliber. (3) Protein kinetics related to 4-1BB To explore the binding kinetics of the humanized antibodies, this example performed affinity rankings using Octet Red 96. 41B01 and 41B02 were tested, as shown in Table 8 below. [Table 8] As shown in Table 8, the anti-4-1BB antibodies tested exhibited high 4-1BB binding affinity. Example 3. Characterization of anti-BCMA / anti-4-1BB bispecific antibodies 3-1. Production of anti-BCMA / anti-4-1BB bispecific antibody Various anti-BCMA / anti-4-1BB bispecific antibody candidates were produced in the full-length IgG (anti-BCMA antibody)-scFv (anti-4-1BB antibody) format shown in Table 9. The constant region of the anti-BCMA antibody contained in the bispecific antibody can be further modified by introducing more than one mutation into human IgG. In one example, an NA mutation (N297A) was introduced. [Table 9] To prepare anti-BCMA / anti-4-1BB bispecific antibodies in the form of IgG-scFv fusions in which an scFv antibody fragment of one antigen is fused to the C-terminus of an IgG fragment of the other antigen, the anti-BCMA IgG clone and anti-4-1BB scFv clone prepared in Examples 1 and 2, respectively, were selected as exemplary antibodies. When BCMA was located in the full-length IgG moiety, an IgG1 with a mutant backbone with reduced ADCC (N297A mutation; Cancer Cell, Vol. 19, Issue 1, pp. 101-113, etc.) was used, and when 4-1BB was located in the full-length IgG moiety, an IgG4 was used. The amino acid sequences of the anti-BCMA IgG and 4-1BB scFv are listed in Tables 10 and 11, respectively. [Table 10] JPEG0007813704000011.jpg175135JPEG0007813704000012.jpg176135JPEG0007813704000013.jpg134135JPEG00078137040 00014.jpg124124JPEG0007813704000015.jpg133131JPEG0007813704000016.jpg125125JPEG0007813704000017.jpg162162 [Table 11] DNA segment 1, containing the nucleotide sequence encoding the heavy chain of the IgG antibody of the anti-BCMA / anti-4-1BB bispecific antibody, was inserted into pcDNA3.4 (Invitrogen, A14697; Plasmid 1), and DNA segment 2, containing the nucleotide sequence encoding the light chain of the IgG antibody of the anti-BCMA / anti-4-1BB bispecific antibody, was inserted into pcDNA3.4 (Invitrogen, A14697; Plasmid 2). DNA segment 3, encoding the scFv, was then fused to the C-terminus of the Fc region of the IgG antibody inserted into Plasmid 1 using DNA segment 4, encoding a 20-amino acid linker peptide consisting of (GGGGS)4 (SEQ ID NO: 98), or DNA segment 5, encoding an 18-amino acid linker peptide consisting of (GS)9 (SEQ ID NO: 99), to prepare a vector for expressing the bispecific antibody. To stabilize the scFv, further modifications were applied to generate disulfide bridges by fusing VL103-VH44 (VL103: VL with a G→C mutation at position 103; VH44: VH with a G→C mutation at position 44) to the C-terminus of the light chain and the C-terminus of the heavy chain, respectively, as described in Example 2. The amino acid sequences of the bispecific antibodies produced are presented in Table 12. [Table 12] JPEG0007813704000020.jpg130130JPEG0007813704000021.jpg165133For purposes of improved stability and efficacy, reduced immunogenicity, etc., one or more point mutations may be applied in one of the amino acid sequences in the antibodies presented below. 3-2. Antigen-binding ability of anti-BCMA / anti-4-1BB antibodies (full-length IgG form) to target proteins (1) Antigen binding measured by DACE (dual antigen capture ELISA) To evaluate antigen-binding activity, antibody candidates were subjected to ELISA testing. Briefly, microtiter plates were coated overnight at 4°C with 100 μl / well of 0.5 μg / mL human BCMA-Fc protein in PBS and blocked with 100 μl / well of 1% (w / v) BSA. Starting at 20 μg / mL, three-fold dilutions of bispecific antibodies were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with 0.8 μg / mL human 4-1BB his protein containing 1% (w / v) BSA for 1 hour at 37°C. The plates were then washed with PBST (0.05% (v / v) Tween 20 in PBS). The plates were then developed using TMB substrate and analyzed by spectrophotometer at OD 450-630 nm. As shown in Figures 2A to 2C, all bispecific antibodies can bind to human BCMA and 4-1BB proteins simultaneously in a dose-dependent manner. The EC50 (nM) values ​​are summarized in Table 13. [Table 13] (2) Cell surface binding test using flow cytometry To evaluate antigen-binding properties, antibody candidates were analyzed for their binding to BCMA-expressing cells by FACS. Briefly, human BCMA-expressing CHOK1 (CHOK1-hBCMA) or endogenously BCMA-expressing H929 cells were treated with 100 nM of the indicated antibodies for 1 hour at 4°C. CHOK1 cells were used as a BCMA-negative control. After washing with FACS buffer (1% (w / v) BSA in PBS), the cells were incubated with FITC-anti-human IgG antibody for 1 hour at 4°C and subjected to FACS analysis. As shown in Figures 2D-2F, the anti-BCMAx4-1BB antibody bound to the BCMA-expressing CHOK1-hBCMA and H929 cell lines but not to the BCMA-negative CHOK1 cells. This result indicates that the anti-BCMA / anti-4-1BB bispecific antibody can specifically bind to the tumor-targeting antigen (BCMA). The results in Figures 2D-2F were quantified and summarized in Table 14. [Table 14] 3-3. In vitro 4-1BB activation test of anti-BCMA / anti-4-1BB bispecific antibodies (4-1BB reporter bioassay) In this assay, GloResponse™ vectors were genetically modified to stably express human 4-1BB and luciferase downstream of the response element. TM The NFκB-luc2 / 4-1BB Jurkat cell line was used as the effector cell, and cancer cells expressing or not expressing BCMA were used as the target cells. Briefly, CHOK1-hBCMA (BCMA-positive, 2.5x10 4 ), or CHOK1 (BCMA negative, 2.5x10 4 ) are plated into a white 96-well assay plate containing 100 μl of culture medium. The plate is then incubated overnight at 37°C in a 5% CO2 humidified incubator. After overnight incubation, 100 μl of culture medium is removed, and 25 μl of assay medium (RPMI 1640 containing 1% (v / v) FBS) is dispensed onto the pre-plated target cells. For suspension cells, H929 (BCMA-positive, 2.5x10 5 ) or Jurkat (BCMA negative, 2.5x10 5 ) was plated into a white 96-well assay plate containing 25 μl of assay medium. 25 μl of each bispecific antibody (starting at 50 nM, 5-fold dilutions, or starting at 200 nM, 4-fold dilutions) was added to the plate. TM NFκB-luc2 / 4-1BB Jurkat cell line was harvested and resuspended in assay medium. 25 μl of GLORESPONSE TM NFκB-luc2 / 4-1BB Jurkat cell line was cultured at 2.5x10 cells per well. 4 Dispense the cells into plates at individual cells. Incubate for 6 hours at 37°C in a 5% CO2 humidified incubator. Reconstitute the BIO-GLO™ Reagent according to the manufacturer's instructions for the incubation period. After the 6-hour incubation, add 75 μl of BIO-GLO™ Reagent per well of the assay plate. TMAdd reagent, wait 5 minutes, and measure luminescence using a microplate reader. GraphPad software was used to evaluate a four-parameter logistic curve. Experimental results using CHOK1-hBCMA cells, CHOK1 cells, H929 cells, and Jurkat cells are shown in Figures 3A to 3D, respectively. As shown in Figures 3A-3D, the BCMAx4-1BB bispecific antibody exhibited stronger 4-1BB signal activation in the presence of tumor antigen (BCMA) than the anti-4-1BB monoclonal antibody alone or cross-linked 4-1BB. Furthermore, the agonistic anti-4-1BB monoclonal antibody BMUR (reference antibody) also activated 4-1BB in vitro in the absence of BCMA (Figures 3B-3D). This means that anti-BCMA / anti-4-1BB antibodies can specifically act in the presence of BCMA-expressing cancer cells, but agonistic anti-4-1BB antibodies cannot. The results in Figures 3A to 3D were quantified and summarized in Tables 15 and 16 (Table 15: CHOK1-hBCMA (EC50, nM); Table 16: H929 (EC50, nM)). [Table 15] [Table 16] 3-4. In vitro 4-1BB activation test of anti-BCMA / anti-4-1BB bispecific antibodies (human PBMC-based test) Human PBMCs were co-cultured with the CHOK1 cell line (which does not express human BCMA) or genetically modified CHOK1-hBCMA (which stably expresses human BCMA) in the presence of anti-human CD3 antibody and the antibody to be tested. Briefly, PBMCs were cultured at 3x10 per well. 4 Plate CHOK1 or CHOK1-hBCMA at 1x10 cells per well. 4The cells were co-plated with 1000 μg of IFN-gamma antibody. Bispecific antibodies (starting at 20 nM and diluted 4-fold) and monoclonal antibodies (starting at 26.67 nM and diluted 4-fold) were added to the plate wells. After incubation, the concentration of IFN-gamma secreted in the supernatant was measured using the Human IFN-gamma Quantitation Kit (R&D system, SIF50). As shown in Figures 4A-4C, the anti-BCMA / anti-4-1BB antibodies induced greater cytokine secretion than either the monoclonal antibodies or BMUR (agonistic anti-4-1BB monoclonal antibody). 3-5. Comparison of target protein binding activity (wild type vs. mutant) To stabilize the anti-BCMA / anti-4-1BB bispecific antibodies, one or more point mutations in the amino acid sequence were introduced into the heavy chain or light chain CDR of the antibodies, as shown in Table 4. To evaluate the antigen-binding activity, the antibody candidates (wild-type clones and mutant clones) were subjected to a dual antigen capture ELISA (DACE) test, as performed in Example 3-2(1). The results are shown in Figures 5A and 5B. As shown in Figures 5A and 5B, all bispecific antibodies could simultaneously bind to human BCMA and 4-1BB proteins in a dose-dependent manner. Numerous mutations were identified that improved target protein binding. Among these mutations, 5D5M4(NA)x41B01 M12 and 5A6M6(NA)x41B01 M12 exhibited superior antigen-binding activity compared to the wild-type clone (Figure 5C). To evaluate their native antigen-binding properties, 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 were analyzed for their binding to mammalian-expressed BCMA by FACS, as performed in Example 2-2(2). As shown in Figure 5D, 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 showed increased native antigen-binding activity compared to wild-type. The results of protein binding and cell binding assays were quantified and are summarized in Tables 17 and 18 (Table 17: Protein Binding (EC50, nM)). [Table 17] [Table 18] 3-6. Binding affinity (SPR) of mutant bispecific antibodies to target proteins BCMA and 4-1BB In SPR experiments, the anti-BCMA / anti-4-1BB bispecific antibodies obtained in Examples 3-5 were individually captured on flow cells 2, 3, and 4 of a Protein A chip on which anti-BCMA / anti-4-1BB bispecific antibodies (5D5M4(NA)X41B01 M12 or 5A6M6(NA)X41B01 M12) had been immobilized by amine coupling, while flow cell 1 was maintained as the reference. Recombinant human BCMA or human 4-1BB protein was flowed across the chip at concentrations ranging from 100 nM to 6.25 nM for human BCMA, or from 250 nM to 15.625 nM and 0.78 nM for human 4-1BB, at 30 μl / min for 300 seconds, followed by a 400-second dissociation phase. Regeneration was performed using 10 mM glycine-HCl (pH 2.0). The results are shown in Tables 19 and 20 below. As shown in Tables 19 and 20, 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 showed high affinity for BCMA and 4-1BB (Table 19: Affinity measurement results for 5D5M4(NA)X41B01 M12, Table 20: Affinity measurement results for 5A6M6(NA)X41B01 M12). [Table 19] [Table 20] 3-7. In vitro 4-1BB activation test for mutant bispecific antibodies Antibody candidates were analyzed for their in vitro 4-1BB activity using the Promega kit system as in Examples 2 and 3. As shown in Figures 6A-6C, the mutant formats of the anti-BCMA / anti-4-1BB bispecific antibody showed improved potency in BCMA-positive cancer cells compared to the wild-type format, and all clones did not activate 4-1BB signaling from BCMA-negative cancer cells (Jurkat) (Table 21: In vitro 4-1BB activation results (EC50, nM)). Thus, 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 demonstrated improved target-mediated 4-1BB activation. [Table 21] 3-8. Monkey cross-reactivity test for mutant bispecific antibodies To evaluate cross-reactivity, antibody candidates (5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12) were applied to ELISA tests. Briefly, microtiter plates were coated with rhesus BCMA-Fc protein (50 ng / well) overnight at 4°C and blocked with 200 μl / well PBSB (1% (w / v) BSA in PBS). Starting from 100 nM, 3-fold dilutions of 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBST (0.05% (v / v) Tween 20 in PBS) for 1 hour and incubated with rhesus 4-1BB-His protein (80 ng / well) for 1 hour at 37°C. The plates were washed with PBST (0.05% (v / v) Tween 20 in PBS) and incubated with HRP (horse radish peroxidase)-conjugated anti-His antibody for 1 hour at 37°C. After washing, the plates were developed using TMB substrate and analyzed spectrophotometrically at 450-630 nm. As shown in Figure 7, 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 simultaneously bound to rhesus BCMA and 4-1BB in a dose-dependent manner (Table 22: Protein Binding (EC50, nM)). [Table 22] 3-9. Tumor growth suppression by mutant bispecific antibodies in H929-bearing humanized NOG mice The anti-tumor effect of anti-BCMA / anti-4-1BB antibodies was tested in humanized NOG mice injected with H929 cells. Briefly, H929 cells were injected into the right flank of 74 non-irradiated female animals with NCI-H929 cells (5x10 6 On day 11, purified T-cells (10x10) from three donors were injected subcutaneously. 6 ) was injected intraperitoneally into mice. The tumors were 154 mm 3 When the average body weight reached 1000 mg / kg (day 12), the animals were randomly divided into four groups of 14 mice per group. The mice were intravenously administered the following antibodies 5 times (5 injections of antibody every 3 days) at Q3D: human IgG control group (7.5 mg / kg, No T-cell injections); isotype control group (7.5 mg / kg, No T-cell injections); T cells ), a human IgG control group (7.5 mg / kg; allogeneic control group_7.5 mg / kg), an anti-BCMA / anti-4-1BB bispecific antibody (5D5M4(NA)X41B01 M12, 10 mg / kg), and an anti-BCMA / anti-4-1BB bispecific antibody (5A6M6(NA)X41B01 M12, 10 mg / kg). Tumor volume was monitored twice weekly during the experimental period by caliper measurement, and the results are shown in Figures 8A to 8C. As shown in Figures 8A and 8B, 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 demonstrated significant anti-tumor effects. The tumor growth inhibition rate (TGI%) was 44.8% for 5D5M4(NA)X41B01 M12 and 49.4% for 5A6M6(NA)X41B01 M12, and the efficacy of the two bispecific antibodies was therefore similar. 3-10. Analysis of tumor-infiltrating lymphocytes (TIL) To evaluate TILs, formalin-fixed, paraffin-embedded tumor tissue sections from hPBMC-bearing H929 mice were immunostained with anti-CD45 antibody (a human leukocyte marker) and anti-CD8 antibody (a human cytotoxic T-lymphocyte marker). An avidin-biotin detection kit was applied to perform immunochemistry. The results are shown in Figures 9A and 9B. As shown in Figures 9A and 9B, the anti-BCMA / anti-4-1BB bispecific antibody effectively increased the infiltration of immune cells, including CD45+ cells and CD8+ T cells, in tumor tissue compared with peritumor tissue. These results indicate that CD45+ cells and CD8+ cells were particularly increased in the tumor compartment in the anti-BCMA / anti-4-1BB bispecific treatment group. Pharmacokinetics of anti-BCMA / anti-4-1BB bispecific antibodies in cynomolgus monkeys Cynomolgus monkeys were injected with 10 mg / kg of anti-BCMA / anti-4-1BB bispecific antibodies (5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12) via the saphenous vein. Blood samples were collected from each animal via the femoral vein before and at designated intervals from 0.05 to 504 hours after dose administration. Serum was obtained by centrifugation of the blood samples. The concentrations of 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 in cynomolgus monkey serum were measured using enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated with human BCMA-Fc protein and blocked with blocking buffer. The plates were then washed, and the standards, quality control samples, and research samples were added to the plates and incubated at 37°C for 2 hours. The plates were then washed again and incubated with human 4-1BB His protein. After washing, bound molecules were detected using an HRP-conjugated anti-His tagged antibody (horse radish peroxidase-conjugated anti-His tagged antibody). The plates were developed with TMB substrate and analyzed spectrophotometrically at OD 450-650 nm. Concentrations from serum samples were determined using a four-parameter algorithm from a standard curve constructed with known amounts of 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 in the appropriate cynomolgus monkey serum. The standard curve range for 5D5M4(NA)X41B01 M12 and 5A6M6(NA)X41B01 M12 was 46 to 300,000 ng / mL, and the lower limit of quantitation (LLOQ) was determined to be 300 ng / mL. Pharmacokinetic parameters were calculated using a non-compartmental model using WinNonlin software (Phoenix WinNonlin 8.0). The results are shown in Figures 10A and 10B.The results in Figures 10A and 10B were quantified and summarized in Tables 23 and 24, respectively (WinNonlin settings, NCA, Linear Trapezoidal Linear Interpolation, IV Bolus, half-life calculation: 24 hours to 240 hours). The results show that 5D5M4(NA)X41B01 M12 has superior PK properties than 5A6M6(NA)X41B01 M12. [Table 23] [Table 24] 3-12. Tumor growth suppression by anti-BCMA / 4-1BB bispecific antibody in 4-1BB knock-in mice with BCMA-overexpressing MC38 The in vivo anti-tumor efficacy of anti-BCMA / anti-4-1BB bispecific antibodies was evaluated using a BCMA-overexpressing MC38-carrying human 4-1BB knock-in mouse system (CRO: Biocytogen). 6 ) cells were inoculated by subcutaneous injection into the right flank of non-irradiated female animals. On day 7, the mean tumor volume was 110 mm 3 Upon reaching the target tumor size, mice were randomly divided into three groups (n=8 / group). Subsequently, 7.5 mg / kg of an isotype control antibody (hIgG1) and 2 or 0.4 mg / kg of an anti-BCMA / anti-4-1BB bispecific antibody (5D5M4(NA)x41B01 M12) were intravenously administered on a Q3D (once every three days) schedule for a total of eight doses. Tumor volume was monitored twice weekly by caliper measurement to obtain tumor growth data. The results are shown in Figure 11 and Table 25. Table 25 summarizes the tumor growth inhibition results of 5D5M4(NA)x41B01 M12 in MC38-hBCMA-bearing 4-1BB knock-in mice (*P<0.05; **P<0.01). In Table 25, Q3D means a dosing frequency of once every three days. [Table 25] As shown in Figure 11 and Table 25, 5D5M4(NA)x41B01 M12 demonstrated anti-tumor efficacy. The tumor growth inhibition rate (TGI%) was approximately 62.2% for G2 (5D5M4(NA)x41B01 M12, 2 mg / kg) and approximately 47.4% for G3 (5D5M4(NA)x41B01 M12, 0.4 mg / kg). Taken together, the anti-BCMA / anti-4-1BB bispecific antibody demonstrated excellent anti-tumor efficacy in human BCMA / MC38 tumors.

Claims

1. an anti-BCMA antibody or antigen-binding fragment thereof; an anti-BCMA (B-cell maturation antigen) / anti-4-1BB bispecific antibody or antigen-binding fragment thereof, comprising an anti-4-1BB antibody or antigen-binding fragment thereof, The anti-BCMA (B-cell maturation antigen) / anti-4-1BB bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 27, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 31, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 54, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 27, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 31, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 55, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 27, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 31, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 56, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 27, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 31, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 57, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 27, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 31, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 58, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 27, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 31, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 25, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 32, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 59, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 32, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 60, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 32, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 25, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 63, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 25, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 64, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 61, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 63, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 61, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 64, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; a bispecific antibody comprising an anti-BCMA antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 62, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 63, and an anti-4-1BB antibody or antigen-binding fragment thereof comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 96; and an anti-BCMA (B-cell maturation antigen) / anti-4-1BB bispecific antibody or antigen-binding fragment thereof selected from the group consisting of bispecific antibodies comprising: an anti-BCMA antibody or antigen-binding fragment thereof, comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 13, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 21, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 62, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 64; and an anti-4-1BB antibody or antigen-binding fragment thereof, comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 81, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 84, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 88, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 92, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 94, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO:

96.

2. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 5, and / or the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, and SEQ ID NOs: 41 to 53.

3. the anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68; and The bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-4-1BB antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

76.

4. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising one or more peptide linkers.

5. The bispecific antibody or antigen-binding fragment thereof of claim 4, wherein the peptide linker comprises a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO:

99.

6. the antibody is IgA, IgD, IgE, IgG or IgM; the antibody is a monoclonal or polyclonal antibody; the antigen-binding fragment is an scFv, (scFv)2, Fv, Fab, Fab', F(ab')2, or a combination thereof; or The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is modified by conjugation or binding, glycosylation, tagging, or a combination thereof.

7. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the anti-BCMA antibody or antigen-binding fragment thereof, and the anti-4-1BB antibody or antigen-binding fragment thereof are each independently a chimeric antibody or a human antibody.

8. The bispecific antibody or antigen-binding fragment thereof may be any of IgG-scFv, triomab (registered trademark), kih (knobs into holes) IgG with a common light chain, crossmab (registered trademark), ortho-Fab IgG, DVD-Ig (dual variable domain immunoglobulin), 2 in 1-IgG, scFv2-Fc, BiTE (bispecific T cell engager) (registered trademark), tandAb, DART (registered trademark) (dual affinity retargeting) antibody, DART-Fc, scFv-HSA (human serum albumin), and scFv-HSA (human serum albumin).

8. The bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 7, in the form of a bispecific antibody comprising a bis-scFv (Bis-albumin), a dock-and-lock (DNL)-Fab3, a minibody, a scFv-Fc, a scFv-zipper, a scFv, a Fab, a Fab2 (bispecific), a Fab3 (trispecific), a scFab, a Bis-scFv (bispecific), a tetrabody, a triabody, a diabody, an IgG, a bispecific construct comprising a knob in hole (KIH), a bispecific construct comprising a DuoBody®, a tetravalent multispecific antibody, a tetravalent construct, a tetravalent DVD (dual variable domain)™ construct, a tetravalent IgGScv construct, or a composite antibody, or a combination thereof.

9. The BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, for use in the prevention or treatment of a disease associated with BCMA (B-cell maturation antigen), 4-1BB, or both.

10. The BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to claim 9, wherein the disease associated with BCMA, 4-1BB, or both is cancer.

11. The BCMA / anti-4-1BB bispecific antibody or antigen-binding fragment thereof of claim 10, wherein the cancer is multiple myeloma.

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