Antibodies and chimeric antigen receptors specific for BCMA

Abstract

One embodiment of the present invention relates to an anti-BCMA binding protein. More specifically, an isolated BCMA binding protein is provided, which includes an antigen binding domain that specifically binds to BCMA, wherein the antigen binding domain includes: i) a complementarity determining domain 1 (VH-CDR1) of a heavy chain variable domain, which includes an amino acid sequence of SEQ ID NO.1 or an amino acid sequence having more than 80% homology to SEQ ID NO.1; ii) a complementarity determining domain 2 (VH-CDR2) of a heavy chain variable domain, which includes an amino acid sequence of SEQ ID NO.2 or an amino acid sequence having more than 80% homology to SEQ ID NO.2; and iii) a complementarity determining domain 3 (VH-CDR3) of a heavy chain variable domain, which includes an amino acid sequence of SEQ ID NO.3 or an amino acid sequence having more than 80% homology to SEQ ID NO.3.

Description

Technical Field

[0001] The present invention relates to a protein that specifically binds to a novel BCMA, and more particularly, to a BCMA binding protein, a nucleic acid encoding the BCMA binding protein, a cell comprising the BCMA binding protein, and a composition having an anti-cancer effect comprising the cell. Background Art

[0002] Modified T cell therapy, which has shown excellent effects on cancer treatment, especially hematological malignancy, is a cancer treatment method that uses genetically modified T cells in a way that can kill tumor cells by specifically binding to tumor cells in a targeting molecule-specific manner. This targeting is usually achieved by introducing a recombinant DNA molecule encoding a chimeric antigen receptor (CAR) into T cells derived from the patient. CAR is a synthetic receptor that includes an extracellular targeting domain, a transmembrane domain, and one or more intracellular signaling domains. In most cases, the extracellular targeting domain is composed of a single-chain Fv fragment (scFv) of an antibody specific for a targeting molecule such as a specified tumor-associated antigen (TAA: tumor-associated antigen). The extracellular targeting domain makes CAR specific to tumors, and the intracellular signaling domain activates T cells when the CAR's targeting molecule binds. These genetically modified T cells are re-injected into cancer patients and specifically bind to cells expressing the CAR's targeting molecule to kill the cells.

[0003] Most clinical studies reporting the efficacy of CAR-T therapy for blood cancers involve the treatment of B-cell malignancies, which target CD19 or CD20. Although CAR-T cell therapy has emerged as an effective treatment, there is still room for improvement in many aspects, such as optimization of CAR signaling, optimization of target antigens, optimization of cell manufacturing methods, improvement of safety, and optimization of combination strategies to enhance the therapeutic potential of CAR-T cells.

[0004] Multiple myeloma (MM) is a cancer characterized by the accumulation of clonal plasma cells. MM is the second most common cancer among blood cancers, and deaths caused by MM account for 2% of all cancer-related deaths. MM is a disease with heterogeneous characteristics among patients, and the degree of aggressiveness and treatment resistance varies greatly. For example, some patients can survive for more than 10 years after diagnosis, but on the contrary, due to the rapid progression of treatment resistance, many patients die within less than 2 years. Although a variety of therapeutic agents have been developed, there is currently no treatment for MM. That is, although existing therapies induce MM remission, the disease relapses in almost all patients in the end, and even death occurs. Traditional treatments such as chemotherapy and radiotherapy have toxic side effects. Therefore, there is a need for more effective therapeutic agents for the treatment of MM.

[0005] Among the potential targets reported for MM treatment, there is B cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor family that is mainly expressed on mature B cells. Although BCMA mediates the survival of plasma cells in order to maintain organ humoral immunity in a healthy body, according to recent reports, the expression of BCMA is associated with many cancers, autoimmune diseases, and infectious diseases. As cancers with increased BCMA expression, there are some blood cancers, such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphomas, various leukemias, and glioblastomas. Therefore, there is a need for effective therapeutic agents that specifically target BCMA-expressing cells. Summary of the invention

[0006] Technical issues

[0007] The present invention is proposed to solve the above-mentioned requirements. The present invention provides an isolated BCMA-binding protein, which is an isolated BCMA-binding protein comprising an antigen-binding domain that specifically binds to BCMA, wherein the antigen-binding domain comprises: i) a complementarity determining domain 1 (VH-CDR1) of a heavy chain variable domain, which comprises an amino acid sequence of SEQ ID NO.1 or an amino acid sequence having more than 80% homology with SEQ ID NO.1; ii) VH-CDR2, which comprises an amino acid sequence of SEQ ID NO.2 or an amino acid sequence having more than 80% homology with SEQ ID NO.2; and iii) VH-CDR3, which comprises an amino acid sequence of SEQ ID NO.3 or an amino acid sequence having more than 80% homology with SEQ ID NO.3.

[0008] Furthermore, the present invention provides isolated nucleic acids encoding the above-mentioned BCMA-binding proteins.

[0009] Furthermore, the present invention provides cells comprising a nucleic acid encoding the above-mentioned BCMA-binding protein.

[0010] Furthermore, the present invention provides cells expressing the above-mentioned BCMA-binding protein.

[0011] Furthermore, the present invention provides a composition having an anti-cancer effect comprising the above-mentioned cells.

[0012] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and ordinary technicians in the technical field to which the present invention belongs can clearly understand other technical problems not mentioned through the following records.

[0013] Technical Solution

[0014] In order to achieve the above technical problem, an isolated BCMA-binding protein according to one aspect of the present invention comprises an antigen-binding domain that specifically binds to BCMA, wherein the antigen-binding domain comprises: i) a complementarity determining domain 1 (VH-CDR1) of a heavy chain variable domain, which comprises an amino acid sequence of SEQ ID NO.1 or an amino acid sequence having more than 80% homology to SEQ ID NO.1; ii) a complementarity determining domain 2 (VH-CDR2) of a heavy chain variable domain, which comprises an amino acid sequence of SEQ ID NO.2 or an amino acid sequence having more than 80% homology to SEQ ID NO.2; and iii) a complementarity determining domain 3 (VH-CDR3) of a heavy chain variable domain, which comprises an amino acid sequence of SEQ ID NO.3 or an amino acid sequence having more than 80% homology to SEQ ID NO.3.

[0015] Among them, the above-mentioned antigen binding domain may also include: i) a complementarity determining domain 1 (VL-CDR1) of the light chain variable domain, which includes the amino acid sequence of SEQ ID NO.4 or an amino acid sequence with more than 80% homology to SEQ ID NO.4; ii) a complementarity determining domain 2 (VL-CDR2) of the light chain variable domain, which includes the amino acid sequence of SEQ ID NO.5 or an amino acid sequence with more than 80% homology to SEQ ID NO.5; and iii) a complementarity determining domain 3 (VL-CDR3) of the light chain variable domain, which includes the amino acid sequence of SEQ ID NO.6 or an amino acid sequence with more than 80% homology to SEQ ID NO.6.

[0016] The BCMA-binding protein may include: a heavy chain variable domain (VH) sequence including an amino acid sequence of SEQ ID NO.7 or an amino acid sequence having more than 80% homology to SEQ ID NO.7; and a light chain variable domain (VL) including an amino acid sequence of SEQ ID NO.8 or an amino acid sequence having more than 80% homology to SEQ ID NO.8. The BCMA-binding protein may also include a linker, and the linker, VH and VL may be arranged in any order selected from VH-linker-VL or VL-linker-VH from the N-terminus to the C-terminus.

[0017] The BCMA-binding protein may include the amino acid sequence of SEQ ID NO.9 or an amino acid sequence having 80% or more homology with SEQ ID NO.9.

[0018] The BCMA-binding protein may be any one selected from the group consisting of the following components: i) an immunoglobulin selected from IgA, IgD, IgE, IgG and IgM; ii) a native antibody fragment selected from Fv, Fab, Fab′, F(ab′)2, VHH, and VNAR; iii) an engineered antibody selected from scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, tetrabody, and pentabody; iv) Fc, one or more heavy chain invariant domains, a multimerization domain, a chemical linker, or ii) or iii) connected to a combination thereof; v) an anti-BCMA chimeric antigen receptor (CAR).The anti-BCMA chimeric antigen receptor further comprises an intracellular signal transduction domain, which may comprise a signal transduction domain derived from a protein selected from CD3ζ, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, CD40, MyD88, DAP10, DAP12, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, IT GAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and any one or more of the CD83-specific ligands. The anti-BCMA chimeric antigen receptor further includes a transmembrane domain, which may be derived from any one selected from the group consisting of T cell receptor (TCR) α chain, TCR β chain, CD3ζ, CD3ε, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278 and CD314.The anti-BCMA chimeric antigen receptor further comprises a hinge domain, which is located between the BCMA binding protein and the transmembrane domain and may be derived from any one selected from CD8, FcγIIIα and IgG1. The anti-BCMA chimeric antigen receptor further comprises a signal peptide at the N-terminus, which may be derived from any one selected from CD8, IgG1 heavy chain, IgK light chain and GM-CSF. The anti-BCMA chimeric antigen receptor may comprise an amino acid sequence of SEQ ID NO.15 or an amino acid sequence having more than 80% homology to SEQ ID NO.15.

[0019] The isolated nucleic acid according to another aspect of the present invention is characterized in that it encodes the aforementioned BCMA-binding protein.

[0020] A cell according to another aspect of the present invention is characterized in that it comprises a nucleic acid encoding the above-mentioned BCMA-binding protein.

[0021] The cell according to another aspect of the present invention is characterized in that the above-mentioned BCMA-binding protein is expressed.

[0022] According to another aspect of the present invention, a composition having an anti-cancer effect comprises a cell comprising a nucleic acid encoding the above-mentioned BCMA-binding protein or a cell expressing the above-mentioned BCMA-binding protein.

[0023] The above solutions to the problems are merely illustrative and should not be interpreted as limiting the present invention. In addition to the above illustrative embodiments, there may be additional embodiments in the drawings and the content of the invention.

[0024] Effects of the Invention

[0025] The present invention initially discloses an isolated BCMA-binding protein, which is a BCMA-binding protein comprising an antigen-binding domain that specifically binds to BCMA, wherein the antigen-binding domain comprises: i) a complementarity determining domain 1 (VH-CDR1) of a heavy chain variable domain, which comprises an amino acid sequence of SEQ ID NO.1 or an amino acid sequence having more than 80% homology to SEQ ID NO.1; ii) VH-CDR2, which comprises an amino acid sequence of SEQ ID NO.2 or an amino acid sequence having more than 80% homology to SEQ ID NO.2; and iii) VH-CDR3, which comprises an amino acid sequence of SEQ ID NO.3 or an amino acid sequence having more than 80% homology to SEQ ID NO.3.

[0026] According to one embodiment of the present invention, the BCMA-binding protein can exhibit specific binding activity to BCMA.

[0027] According to one embodiment of the present invention, a chimeric antigen receptor (CAR) comprising an antigen binding domain that specifically binds to the above-mentioned BCMA can be successfully expressed in immune cells. In this way, immune cells expressing the above-mentioned CAR specific to BCMA can exhibit specific cytotoxicity against cells expressing BCMA, and when introduced into a living body, can effectively treat tumors expressing BCMA.

[0028] The BCMA-binding protein according to one embodiment of the present invention can be used in the diagnosis, prevention, prognosis prediction and treatment of many cancers, autoimmune diseases and infectious diseases known to be associated with BCMA, especially cancer.

[0029] The effects of the present invention are not limited to the above-mentioned effects, and should be understood to include all effects that can be derived from the inventive content of the present invention or the structure of the invention described in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the FACS data for confirming the BCMA-specific binding of the anti-BCMA scFv antibody candidate substances.

[0031] Figure 2 FIG. 1 is a schematic structural diagram of an anti-BCMA CAR and a vector comprising the same according to one embodiment of the present invention.

[0032] Figure 3 Detailed description of the invention Shows the sequence and structure of the anti-BCMA CAR according to one embodiment of the present invention.

[0033] Figure 4 The figure shows data of analyzing the cell expression of anti-BCMA CART according to one embodiment of the present invention using biotinylated BCMA.

[0034] Figure 5 The figure shows data of analyzing the cell expression of anti-BCMACAR according to one embodiment of the present invention using biotinylated anti-human Fab antibody.

[0035] Figure 6 The figure shows the IFN-γ secretion activity analysis data of anti-BCMACAR-T cells according to one embodiment of the present invention.

[0036] Figure 7 Figure 2 shows the data of each anti-BCMA CAR structure prepared by anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv and their expression on T cells.

[0037] Figure 8 The data show the cytotoxic properties of each anti-BCMA CART-T prepared by anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv.

[0038] Fig. 9 Representative images showing the in vivo anticancer efficacy of anti-BCMA CAR-T prepared by anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv (CAR-T administration dose 1X10 6 indivual).

[0039] Fig.10 Representative images showing the in vivo anticancer efficacy of anti-BCMA CAR-T prepared by anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv (CAR-T dosage 0.5X10 6 indivual).

[0040] Fig.11 Representative images showing the in vivo anticancer efficacy of anti-BCMA CAR-T prepared by anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv (CAR-T dosage 0.25×10 6 indivual).

[0041] Fig.12 The data show the in vivo anticancer efficacy of different dosages of each anti-BCMA CAR-T prepared by anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv. DETAILED DESCRIPTION

[0042] The present invention is described below with reference to the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. In addition, in order to clearly illustrate the present invention, parts not related to the description are omitted in the accompanying drawings, and similar parts are marked with similar reference numerals throughout the specification.

[0043] Throughout the specification, when some parts are "connected (connected, in contact with, combined)" to another part, this includes not only the case of "direct connection" but also the case of "indirect connection" with other parts in between. Furthermore, when some parts "include" certain structural elements, this means that other structural elements may also be present, rather than excluding other structural elements, unless there is any special description to the contrary.

[0044] The terms used in this specification are used only to illustrate specific embodiments and do not limit the present invention. Singular expressions include multiple expressions as long as they are not clearly different in context. In this specification, terms such as "including" or "having" specify the presence of features, numbers, steps, actions, structural elements, parts or combinations thereof recorded in the specification, and should be understood as not excluding the existence or additional possibility of one or more other features or numbers, steps, actions, structural elements, parts or combinations thereof in advance.

[0045] In this specification, "domain" is used as a term to represent a unit with functional or structural characteristics. For example, a binding domain refers to any domain that has the function of recognizing a target molecule (e.g., an antigen) or interacting with the target molecule and capable of specific binding. The BCMA-binding protein according to the present invention includes a binding domain specific for BCMA as an antigen. The region according to the present invention is preferably a polypeptide. Such a polypeptide may be a single polypeptide chain or a module formed by the aggregation of parts of multiple polypeptide chains. The above-mentioned polypeptide may include proteinaceous parts and non-proteinaceous parts. In this case, the above-mentioned non-protein part may be, for example, a chemical cross-linking agent or a chemical linker such as glutaraldehyde.

[0046] "VH", "VL", "CDR", "Fv", "Fab", "Fab'", "F(ab')2", "Fc", "VHH" and "VNAR" used in the present invention are generally used as a part (i.e., fragment) of a native antibody, which is a term that can be replaced with immunoglobulin. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (light chain or L chain) and two identical heavy chains (heavy chain or H chain). Each light chain is connected to the heavy chain by a conjugated disulfide bond, and the two heavy chains are connected by one or more disulfide bonds depending on the heavy chain isotype. Each heavy chain and light chain also has regularly spaced inter-chain disulfide bridges. Each heavy chain includes a variable domain (VH) and an invariant domain (CH) connected thereto in the order from N-terminus to C-terminus. At this time, for mammalian immunoglobulins, there are 3 α, δ and γ chains respectively, and 4 CHs are respectively provided for μ and ε isotypes. Each light chain has a variable domain (VL) and an invariant domain (CL) connected thereto in the order from N-terminus to C-terminus. VL is aligned with VH and sorted, and CL is aligned with the first invariant domain (CH1) of the heavy chain and sorted. It is reported that specific amino acid residues form an interface between the light chain and the heavy chain variable domain, and a single antigen binding domain is formed by pairing VH and VL. Immunoglobulins can be classified according to the type of heavy chain included. For example, mammalian immunoglobulins including α, δ, ε, γ, and μ as heavy chains are respectively divided into IgA, IGD, IgE, IgG, and IgM in order. In some animals of the system, there are also other forms of immunoglobulins. For example, in birds, amphibians, bony and cartilaginous fish, there are immunoglobulins with different types and / or numbers of heavy chain variable domains, in camelids and sharks, there are immunoglobulins consisting only of heavy chains, with the number of invariant domains being 2 and 5, respectively, and comprising heavy chain variable domains called VHH and VNAR, respectively.

[0047] In the above-mentioned "variable", the defined sections of the variable domain are related to the fact that the sequences between antibodies differ greatly. The variable domain is the part that mediates antigen binding, which defines the specificity of a specific antibody for a specific antigen. This variability appears unevenly throughout the variable domain, and is concentrated in three sections called hypervariable regions (HVR (hypervariable region) or complementarity determining regions (CDR (complementarity-determining region)) in both the light chain and the heavy chain variable domains. In contrast, the relatively highly conserved parts of the variable domain are called framework regions (FR (framework regions)). The variable domains of the heavy and light chains of mammalian immunoglobulins include four FRs connected by three CDRs, respectively. In each chain, the CDRs are arranged close to each other according to the FR domain, thereby forming the antigen-binding domain of the antibody together with the CDRs of the other chain. Although the invariant domain is not directly related to the binding of the antibody to the antigen, it has the function of becoming a site for recognizing the antibody when inducing antibody-dependent cellular cytotoxicity.

[0048] Fv is the minimum fragment that completely includes the antigen recognition and binding site of the antibody, and is composed of a dimer of VH and VL. Fab refers to a fragment that includes the constant domain of the light chain and CH1 in addition to Fv, which can be obtained by treating IgG antibodies with papain. Fab' includes a part of the hinge (between CH1 and CH2) in addition to Fab, and F(ab')2 that can be obtained when IgG antibodies are treated with pepsin includes a hinge part connected by a disulfide bond, and therefore includes a divalent binding site. The Fc domain is an antibody that does not include an antigen-specific binding domain and can be crystallized. It is composed of two identical protein fragments. The Fc domain of IgG, IgA and IgD isotypes includes the second and third constant domains (CH2-CH3) of the antibody heavy chain, and the Fc domain of IgM and IgE isotypes includes the second, third and fourth constant domains (CH2-CH3-CH4) of the antibody heavy chain.

[0049] The amino acid sequences included in this specification may include sequences having more than 80% homology with the sequence, preferably more than 90%, more preferably more than 95%, and most preferably more than 99% homology, while maintaining the binding properties of the binding protein according to the present invention. The above changes may be selected from any one or more of the addition, substitution or deletion of a portion of amino acid residues.

[0050] According to one aspect of the present invention, an isolated BCMA-binding protein comprises an antigen-binding domain that specifically binds to BCMA, wherein the antigen-binding domain comprises: i) a complementarity determining domain 1 (VH-CDR1) of a heavy chain variable domain, which comprises an amino acid sequence of SEQ ID NO.1 or an amino acid sequence having more than 80% homology to SEQ ID NO.1; ii) VH-CDR2, which comprises an amino acid sequence of SEQ ID NO.2 or an amino acid sequence having more than 80% homology to SEQ ID NO.2; and iii) VH-CDR3, which comprises an amino acid sequence of SEQ ID NO.3 or an amino acid sequence having more than 80% homology to SEQ ID NO.3. Preferably, the BCMA-binding protein comprises: i) a complementarity determining domain 1 (VH-CDR1) of a heavy chain variable domain, which comprises an amino acid sequence of SEQ ID NO.1; ii) VH-CDR2, which comprises an amino acid sequence of SEQ ID NO.2; and iii) VH-CDR3, which comprises an amino acid sequence of SEQ ID NO.3.

[0051] Wherein, the above-mentioned BCMA-binding protein may further include: i) complementarity determining domain 1 (VL-CDR1) of the light chain variable domain, which includes the amino acid sequence of SEQ ID NO.4 or an amino acid sequence with more than 80% homology to SEQ ID NO.4; ii) VL-CDR2, which includes the amino acid sequence of SEQ ID NO.5 or an amino acid sequence with more than 80% homology to SEQ ID NO.5; and iii) VL-CDR3, which includes the amino acid sequence of SEQ ID NO.6 or an amino acid sequence with more than 80% homology to SEQ ID NO.6. Preferably, the above-mentioned BCMA-binding protein may further include: i) complementarity determining domain 1 (VL-CDR1) of the light chain variable domain, which includes the amino acid sequence of SEQ ID NO.4; ii) VL-CDR2, which includes the amino acid sequence of SEQ ID NO.5; and iii) VL-CDR3, which includes the amino acid sequence of SEQ ID NO.6.

[0052] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor (TNFR) superfamily. BCMA binds to B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). BCMA is reported to be expressed in non-malignant cells, mainly in plasma cells and mature B-cell subsets.

[0053] BCMA is expressed or overexpressed in a variety of human cancers. Examples of cancers expressing or overexpressing BCMA include Burkitt's lymphoma, diffuse large-B cell lymphoma (Diffuse large B-cell lymphoma or DLBCL), acute lymphocytic leukemia (acute lymphocytic leukemia or ALL) lymphoma, Hodgkin lymphoma, and multiple myeloma (multiple myeloma or MM). The BCMA binding protein of the present invention specifically recognizes BCMA and binds to it, thereby targeting cancer cells that express BCMA. It can be predicted that such a polypeptide or protein including a BCMA binding protein can be used in the detection of cancer cells expressing BCMA, the targeting and destruction of cancer cells expressing BCMA, the reduction and removal of cancer cells, the promotion of penetration into tumor sites of immune cells or effector molecules or both, the improvement of anticancer responses, the extension of anticancer responses, and the entire process of diagnosis, prevention, prognosis prediction, and treatment of diseases. When the polypeptide or protein including the BCMA-binding protein is used as described above, it may be combined with additional polypeptides, proteins or compounds to improve its efficacy. For example, the BCMA-binding protein may additionally include Fc (crystallizable fragment), conjugated drugs, molecular tags (such as fluorescent markers, protein tags, etc.) that can be easily tracked, etc., alone or in combination.

[0054] The BCMA-binding protein may include a heavy chain variable domain (VH), which includes the amino acid sequence of SEQ ID NO.7 or an amino acid sequence having more than 80% homology to SEQ ID NO.7, and preferably may include an amino acid sequence including SEQ ID NO.7. The BCMA-binding protein may also include a light chain variable domain (VL), which includes the amino acid sequence of SEQ ID NO.8 or an amino acid sequence having more than 80% homology to SEQ ID NO.8, and preferably may also include an amino acid sequence including SEQ ID NO.8.

[0055] The above-mentioned BCMA-binding protein may include: a heavy chain variable domain (VH), which includes the amino acid sequence of SEQ ID NO.7 or an amino acid sequence with more than 80% homology to SEQ ID NO.7; and a light chain variable domain (VL), which includes the amino acid sequence of SEQ ID NO.8 or an amino acid sequence with more than 80% homology to SEQ ID NO.8, preferably, it may include VH including the amino acid sequence of SEQ ID NO.7 and VL including the amino acid sequence of SEQ ID NO.8.

[0056] The BCMA-binding protein further comprises a linker, and the linker, VH and VL may be arranged in any order selected from VH-linker-VL or VL-linker-VH along the direction from the N-terminus to the C-terminus, and preferably may be arranged in the order of VH-linker-VL. The linker is a part that connects different domains, and may comprise 2 to 500 amino acids. The linker may be composed of glycine and serine, and preferably, the linker may comprise (Gly x Ser y ) n (n sequences consisting of x Glycine and y Serine connected, x, y and n are integers of 1 to 10 respectively), more preferably including (Gly4-Ser1) n The sequence, wherein more preferably, n may be 1, 2, 3, 4, 5 or 6, most preferably, n is 3 or 4, for example, may include (Gly4-Ser1)3 (ie, GGGGSGGGGSGGGGS) and the like.

[0057] The BCMA-binding protein may include the amino acid sequence of SEQ ID NO.9 or an amino acid sequence having more than 80% homology with SEQ ID NO.9, and preferably may include the amino acid sequence of SEQ ID NO.9.

[0058] The BCMA-binding protein may be any one selected from the group consisting of the following components: i) an immunoglobulin selected from IgA, IgD, IgE, IgG and IgM; ii) a native antibody fragment selected from Fv, Fab, Fab′, F(ab′)2, VHH, and VNAR; iii) an engineered antibody selected from scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, tetrabody, and pentabody; iv) Fc, one or more heavy chain invariant domains, a multimerization domain, a chemical linker, or ii) or iii) connected to a combination thereof; v) an anti-BCMA chimeric antigen receptor (CAR).

[0059] The BCMA-binding protein may have antigen specificity only for BCMA (i.e., monospecific), and may have antigen specificity for more than one epitope on BCMA other than the epitope bound by the BCMA antigen-binding domain included in the BCMA-binding protein; or more than one antigen other than BCMA (i.e., multispecific). The BCMA-binding protein may include one antigen-binding domain (monovalent), and may include more than one antigen-binding domain (multivalent). The BCMA-binding protein may be an engineered antibody including more than one sequence described in the present invention. scFv is a connecting peptide connecting the VH and VL of the antibody, dsFv is a disulfide-stabilized Fv, ds-scFv is a disulfide-stabilized scFv, and (scFv)2 is bound to two scFvs. Diabodies, tribodies, tetrabodies, and pentabodies are dimers, trimers, tetramers, and pentamers formed by scFv, respectively.

[0060] The BCMA-binding protein of the present invention can be any type of immunoglobulin known in the art. For example, the BCMA-binding protein can be an antibody of any isotype, such as IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgM, etc. The BCMA-binding protein can be monoclonal or polyclonal. The BCMA-binding protein can be a natural antibody isolated or purified from animals such as mice, rabbits, goats, horses, chickens, hamsters, and humans, or can be a synthetic antibody designed and genetically synthesized with reference to the sequence characteristics of natural antibodies. The BCMA-binding protein can be an antibody manufactured by a humanized antibody or a chimeric antibody. The BCMA-binding protein can be in monomeric or polymeric form. The BCMA-binding protein may include chemical deformation, for example, disulfide bond addition, glycosylation, side chain deformed amino acids, methionine oxidation, asparagine or glutamine deamidation, γ-carboxylation, β-hydroxylation, sulfation, etc. The BCMA-binding protein may have any level of affinity or avidity for BCMA. The BCMA-binding protein may be any one selected from mouse, rabbit, human, humanized or chimeric proteins.

[0061] According to the present invention, the anti-BCMA chimeric antigen receptor (CAR) molecule includes, in addition to the aforementioned BCMA binding domain, a transmembrane domain and an intracellular signal transduction domain.

[0062] Usually, CAR is a group of polypeptides that can be connected to the intracellular signaling activation of the above-mentioned immune cells when it is expressed on immune cells. In addition to cells that recognize targeted antigens, CAR includes at least an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The above-mentioned intracellular signaling domain is preferably derived from a stimulatory molecule or a co-stimulatory molecule. The group consisting of the above-mentioned polypeptides can be a single-chain polypeptide form, and can be a multi-chain (polychain) form that has a switch that is adhered to each other by stimulating dimerization or multimerization. Stimulatory molecules refer to molecules related to signaling that specifically activates immune responses, and co-stimulatory molecules refer to molecules that non-specifically amplify or reduce the initial signals of immune cells. The above-mentioned intracellular signaling domain may include one or more signaling domains derived from stimulatory molecules or co-stimulatory molecules. The activation of immune cells expressing the domain can be preferably induced by this signal transduction domain, wherein the activation may include starting and / or increasing cell proliferation, differentiation to effector cells, cytokine secretion, cytotoxic substance secretion, phagocytosis, targeted cell apoptosis induction, or more. CAR may be a fusion protein including an antigen binding domain, the transmembrane domain, and the intracellular signal transduction domain in addition to the above-mentioned cells. The above-mentioned CAR may also include a signal peptide (signal peptide or leader sequence) at the N-terminus, and the above-mentioned signal peptide may be cut off during the process of CAR expression and placement on the cell membrane.

[0063] The intracellular signal transduction domain may include a signal transduction domain selected from CD3ζ, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, CD40, MyD88, DAP10, DAP12, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, I TGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and the signal transduction domain of any one of the CD83-specific ligands. Preferably, the intracellular signal transduction domain may include any one or more selected from CD3ζ and 4-1BB, and most preferably may include both CD3ζ and 4-1BB.

[0064] The intracellular signal transduction domain may include any one or more selected from the group consisting of an amino acid sequence of SEQ ID NO.10 or an amino acid sequence having 80% or more homology to SEQ ID NO.10 and an amino acid sequence of SEQ ID NO.11 or an amino acid sequence having 80% or more homology to SEQ ID NO.11. Preferably, the intracellular signal transduction domain may include any one or more selected from the group consisting of an amino acid sequence of SEQ ID NO.10 and an amino acid sequence of SEQ ID NO.11, and more preferably, both may include an amino acid sequence of SEQ ID NO.10 and an amino acid sequence of SEQ ID NO.11.

[0065] The transmembrane domain may be derived from any one selected from T cell receptor (TCR) α chain, TCR β chain, CD3ζ, CD3ε, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278 and CD314. Preferably, the transmembrane domain may be selected from a domain that can promote dimerization of the anti-BCMACAR molecule, for example, it may be derived from CD3ζ, CD28 or CD8, more preferably from CD8, and most preferably from CD8α. The transmembrane domain may include the amino acid sequence of SEQ ID NO.12 or an amino acid sequence having more than 80% homology in SEQ ID NO.12, preferably may include the amino acid sequence of SEQ ID NO.12.

[0066] The anti-BCMA CAR molecule further includes a hinge domain, which may be located between the BCMA binding protein and the transmembrane domain. The hinge domain may be derived from any one selected from CD8, FcγRIIIα and IgG1, preferably from CD8, and more preferably from CD8α. The hinge domain may include an amino acid sequence of SEQ ID NO.13 or an amino acid sequence having more than 80% homology in SEQ ID NO.13.

[0067] The anti-BCMA CAR molecule may further include a signal peptide at the N-terminus. The signal peptide may be derived from any one selected from CD8, IgG1 heavy chain, IgK light chain and GM-CSF, preferably from CD8, and most preferably from CD8α. The signal peptide may include the amino acid sequence of SEQ ID NO.14 or an amino acid sequence having more than 80% homology with SEQ ID NO.14, preferably may include the amino acid sequence of SEQ ID NO.14.

[0068] The anti-BCMACAR may include the amino acid sequence of SEQ ID NO.15 or an amino acid sequence having 80% or more homology with SEQ ID NO.15, and preferably may include the amino acid sequence of SEQ ID NO.15.

[0069] The anti-BCMA CAR may further include a linker connecting different peptide modules, domains, etc., wherein the linker may include 2 to 500 amino acids as described above. The linker may be any one selected from the above linker and the sequence in the following table.

[0070] [Table 1]

[0071]

[0072]

[0073] According to another aspect of the present invention, the isolated nucleic acid is characterized in that the aforementioned BCMA-binding protein is encoded. The above-mentioned nucleic acid may include a nucleic acid that operably connects an expression regulatory sequence to a sequence encoding the above-mentioned BCMA-binding protein in a cell. The above-mentioned expression regulatory sequence may include one or more promoters, enhancers, polyadenylation signals, internal ribosome entry sites (IRES, internal ribosome entry site), introns, other post-transcriptional regulatory elements, etc. For the above-mentioned expression regulatory sequence, as described below, the isolated nucleic acid encoding the above-mentioned BCMA-binding protein is included in the cell, and when the above-mentioned BCMA-binding protein is expressed, it can be selected in a manner suitable for the expression regulation of the cell.

[0074] The nucleic acid can be included in the cell in the form of a vector. The vector can be selected from DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector. Preferably, it can be selected from lentiviral vector and retroviral vector, because the gene entering the lentiviral vector and retroviral vector is inserted into the genomic DNA of the immune cell, thereby stably expressing the factor. Most preferably, it can be a lentiviral vector.

[0075] Regarding the use of the above-mentioned nucleic acid in the form of the vector as described above, changes within the vector, namely, the addition, deformation and loss of base sequences that may occur during the cloning process of introducing a specific sequence into the vector, or the change and introduction of structural elements that improve the convenience or performance of the vector, etc., are obvious to ordinary technicians, so detailed description is omitted.

[0076] The cell according to another aspect of the present invention is characterized in that it encodes the aforementioned BCMA-binding protein.

[0077] The cell according to another aspect of the present invention is characterized in that the cell expresses the aforementioned BCMA-binding protein.

[0078] The above-mentioned cells include eukaryotic cells and prokaryotic cells including yeast, mold, insect cells and mammalian cells. For example, the above-mentioned cells can be bacteria such as E. coli, which can be eukaryotic microorganisms such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia, Pichia pastoris, Candida, Trichoderma reesei, Neurospora, Schwanniomyces, Kluyveromyces, Penicillium, Tolypocladium and Aspergillus hosts. In order to increase the post-translational modification process such as glycosylation to be suitable for expression in the form of antibodies, the above-mentioned cells can be derived from multicellular organisms, and host cells suitable for vectors containing nucleic acids encoding the above-mentioned BCMA-binding proteins can be selected. For example, when using a baculovirus vector, the cells may be derived from an organism appropriately selected from Spodoptera frugiperda, Aedes aegypti, Aedes albopictus, Drosophila melanogaster, Bombyx mori, etc. The BCMA-binding protein expressed in the cells may be used by separation and purification. After expressing the BCMA-binding protein in the cells, it may be preferably separated from the soluble fraction of the cells and then purified by affinity chromatography, size exclusion chromatography, etc., but is not limited thereto. The expression (biosynthesis), separation, and purification of antibodies, fusion proteins, etc. are matters that can be obviously selected by ordinary technicians, so a specific description thereof is omitted.

[0079] The above-mentioned cells may preferably be immune cells, and the above-mentioned immune cells may be any one selected from T cell precursors, NK cell precursors, T cells, natural killer (NK, Natural Killer) cells, NKT (Natural Killer T) cells, B cells, monocytes, macrophages and dendritic cells, more preferably may be T cells, and most preferably may be cytotoxic T cells (cytotoxic T lymphocyte).

[0080] When the above cells are administered to a patient, they can be any of autologous, allogeneic or heterologous cells of the subject patient, and heterologous cells can be obtained from mammals. In order to reduce the possibility of immune rejection, allogeneic cells can be preferably used, and autologous cells can be most preferably used.

[0081] The above-mentioned cells may also include another agent for the activity, suitability, efficiency and / or safety improvement of the cells expressing the above-mentioned BCMA binding protein. The above-mentioned agent may be a nucleic acid or a protein. The above-mentioned agent may be expressed by the above-mentioned BCMA binding protein or a molecule different from the nucleic acid encoding it, and may be included in the form of the same molecule. The above-mentioned agent may be, for example, one or more additional CARs, and the above-mentioned additional CARs have the same targeting molecule, that is, another epitope of BCMA can be used as a target, and different targeting molecules can be used as targets. The above-mentioned different targeting molecules are selected from 5T4, α5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr-abl, MN / CIX antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD44 variant domain 6. CD123, CD135, CD138, CD269, CDC27 / m, CDK4 / m, CEA, CLEC12A, CT, Cyp-B, DAM, EGFR, ErbB3, ELF 2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / new, HLA-A*0201-R170I 、HPV-E7、HSP70-2M、HST-2、hTERT(or hTRT)、iCE、IGF-1R、IL-2R、IL-5、KIAA0205、LAGE、LDLR / FUT、MAGE、MART-1 / melan-A、MART-2 / Ski、MC1R、myosin / m、MUC1、MUM-1、MUM-2、MUM-3、NA88-A、natural killer group 2D, Lewis Y antigen, PAP, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1, SART-3, survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, NY-Eso-1 and NY-Eso-B, which may be a molecule specifically expressed in addition to BCMA in the BCMA-expressing cancer of the above-mentioned patient. The above-mentioned agent may be one or more agents that adjust or modulate the function of immune cells. The molecule that adjusts or modulates the function of the above-mentioned immune cells may be an inhibitory molecule, and the above-mentioned agent may be a substance that reduces the expression, function or both of the above-mentioned molecules.The above-mentioned inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and CEACAM-5, etc.), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFRβ, etc. The above-mentioned agents are inhibitory nucleic acids such as dsRNA, siRNA, shRNA, etc., clustered regularly interspaced short palindromic repeats (CRISPR, clustered regularly interspaced short palindromic repeat, CRISPR), transcription activator-like effector nucleases (TALEN, transcription activator-like effector nucleases) or zinc finger (zincfinger) endonuclease (ZFN) and other inhibitory proteins or systems, or antibodies or binding sites for the above-mentioned inhibitory molecules. Proteins or small molecules (small molecules), which can prevent the above-mentioned inhibitory molecules by binding. The above-mentioned agents can regulate the survival of the above-mentioned cells. For example, when the above-mentioned cells are administered to a patient, in the case of side effects such as cytokine storm, the cells injected in reaction with a specific substance or molecules that can induce cell apoptosis can be removed. Examples thereof include U.S. Terminated Patent No. 5,358,866, U.S. Granted Patent No. 9,393,292, and European Granted Patent No. 2,836,511.

[0082] According to another aspect of the present invention, a composition having an anti-cancer effect comprises a cell comprising a nucleic acid encoding the aforementioned BCMA-binding protein or a cell expressing the aforementioned BCMA-binding protein.

[0083] The above composition can be used for the treatment of cancers with increased BCMA expression. The above composition can preferably be used for the treatment of multiple myeloma, Hodgkin's and non-Hodgkin's lymphomas, various leukemias and glioblastoma.

[0084] In the composition having the above-mentioned anti-cancer effect, in addition to the above-mentioned immune cells, various pharmaceutically acceptable salts, carriers, excipients, solvents and other additives that can further enhance the therapeutic effect can be added. This is obvious to ordinary technicians, so specific descriptions thereof are excluded.

[0085] In the present invention, the preferred pharmaceutical dosage form can be determined according to the general knowledge of the disclosure and formulation technology according to the intended route of administration, delivery form and the intended dosage. Whether it is the mode of administration, or the effective capacity can be calculated according to the patient's weight, surface area or organ size. The calculation and additional refinement for determining the appropriate dosage for the treatment of each dosage form recorded in this specification sheet are carried out daily by ordinary personnel and are within the area of ​​the work performed daily in this field. The appropriate dosage can be confirmed by using appropriate capacity-response data.

[0086] The following describes the embodiments of the present invention in detail so that those skilled in the art can easily implement the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[0087] Example 1. Construction of scFv library and derivation of anti-BCMA scFv candidate group

[0088] <Example 1-1> Construction of scFv library

[0089] The scFv library was constructed according to the method of Korean Patent No. 10-0961392.

[0090] In brief, the human VH3-23 / VL1-g gene is used as a skeleton, and a random sequence is inserted into the complementarity determining regions (CDR) to design a library. In a plasmid vector having a β-lactamase gene as a selection marker, a restriction enzyme cleavage sequence is introduced after the leader sequence of the β-lactamase gene to prepare a pFDV plasmid vector, and after inserting the scFv gene library, the vector is transfected into Escherichia coli and then cultured to construct a library. Through this process, most of the scFv gene sequences with abnormal stop codons or frameshifts are removed from the library, and the quality of the library can be improved. After removing the wrong sequence from the cultured library by synthase chain reaction amplification, the scFv gene library is recombined therefrom and inserted into the pComb3X phagemid vector, and the final library is obtained by transfecting the ER2537 strain of Escherichia coli. The library was cultured in 400 mL of super broth (SB) medium containing carbenicillin. When the absorbance at 600 nm was 0.5, 1013 CFU of VCSM13 helper phage was added, stirred at 80 rpm, and infected at 37 degrees Celsius for 1 hour. A final 70 ug / mL of kanamycin antibiotic was added, stirred at 30 degrees Celsius and 200 rpm, and cultured overnight to produce phages displaying scFv. The next morning, the culture solution was centrifuged, and 4% polyethylene glycol-8000 and 3% sodium chloride were added to precipitate the phages in the culture solution. The precipitated phages were dissolved in 50 mL of PBS buffer solution, reprecipitated as above, and finally dissolved in 2 mL of PBS buffer solution. It was centrifuged to remove foreign matter, thereby obtaining a phage scFv library. Generally speaking, the final phage library includes 10 13 CFU / mL or more of phage particles.

[0091] <Example 1-2> Screening of anti-BCMA scFv

[0092] Add 10ug / ml BCMA to the immunotube. After the protein is adsorbed on the surface of the test tube for 1 hour, add 3% skim milk solution to protect the surface that is not adsorbed with BCMA. After emptying the test tube, put 10ug / ml of BCMA dispersed in 3% skim milk solution into it. 12 The antibody phage library of CFU was bound to the antigen. The non-specifically bound phages were removed using TBST (tris buffered saline-tween 20) solution, and the remaining antigen-specific phage antibodies were eluted using 100 mM triethylamine solution.

[0093] After neutralizing the eluted phage with 1.0 M Tris-HCl buffer (pH 7.8), the phage was infected with ER2537 Escherichia coli at 37° C. for 1 hour, and the infected Escherichia coli was spread on Luria-Bertani (LB) agar medium containing carbenicillin and cultured overnight at 37° C. The cultured Escherichia coli was suspended in 3 mL of SB (super broth)-carbenicillin culture solution, 15% glycerol was added, a portion was stored at -80° C., and 50 μl of the remaining portion was inoculated in 20 mL of SB-carbenicillin-2% glucose solution and cultured at 37° C. If the absorbance of the culture medium at 600 nm is 0.5, separate only the bacteria by centrifugation, resuspend them in 20 mL of SB-carbenicillin culture medium, add 1012 PFU (plaque forming units) of VCSM13 helper phage, stir slowly, and culture at 37°C.

[0094] After 1 hour, kanamycin 70 μg / ml was added and cultured overnight at 250 rpm with rapid stirring at 30°C. The next day, the culture solution was centrifuged and 1 mL of the supernatant containing phage particles was used as a library, and the above panning process was repeated to concentrate antigen-specific clones.

[0095] Example 2. Expression and purification of anti-BCMA scFv antibody

[0096] After 3-4 times of repeated panning, four antigen-specific candidate clones (OPAL A1, OPALB10, OPALS-L A5 and OPALS-K H6) were ensured. Escherichia coli containing each antibody gene was spread on LB agar medium containing carbenicillin to obtain a single colony, which was inoculated in 200 μL SB-carbenicillin solution for culture, and then induced by IPTG to express the scFv protein in the periplasm of the Escherichia coli cells. After suspending the Escherichia coli in 40 μL of 1X TES (50 mM Tris, 1 mM EDTA, 20% Sucrose, pH 8.0) solution, 60 μL of 0.2X TES solution was added, mixed, treated at 4 degrees Celsius for more than 30 minutes, centrifuged, and the periplasm was extracted as the supernatant.

[0097] <Example 2-1> Expression of scFv antibodies for BCMA screening

[0098] The scFv-positive single colony clones of the four BCMAs screened were cultured in 5 ml of SB medium (30 g Bactotrytone, 20 g yeast extract, 10 g / L MOPS buffer) containing carbenicillin to start seed culture. After overnight culture, the clones were transferred to 500 ml of SB medium containing carbenicillin. When the OD600 was about 0.5, 1 mM IPTG was added and cultured overnight at 30 degrees Celsius to express the scFv antibody in the periplasm of E. coli cells. The next day, the E. coli obtained by centrifugation was suspended in 1X TES buffer (50 mM Tris, 1 mM EDTA, 20% Sucrose, pH 8.0), and 0.2X TES was added 1.5 times, mixed, and centrifuged to extract the periplasm.

[0099] <Example 2-2> Purification of screened scFv antibodies

[0100] The scFv antibody extracted from the periplasm of the cells was added with a final 5mM MgSO4, mixed with Ni-NTA beads pre-equilibrated with PBS, and refrigerated for 1 hour. After binding to Ni-beads, affinity chromatography was performed and unbound proteins were fully washed with PBS. After more thorough washing with a buffer containing 5mM imidazole, the bound scFv antibody was eluted with a 200mM imidazole buffer. The eluted antibody was dialyzed, the purity was confirmed by electrophoresis, and the protein was quantified by the BCA method. After recording the amount of purified antibody, a prescribed amount was dispensed and stored frozen.

[0101] <Example 2-3> FACS and sequencing for screening anti-BCMA scFv

[0102] For scFv antibodies extracted from the periplasm of cells, fluorescence-activated cell sorting (FACS) technology was used to confirm whether BCMA on the cell surface binds to the scFv antibody. The cells used were Raji cells, a B-cell lymphoma cell line known to express BCMA. 2X10 6After culturing Raji cells on the well plate, they were treated with trypsin, separated from the well plate, and centrifuged. After blocking the cells prepared in this way with PBS containing % BSA (w / v) at 4°C for 1 hour, they were washed once with 1XPBS, centrifuged at 3000 rpm for 3 minutes, and then reacted with the first antibody (each experimental group: OPAL A1 is 6.2μg / mL, OPAL B10 is 13.2μg / mL, OPALS-L A5 is 21μg / mL, and OPALS-K H6 is 20μg / mL; positive control group: anti-BCMA polyclonal antibody (biotinylated) 0.5μg / mL), washed three times with 1XPBS, and then reacted with the second antibody (each experimental group: anti-HA-PE; positive control group: Avidin-FITC), and washed twice with 1XPBS. After diluting with specified dilution water, fluorescence-activated cell sorting (FACS) analysis (FACSCalibur, BD Bioscience) was performed ( Figure 1 ).

[0103] One BCMA antigen-specific antibody clone (OPAL B10, hereinafter referred to as B10) finally screened in this way was cultured overnight in 10 ml of SB medium containing carbenicillin, and plasmid DNA was extracted using a plasmid miniprep kit, and the base sequence was obtained by capillary sequencing service (Macrogen Co). After converting it into a protein sequence, the CDR sequence was analyzed according to the Kabat protein sequence database and the international ImMunoGeneTics information system (IMGT) analysis method.

[0104] The cell lines and culture media used in the following examples and experimental examples are as follows: 293T cells were cultured in a culture medium containing 10% fetal bovine serum (FBS), 2mM L-Glutamine, 100U / mL penicillin, and 100μg / mL streptomycin in DMEM (Thermofisher Scientific). NCI-H929, RPMI 8226, and U266B1 cells were cultured in a culture medium containing 10% FBS, 2mM L-Glutamine, 100U / mL penicillin, and 100μg / mL Streptomycin (Thermofisher Scientific) in RPMI1640. PBMCs were cultured in a culture medium containing 5% Immune Cell SR (Thermofisher Scientific) in AIM-V medium.

[0105] Example 3, Preparation of pL-B10-BBz plasmid

[0106] Gene fragments SEQ ID#1 and SEQ ID#2 (Table 2) containing the B10 base sequence were ordered from Integrated DNA Technologies for synthesis. Overlap extension PCR was performed using the two gene fragments and primers SEQ ID#1 and #2 (Table 3) to obtain PCR products. The PCR products treated with restriction enzymes Xho1 and EcoRV were inserted into the pLV-CD19-BBz plasmid (PNAS, 2016, Ma JSY) treated with restriction enzymes Xho1 and EcoRV to prepare pLV-B10-BBz. ( Figure 2 and Figure 3 )

[0107] [Table 2]

[0108]

[0109]

[0110] [Table 3]

[0111]

[0112] Example 4. Preparation of lentiviral vector

[0113] 293T cells were placed in a 150 mm culture dish (SPL) coated with poly-D-Lysine and cultured for 24 hours. The next day, after changing to a new 293T cell culture medium, pMDL g / p (Gag / Pol expression), pRSV-Rev (Rev expression), pMDG.1 (Envelope expression) and pLV-B10-BBz plasmids and Lipofectamine 2000 (Thermofisher Scientific) were added for transfection. After 48 hours, the culture medium was recovered and concentrated using a Lenti-X Concentrator (Takara Inc.) produced by the Lenti-X Concentrator kit, and then the concentrated Lenti-X Concentrator was suspended in a 4% lactose solution. Then, the solution was filtered using a 0.2 μm syringe filter (Millipore), and then stored at -80°C.

[0114] Example 5. T cell transformation and expansion

[0115] After thawing, frozen human peripheral blood mononuclear cells (Human PBMC) were washed twice with a washing solution including CliniMACS PBS / EDTA buffer (Miltenyi Biotec) and 0.5% albumin (Green Cross). MACS CD4 microbeads and MACS CD8 microbeads (Miltenyi Biotec) were placed in the washed peripheral blood mononuclear cells (PBMC), and after binding for 30 minutes, T cells were separated by QuadroMACSTM Separator (QuadroMACSTM Separator) (Miltenyi Biotec). After washing the separated T cells, they were suspended in culture medium and placed in T cell activation reagent (T Cell TransAct TM) (MiltenyiBiotec) and 100IU / mL recombinant human interleukin-2 (recombinant human IL-2) (BMI Korea) were used for activation. After 24 hours, the lentiviral vector was placed in the activated T cells to an MOI of 3 and cultured overnight. The next day, after washing the transduced T cells, 100IU / mL recombinant human IL-2 was placed and placed in a G-Rex 6M Plate (WilsonWolf) to culture CAR-T cells in large quantities. The cell number was confirmed by the change in the glucose concentration of the culture medium.

[0116] Experimental Example 1: Anti-BCMACAR expression analysis

[0117] CAR expression on the cell surface was confirmed by flow cytometry. Wash twice with D-PBS 1x10 6 After 10 cells were added, human BD Fc Block (BD Bioscience) was mixed and reacted at room temperature for 10 minutes. Biotinylated BCMA (Argo) or biotinylated anti-human Fab antibody was added together with PE-Cy7 mouse anti-human CD3 (BD ​​Bioscience) antibody and reacted at room temperature for 15 minutes. After washing the sample with D-PBS for more than 2 times, Fixable Viability Dye eFlour 780 (eBioscience) and streptavidin-Alexa were added. 647 (Streptavidin-Alexa 647) (Biolegend), and then reacted at room temperature for 15 minutes. After washing with D-PBS twice, the cells were resuspended with D-PBS and then passed through BD FACSVer TM Flow cytometer (BD FACSVerse TM CAR expression was confirmed by analysis with FlowCytometer.

[0118] The results of using biotinylated BCMA are as follows Figure 4 As shown, the results of using biotinylated anti-human Fab antibody are as follows Figure 5 As shown, the expression of the anti-BCMACAR described above was confirmed in the T cell (B10-BBZ CAR-T) group transformed with the vector including the anti-BCMACAR according to an example of the present invention.

[0119] Experimental Example 2: IFN-γ Release Assay

[0120] 5.0x10 cells were cultured individually in a 37°C, 5% CO2 cell culture incubator. 4 T cells, and 24 hours of co-culture (co-cultivation) 5.0x10 4 The amount of IFN-γ contained in the supernatant obtained by centrifuging each sample at 500×g for 5 minutes was measured using DuoSet ELISA Ancillary Reagent Kit 2 (R&D system) and Human IFN-gamma DuoSet ELISA (R&D system). The results are shown in Table 1. Figure 6 shown. Figure 6 The data separately represent the case where T cells were cultured alone (Single), the case where T cells were co-cultured with K562 cells that do not express BCMA (K562-Mock), the case where T cells were treated with stimulators (PMA / Ionomycin) (Stimulation), the case where T cells were co-cultured with K562 cells that express BCMA (K562-BCMA), and the case where the amount of interferon gamma (IFN-gamma) measured in the K-562-BCMA group was corrected to the ratio of CAR-positive T cells (CAR+correction, %IFN-gamma / ViableCAR-T).

[0121] As a result, for the cell group into which anti-BCMACAR was introduced, the secretion of interferon gamma (IFN-gamma), which is an indicator of cytotoxic activity, was significantly higher in all groups compared to the control group into which anti-BCMACAR was not introduced. In particular, it was confirmed that the secretion of interferon gamma (IFN-gamma) was specifically high in BMCA, the targeting molecule of the above-mentioned CAR.

[0122] Experimental Example 3: Confirmation of T cell transduction of CAR composed of anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv.

[0123] In order to evaluate the functional differences between the known anti-BCMA CAR (C11D5.3) and anti-BCMA (B10) CAR, based on the C11D5.3 scFv base sequence described in US20150051266A1 patent, as Figure 7 CAR was prepared as shown. On the 4th day after transduction with CAR composed of anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv, transduced T cells were screened using LNGFR magnetic beads. On the 6th day after screening, APC-labeled LNGFR Ab (Miltenyi biotec, clone: ​​ME20.4-1.H4, Cat No, 130-113-418), biotinylated human BCMA / TNFRSF17 protein (Biotinylated Human BCMA / TNFRSF17 Protein), FC, Avitag TM After staining T cells with Streptavidin-PE-Cy7, the percentage of transduced cells was confirmed by flow cytometry. As a result, it was confirmed that the purity of transduced T cells was more than 70%, and LNGFR and CAR were expressed in an almost 1:1 ratio ( Figure 7 ).

[0124] Experimental Example 4: T cells transduced with CAR composed of anti-BCMA (C11D5.3) and anti-BCMA (B10) scFv Cytotoxicity evaluation.

[0125] Co-cultured multiple myeloma cell line RPMI8226 cells (RPMI8226-GL) expressing BCMA and GFP at 37°C and 5% CO2 for 48 hours 1x10 5 and Mock T cells, anti-BCMA (B10) CAR T cells, anti-BCMA (C11D5.3) CAR T cells 0.5x10 5 pcs, 0.1x10 5Within 48 hours, the GFP fluorescence value was measured every 2 hours, and normalized to 0 hours to evaluate the cytotoxicity. The results showed that anti-BCMA (B10) CAR T cells showed similar cytotoxicity to anti-BCMA (C11D5.3) CAR T cells ( Figure 8 ).

[0126] Experimental Example 5. Evaluation of the in vivo anticancer efficacy of anti-BCMA (C11D5.3) CAR-T and anti-BCMA (B10) CAR-T price.

[0127] 1x10 7 After RPMI8226-GL was injected with S.Ceh, on day 12, 1x10 6 , 0.5x10 6 , 0.25x10 6 CAR-T cells were injected, and the anti-cancer efficacy was evaluated by measuring the luciferase activity expressed by tumor cells every other week. The experimental results showed that cytotoxicity was significantly observed in the amount of injected CAR-T cells, and the anti-cancer efficacy between anti-BCMA (C11D5.3) CAR-T cells and anti-BCMA (B10) CAR-T cells was similar ( Figures 9 to 12 ).

[0128] In summary, these results show that the anti-BCMA CAR-expressing T cells according to the present invention have high cytotoxic activity specific to BCMA and exhibit high in vitro and in vivo anti-cancer efficacy comparable to that of the existing BCMA CAR with outstanding efficacy.

[0129] The above description of the present invention is for illustration, and those skilled in the art should understand that the present invention can be easily deformed in other specific ways without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. For example, each structural element described in a single type can also be implemented in a dispersed manner, and similarly, the structural elements described in a dispersed manner can also be implemented in a combined form.

[0130] The scope of the present invention is determined by the appended claims, and it should be interpreted that all changes or modified embodiments derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. <110> Cool Rosell Ewha Womans University Industry-Academic Cooperation Group <120> Anti-BCMA binding domain, fusion protein containing the same and composition containing the same <130> P20190428KR <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH-CDR1 <400> 1 Asp Tyr Tyr Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH-CDR2 <400> 2 Ala Ile Ser His Ser Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VH-CDR3 <400> 3 Ala Lys Asn Asn Trp Gly Phe Asp Tyr 1 5 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> VL-CDR1 <400> 4 Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Thr 1 5 10 <210> 5 <211> 7 <212> PRT <213> artificial sequence <220> <223> VL-CDR2 <400> 5 Ser Asp Ser Lys Arg Pro Ser 1 5 <210> 6 <211> 11 <212> PRT <213> artificial sequence <220> <223> VL-CDR3 <400> 6 Ala Ala Trp Asp Ala Ser Leu Ser Gly Tyr Val 1 5 10 <210> 7 <211> 116 <212> PRT <213> artificial sequence <220> <223> VH <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Leu Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser His Ser Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Asn Trp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 110 <212> PRT <213> artificial sequence <220> <223> VL <400> 8 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asp Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Ala Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> scFv <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Leu Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser His Ser Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Asn Trp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Ser Asn Asp Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Ser Asp Ser Lys Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 210 215 220 Ala Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3zeta Signaling Domain <400> 10 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 11 <211> 42 <212> PRT <213> artificial sequence <220> <223> 41BB signal transmission structure <400> 11 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 12 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Transmembrane domain <400> 12 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 13 <211> 45 <212> PRT <213> Artificial sequence <220> <223> Hinge domain <400> 13 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 14 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> signal peptide <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 15 <211> 485 <212> PRT <213> artificial sequence <220> <223> a-BCMA CAR <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Asp Tyr Tyr Met Ser Trp Val Arg Leu Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ala Ile Ser His Ser Ser Gly Asn Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Lys Asn Asn Trp Gly Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro 145 150 155 160 Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly 165 170 175 Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Thr Trp Tyr Gln Gln Leu 180 185 190 Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Ser Lys Arg Pro 195 200 205 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala 210 215 220 Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr 225 230 235 240 Cys Ala Ala Trp Asp Ala Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly 245 250 255 Thr Lys Leu Thr Val Leu Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485

Claims

1. An isolated BCMA-binding protein, comprising an antigen-binding domain that specifically binds to BCMA, characterized in that: The antigen binding domain comprises: i) a complementarity determining domain 1 of the heavy chain variable domain, which consists of the amino acid sequence of SEQ ID NO.1; ii) complementarity determining domain 2 of the heavy chain variable domain, which consists of the amino acid sequence of SEQ ID NO.2; iii) complementarity determining domain 3 of the heavy chain variable domain, which consists of the amino acid sequence of SEQ ID NO.3; iv) a complementarity determining domain 1 of the light chain variable domain, which consists of the amino acid sequence of SEQ ID NO.4; v) a complementarity determining domain 2 of the light chain variable domain, which consists of the amino acid sequence of SEQ ID NO.5; and vi) the complementarity determining domain 3 of the light chain variable domain, which comprises the amino acid sequence of SEQ ID NO.

6.

2. The isolated BCMA-binding protein according to claim 1, characterized in that The BCMA binding protein includes: A heavy chain variable domain sequence consisting of the amino acid sequence of SEQ ID NO.7; and The light chain variable domain consists of the amino acid sequence of SEQ ID NO.

8.

3. The isolated BCMA-binding protein according to claim 2, characterized in that The BCMA-binding protein further comprises a linker, wherein the linker, VH and VL are arranged in an order selected from VH-linker-VL or VL-linker-VH from the N-terminus to the C-terminus.

4. The isolated BCMA-binding protein according to claim 1, characterized in that The BCMA binding protein consists of the amino acid sequence of SEQ ID NO.

9.

5. The isolated BCMA-binding protein according to claim 1, characterized in that The BCMA binding protein is selected from the group consisting of: i) an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG and IgM; ii) a natural antibody fragment selected from Fv, Fab, Fab′, and F(ab′)2; iii) an engineered antibody selected from scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, tetrabody, pentabody; iv) Fc, one or more heavy chain constant domains, multimerization domains, chemical linkers or combinations thereof connected to ii) or iii); v) an anti-BCMA chimeric antigen receptor consisting of the amino acid sequence of SEQ ID NO.

15.

6. An isolated nucleic acid, characterized in that Encodes the BCMA-binding protein of claim 1.

7. A cell, characterized in that It includes a nucleic acid encoding the BCMA-binding protein of claim 6.

8. A cell, characterized in that The BCMA-binding protein of claim 1 is expressed.

9. A composition having an anti-cancer effect, characterized in that: Comprising the cell of claim 7 or 8.

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