Anti-BCMA antibodies and chimeric antigen receptors

Abstract

Anti-BCMA antibodies and chimeric antigen receptors (CARs) are provided. Immune cells expressing the anti-BCMA CARs can be used to treat cancer. The anti-BCMA antibodies and CARs can recognize the extracellular domain of human BCMA. Anti-BCMA CAR T cells exhibit specific cytotoxicity against BCMA-positive target cells.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy, created on 7 May 2021, is named 11299-009938-WO0_ST25.txt and has a size of 91KB.

[0002] This invention relates to anti-BCMA antibodies and chimeric antigen receptors (CARs), as well as their preparation and use. [Background technology]

[0003] Multiple myeloma (MM) is the second most common hematological malignancy after non-Hodgkin lymphoma. While significant advances have been made in recent years with chemotherapy, proteasome inhibitors, immunomodulators (thalidomide derivatives), and CD38-targeted antibodies, almost all patients eventually experience relapse. Therefore, new treatment regimens are urgently needed.

[0004] B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17, is a membrane protein widely expressed on mature B cells. A key characteristic of BCMA is that it is highly expressed in all MM cells and not in other normal tissues (except plasma cells). Therefore, BCMA has become a common target for many pharmaceutical companies and research institutions to treat relapsed or refractory multiple myeloma (R / R MM patients).

[0005] Currently, immunotherapies developed against BCMA targets are mainly classified into three categories: chimeric antigen receptor T cell (CAR-T) therapy, bispecific antibodies (BsAbs), and antibody-drug conjugates (ADCs). CAR-T (bb2121), jointly developed by Celgene and Bluebird Bio, has entered Phase 3 clinical trials. Current bispecific antibodies targeting BCMA have one end that binds to BCMA and the other end that binds to the CD3 T cell receptor on the surface of T cells, thus recruiting T cells to tumor cells and subsequently killing them. The optimal dose of AMG-420 (Amgen) has been reported to achieve an ORR of 70% when treating R / R MM patients. In terms of ADCs, GSK-2857916 from GlaxoSmithKline (GSK) has been used in numerous clinical trials to treat various types of MM patients. In particular, a major clinical trial treating R / R MM patients as a third / fourth-line therapy has yielded interim results. Recent results published in the Blood Cancer Journal showed that patients treated with GSK-2857916 had an ORR of 60%, a complete response rate (CRR) of 15%, and a progression-free survival (PFS) of 12 months.

[0006] Nevertheless, therapies available for treating R / R MM patients are still limited, and there is still a need to develop new drugs and methods for treating R / R MM patients in this field. [Overview of the project]

[0007] This disclosure relates to the light chain variable region (V L ) and heavy chain variable region (V H The present invention provides an anti-BCMA antibody or its antigen-binding moiety, which includes ).

[0008] This disclosure relates to the light chain variable region (V L ) and heavy chain variable region (V HThe present invention provides a chimeric antigen receptor (CAR) that includes an anti-BCMA antigen-binding region.

[0009] The light chain variable region may include CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are approximately 80% to approximately 100% identical to the amino acid sequences shown in (i) SEQ ID NOs. 76, 77, and 78, respectively; (ii) SEQ ID NOs. 82, 83, and 84, respectively; (iii) SEQ ID NOs. 88, 89, and 90, respectively; (iv) SEQ ID NOs. 94, 95, and 96, respectively; (v) SEQ ID NOs. 100, 101, and 102, respectively; (vi) SEQ ID NOs. 106, 107, and 108, respectively; (vii) SEQ ID NOs. 112, 113, and 114, respectively; (viii) SEQ ID NOs. 118, 119, and 120, respectively; or (ix) SEQ ID NOs. 124, 125, and 126, respectively.

[0010] The heavy chain variable region may include three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences that are approximately 80% to approximately 100% identical to the amino acid sequences shown in (i) SEQ ID NOs. 79, 80, and 81, respectively; (ii) SEQ ID NOs. 85, 86, and 87, respectively; (iii) SEQ ID NOs. 91, 92, and 93, respectively; (iv) SEQ ID NOs. 97, 98, and 99, respectively; (v) SEQ ID NOs. 103, 104, and 105, respectively; (vi) SEQ ID NOs. 109, 110, and 111, respectively; (vii) SEQ ID NOs. 115, 116, and 117, respectively; (viii) SEQ ID NOs. 121, 122, and 123, respectively; or (ix) SEQ ID NOs. 127, 128, and 129, respectively.

[0011] V L and V H (i)V Lmay include CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively, and V H may include CDR1, CDR2, and CDR3 of three complementarity-determining regions (CDRs) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively, or (ii) V L may include CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively, and V H may include CDR1, CDR2, and CDR3 of three complementarity-determining regions (CDRs) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively, or (iii) V L may include CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively, and V H may include CDR1, CDR2, and CDR3 of three complementarity-determining regions (CDRs) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, or (iv) V L may include CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively, and V H may include CDR1, CDR2, and CDR3 of three complementarity-determining regions (CDRs) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively, or (v) V LThis may include three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively. H (vi)V L This may include three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively. H (vii)V L This may include three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 112, 113, and 114, respectively. H (vii)V L This may include three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, and V H This may include three complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, each having an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 121, 122, and 123, respectively, or (xi)V LThis may include three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126, respectively. H This may include three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively.

[0012] V L and V H It may have an amino acid sequence that is approximately 80% to approximately 100% identical to the amino acid sequences shown in (i) SEQ ID NOs. 53 and 54, respectively, (ii) SEQ ID NOs. 55 and 56, respectively, (iii) SEQ ID NOs. 57 and 58, respectively, (vi) SEQ ID NOs. 59 and 60, respectively, (v) SEQ ID NOs. 61 and 62, respectively, (vi) SEQ ID NOs. 63 and 64, respectively, (vii) SEQ ID NOs. 65 and 66, respectively, (viii) SEQ ID NOs. 67 and 68, respectively, or (ix) SEQ ID NOs. 69 and 70, respectively.

[0013] V L This may include an amino acid sequence that is approximately 80% to approximately 100% identical to any one of the amino acid sequences shown in SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69. H This may include an amino acid sequence that is approximately 80% to approximately 100% identical to any one of the amino acid sequences shown in SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 70.

[0014] The antibody or its antigen-binding portion may be (a) a whole immunoglobulin molecule, (b) scFv, (c) a Fab fragment, (d) F(ab')2, or (e) a disulfide-bonded Fv.

[0015] The antibody or its antigen-binding moiety may include at least one constant domain selected from (a) an IgG constant domain and (b) an IgA constant domain.

[0016] This antibody or its antigen-binding moiety may contain at least one human constant domain.

[0017] This disclosure provides an antibody conjugate comprising the antibody or its antigen-binding moiety linked to a conjugate moiety. The conjugate moiety may be a detectable marker, drug, toxin, cytokine, radionuclide, enzyme, targeting moiety, or a combination thereof. In one embodiment, the antibody conjugate is an antibody-drug conjugate (ADC).

[0018] The disclosure also includes compositions comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier.

[0019] This disclosure provides a composition comprising the CAR or immune cells and a pharmaceutically acceptable carrier.

[0020] This disclosure provides nucleic acids encoding the antibody or its antigen-binding portion (or encoding a CAR), vectors containing the nucleic acid, and cells containing the vector.

[0021] In CARs, the anti-BCMA antigen-binding region may be a single-chain variable fragment (scFv) that specifically binds to BCMA.

[0022] CAR is as follows: (a) signal peptide, (b) Hinge region, (c) Transmembrane domain, (d) Co-stimulation areas, and, (e) Cytoplasmic signaling domain, It may include one or more of these.

[0023] The co-stimulatory regions of CAR may include the co-stimulatory regions of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0024] The cytoplasmic signaling domain of CAR may include the cytoplasmic signaling domain of CD3ζ.

[0025] The hinge region of CAR may include the hinge regions of Ig4, CD8, CD28, CD137, or a combination thereof.

[0026] The transmembrane domain of CAR may include the transmembrane domains of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

[0027] This disclosure provides immune cells that express CARs. The immune cells may be T cells, natural killer (NK) cells, natural killer T cells, lymphoid progenitor cells, hematopoietic stem cells, stem cells, macrophages, or dendritic cells.

[0028] This disclosure provides a method for treating cancer. This method may include administering the immune cells or composition to a subject in need.

[0029] Cancer can be a blood cancer. Cancer can be a plasma cell malignancy. Cancer can be a BCMA-positive malignancy. Cancer can be multiple myeloma (MM) or plasma cell leukemia.

[0030] Immune cells can be administered by infusion, injection, infusion, implantation, and / or transplantation. Immune cells can be administered intravenously, subcutaneously, intradermally, intralymphatically, intratumorally, intramedullarily, intramuscularly, or intraperitoneally. In one embodiment, immune cells are administered via intravenous infusion.

[0031] Immune cells can be of allogeneic or autologous origin to the subject.

[0032] The test subject may be human or mammal. This antibody, its antigen-binding moiety, CAR, composition, and method may be used in all vertebrates, including mammals and non-mammals, such as humans, mice, rats, guinea pigs, hamsters, dogs, cats, cattle, horses, goats, sheep, pigs, monkeys, apes, gorillas, chimpanzees, rabbits, ducks, geese, chickens, amphibians, reptiles, and other animals.

[0033] The purpose of this disclosure is to provide BCMA antibodies, as well as their preparation and use.

[0034] In a first embodiment of this disclosure, the heavy chain variable (V) is encoded by the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 37. H ) region and light chain variable (V) encoded by the nucleotide sequences shown in SEQ ID NOs: 2, 4, 6, 8, 13, 18, 23, 25, 27, 29, 31, 33, 35, 36, and 38. L A BCMA antibody containing the ) region is provided.

[0035] On the other hand, the antibody comprises a heavy chain having the amino acid sequences shown in SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49, and a light chain having the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 51.

[0036] On the other hand, the antibody has the amino acid sequence shown in SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 70. H V has a region and the amino acid sequence shown in SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69. L Includes the region.

[0037] Any of the above-mentioned amino acid sequences may include derivative sequences in which at least one amino acid is optionally added, deleted, modified, and / or substituted, while maintaining the binding affinity of BCMA.

[0038] In certain embodiments, the antibody is encoded by the nucleotide sequence shown below. H Region and V L Includes area: [Table 1]

[0039] In certain embodiments, the antibody is a mouse antibody.

[0040] In certain embodiments, the antibody comprises a heavy chain and a light chain having the following amino acid sequences: [Table 2]

[0041] In certain embodiments, the antibody is a chimeric antibody.

[0042] In certain embodiments, the antibody has the following amino acid sequence V H Region and V L Includes area: [Table 3]

[0043] In certain embodiments, the antibody is a single-chain antibody.

[0044] In certain embodiments, the antibody binds to human BCMA protein and / or cynomolgus monkey BCMA protein.

[0045] In certain embodiments, the antibody binds to the extracellular domain of the BCMA protein.

[0046] In certain embodiments, the antibody does not bind to tumor necrosis factor receptor superfamily 13B.

[0047] In certain embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0048] In certain embodiments, the heavy chain constant region is derived from humans or mice.

[0049] In certain embodiments, the heavy chain constant region is the heavy chain IgG1 constant region of a human antibody.

[0050] In certain embodiments, the light chain constant region is derived from humans or mice.

[0051] In certain embodiments, the light chain constant region is the light chain κ constant region of a human antibody.

[0052] In certain embodiments, the number of added, deleted, modified, and / or substituted amino acids is 1 to 5 (e.g., 1 to 3, 1 to 2, or 1).

[0053] In certain embodiments, derivative sequences that can be modified and / or substituted with at least one amino acid to maintain BCMA binding affinity are amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or sequence similarity.

[0054] In certain embodiments, the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or a combination thereof.

[0055] In certain embodiments, the ratio (Z1 / Z0) of immunogenicity Z1 of a human chimeric antibody to immunogenicity Z0 of a human non-chimeric antibody (such as a mouse antibody) is 0 to 0.5, 0 to 0.2, or 0 to 0.05 (e.g., 0.001 to 0.05).

[0056] In certain embodiments, the antibody is a partially or fully humanized monoclonal antibody, or a fully human monoclonal antibody.

[0057] In certain embodiments, the antibody is either a double-chain antibody or a single-chain antibody.

[0058] In certain embodiments, the antibody is either a full-length antibody protein or an antigen-binding fragment.

[0059] In certain embodiments, the antibody is a bispecific antibody or a multispecific antibody.

[0060] In certain embodiments, the antibody is either a monoclonal antibody or a polyclonal antibody.

[0061] In the second aspect of this disclosure, (i) an antibody as described in the first aspect of this disclosure, (ii) Any tag sequence that assists expression and / or purification, Recombinant proteins containing the following are provided.

[0062] In certain embodiments, the tag array may include 6xHis tags.

[0063] In certain embodiments, the recombinant protein (or polypeptide) includes a fusion protein.

[0064] In certain embodiments, the recombinant protein may be a monomer, a dimer, or a polymer.

[0065] In a third aspect of this disclosure, from the N-terminus to the C-terminus, (i) scFv targeting BCMA, (ii) Transmembrane domain and (iii) at least one co-stimulatory domain, (iv) Activation domain and A chimeric antigen receptor (CAR) fusion protein is provided, which includes [the specified element].

[0066] scFv-targeted BCMA has the amino acid sequence shown in SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 70. H V has a region and the amino acid sequence shown in SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69. L Includes the region.

[0067] In certain embodiments, scFv is V having the amino acid sequence shown below. H Region and V L Includes area: [Table 4]

[0068] In certain embodiments, the CAR fusion protein is expressed by the following formula I: L-scFv-H-TM-C-CD3ζ (I) (In the formula, Each "-" independently represents a linked peptide or peptide bond. L is any signal peptide sequence, scFv is an scFv that targets BCMA, H is an arbitrary hinge region, TM is a transmembrane domain, C is a co-stimulus signaling molecule, and CD3ζ has the structure shown in the cytoplasmic signaling sequence of CD3ζ.

[0069] In certain embodiments, L is a signal peptide of a protein selected from the group consisting of CD8, GM-CSF, CD4, and CD137, or a combination thereof.

[0070] In certain embodiments, L is a signal peptide derived from CD8.

[0071] In certain embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 52.

[0072] In certain embodiments, scFv is given by the following formula II or III: V L -L1-V H (II) V H -L2-V L (III) (In the formula, V L This is the light chain variable region, V H This is the heavy chain variable region, L1 and L2 each have the structure shown in (the linked peptides, independently of each other).

[0073] In certain embodiments, the amino acid sequence of the linked peptide is as shown in SEQ ID NO: 71.

[0074] In certain embodiments, H is a hinge region of a protein selected from the group consisting of CD8, CD28, and CD137, or a combination thereof. In certain embodiments, H is a hinge region derived from CD8. In certain embodiments, the amino acid sequence of the hinge region is as shown in SEQ ID NO: 72.

[0075] In certain embodiments, TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0076] In certain embodiments, the TM is a transmembrane region derived from CD8. In certain embodiments, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 73.

[0077] In certain embodiments, C is a co-stimulatory signaling molecule of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof. In certain embodiments, C is a co-stimulatory signaling molecule derived from 4-1BB. In certain embodiments, the amino acid sequence of the co-stimulatory signaling molecule is as shown in SEQ ID NO: 74.

[0078] In certain embodiments, the cytoplasmic signaling sequence of CD3ζ is as shown in SEQ ID NO: 75.

[0079] In the fourth aspect of this disclosure, (1) The antibody described in the first aspect of this disclosure (2) Recombinant proteins as described in a second aspect of this disclosure, (3) CAR fusion proteins as described in the third aspect of this disclosure Polynucleotides are provided that encode polypeptides such as the following.

[0080] In certain embodiments, the V of the antibody H The polynucleotides encoding the region are as shown in SEQ ID NOs: 1, 3, 5, 7, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 26, 28, 30, 32, 34, and 37, and / or the V of the antibody. L The polynucleotides encoding the region are as shown in SEQ ID NOs: 2, 4, 6, 8, 13, 18, 23, 25, 27, 29, 31, 33, 35, 36, and 38.

[0081] In certain embodiments, V H Polynucleotides and V that code for the region L The polynucleotides encoding the region are shown below: [Table 5]

[0082] A fifth aspect of this disclosure provides a vector comprising any of the polynucleotides in the fourth aspect of this disclosure.

[0083] In certain embodiments, the vector may be a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, adenovirus, or mammalian cell virus such as a retrovirus, or any other vector.

[0084] In certain embodiments, the vector may be a lentiviral vector.

[0085] A sixth aspect of this disclosure provides a genetically engineered host cell that includes a vector as described in the fifth aspect of this disclosure, or is incorporated into the genome together with an exogenous polynucleotide as described in the fourth aspect of this disclosure, or expresses an antibody as described in the first aspect of this disclosure, a recombinant protein as described in the second aspect of this disclosure, or a CAR fusion protein as described in the third aspect of this disclosure.

[0086] In certain embodiments, cells are isolated cells and / or genetically modified cells.

[0087] In certain embodiments, the immune cells are derived from humans or non-human mammals (such as mice).

[0088] In certain embodiments, the cells include T cells and NK cells.

[0089] In certain embodiments, the host cells are engineered immune cells.

[0090] In certain embodiments, the manipulated immune cells include T cells or NK cells, for example (i) chimeric antigen receptor T cells (CAR-T cells), or (ii) chimeric antigen receptor NK cells (CAR-NK cells).

[0091] In the seventh aspect of this disclosure, (a) an antibody portion selected from the group consisting of antibodies described in the first aspect of the present invention, (b) A conjugate moiety conjugated to the antibody moiety, selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof, An antibody conjugate containing the above is provided.

[0092] In certain embodiments, the antibody portion is conjugated to the conjugate portion via a chemical bond or linker.

[0093] In certain embodiments, the antibody conjugate is an antibody-drug conjugate.

[0094] An eighth aspect of this disclosure provides a method for preparing engineered cells. The engineered cells express an antibody as described in the first aspect of this disclosure, a recombinant protein as described in the second aspect of this disclosure, or a CAR fusion protein as described in the third aspect of this disclosure. The steps include: transducing a polynucleotide as described in the fourth aspect of this disclosure or a vector as described in the fifth aspect of this disclosure into a host cell to obtain engineered cells.

[0095] In certain embodiments, the method further includes a step of detecting the function and effectiveness of the resulting manipulated cells.

[0096] A ninth aspect of this disclosure provides a pharmaceutical composition comprising an antibody as described in a first aspect of this disclosure, a recombinant protein as described in a second aspect of this disclosure, a CAR fusion protein as described in a third aspect of this disclosure, a vector as described in a fifth aspect of this disclosure, a host cell as described in a sixth aspect of this disclosure, or an antibody conjugate as described in a seventh aspect of this disclosure, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0097] In certain embodiments, the pharmaceutical composition is a liquid preparation.

[0098] In certain embodiments, the pharmaceutical composition is an injectable preparation.

[0099] In certain embodiments, the pharmaceutical composition further comprises a second antitumor active ingredient, such as a secondary antibody or a chemotherapeutic agent.

[0100] In certain embodiments, the chemotherapeutic agent is docetaxel, carboplatin, or a combination thereof.

[0101] A tenth aspect of this disclosure provides the use of an active ingredient. The active ingredient is selected from the group consisting of an antibody as described in the first aspect of this disclosure, a recombinant protein as described in the second aspect of this disclosure, a CAR fusion protein as described in the third aspect of this disclosure, a vector as described in the fifth aspect of this disclosure, a host cell as described in the sixth aspect of this disclosure, or an antibody conjugate as described in the seventh aspect of this disclosure, and is used (a) to prepare a diagnostic reagent or kit, and / or (b) to prepare a drug or preparation for the prophylaxis and / or treatment of cancer or tumor.

[0102] In certain embodiments, the tumor is a BCMA-positive tumor.

[0103] In certain embodiments, the tumor is a hematological malignancy, a solid tumor, or a combination thereof.

[0104] In certain embodiments, hematological malignancies include acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0105] In certain embodiments, solid tumors include gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumors, lung cancer, small intestine carcinomas, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumors, thyroid cancer, or combinations thereof.

[0106] In certain embodiments, the tumor is multiple myeloma.

[0107] In certain embodiments, the diagnostic reagent is a test specimen or test plate.

[0108] In certain embodiments, the diagnostic reagent or kit is used to (1) detect BCMA protein in a sample, (2) detect endogenous BCMA protein in tumor cells, and / or (3) detect tumor cells expressing BCMA protein.

[0109] An eleventh aspect of this disclosure provides a method (including diagnostic or non-diagnostic) for detecting BCMA protein in a sample in vitro, comprising the steps of (1) contacting the sample in vitro with an antibody as described in the first aspect of this disclosure, a recombinant protein as described in the second aspect of this disclosure, or an antibody conjugate as described in the seventh aspect of this disclosure, and (2) detecting whether an antigen-antibody complex has been formed (if formed, this means that BCMA protein is present in the sample).

[0110] In a twelfth aspect of this disclosure, a test plate is provided, comprising a substrate (support plate) and a test strip. The test strip comprises an antibody as described in a first aspect of this disclosure, a recombinant protein as described in a second aspect of this disclosure, or an antibody conjugate as described in a seventh aspect of this disclosure, or a combination thereof.

[0111] A thirteenth aspect of this disclosure provides a kit which may include (1) a first container containing the antibody described in the first aspect, and / or (2) a second container containing a secondary antibody against the antibody described in the first aspect of this disclosure.

[0112] The kit may include a test plate as described in a twelfth aspect of this disclosure.

[0113] A fourteenth aspect of this disclosure provides a method for preparing a recombinant polypeptide, comprising the steps of (a) culturing a host cell as described in a sixth aspect of this disclosure under conditions suitable for expression, and (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody as described in a first aspect of this disclosure or a recombinant protein as described in a second aspect of this disclosure.

[0114] A fifteenth aspect of this disclosure provides a method for treating a disease. This method may include administering to a subject in need an effective dose of an antibody as described in the first aspect of this disclosure, a recombinant protein as described in the second aspect of this disclosure, an antibody conjugate as described in the seventh aspect of this disclosure, a host cell as described in the sixth aspect of this disclosure, or a pharmaceutical composition as described in the ninth aspect of this disclosure, or a combination thereof.

[0115] In certain embodiments, the disease is BCMA-positive cancer / tumor. [Brief explanation of the drawing]

[0116] [Figure 1A] This figure shows the ELISA analysis of the binding titer to hBCMA-ECD-Fc in mouse serum after the first booster immunization. [Figure 1B] This figure shows the ELISA analysis of the binding titer to cynoBCMA-ECD-Fc in mouse serum after the first booster immunization. [Figure 2] This figure shows flow cytometry analysis of the binding titers of mouse serum to K562-BCMA+ and K562 cells after the first booster immunization. [Figure 3A] This figure shows the ELISA analysis of the binding titer to hBCMA-ECD-Fc in mouse serum after a second booster immunization. [Figure 3B] This figure shows the ELISA analysis of the binding titer to cynoBCMA-ECD-Fc in mouse serum after a second booster immunization. [Figure 4]This figure shows flow cytometry analysis of the binding titers of K562-BCMA+ and K562 cells in mouse serum after a second booster immunization. [Figure 5A] This figure shows the ELISA analysis of the binding titer to hBCMA-ECD-Fc in mouse serum after a third booster immunization. [Figure 5B] This figure shows the ELISA analysis of the binding titer to cynoBCMA-ECD-Fc in mouse serum after a third booster immunization. [Figure 6] This figure shows flow cytometry analysis of L-BCMA+ and L cell binding titers in mouse serum before and after the third booster immunization. [Figure 7A] This diagram shows the screening process for hybridoma cells (all selected clones do not bind to NC-Fc). [Figure 7B] This diagram shows the screening process for hybridoma cells (all selected clones do not bind to NC-Fc). [Figure 7C] This diagram shows the screening process for hybridoma cells (all selected clones do not bind to NC-Fc). [Figure 7D] This diagram shows the screening process for hybridoma cells (all selected clones do not bind to NC-Fc). [Figure 8] This figure shows SDS-PAGE analysis for the production and purification of small batches of antibodies. [Figure 9A] This figure shows the ELISA analysis of the binding ability of hybridoma antibodies to hBCMA-ECD-Fc. [Figure 9B] This figure shows the ELISA analysis of the binding ability of hybridoma antibodies to hBCMA-ECD-Fc. [Figure 10A] This figure shows the ELISA analysis of the binding ability of hybridoma antibodies to cynoBCMA-ECD-Fc. [Figure 10B] This figure shows the ELISA analysis of the binding ability of hybridoma antibodies to cynoBCMA-ECD-Fc. [Figure 11A] This figure shows the ELISA analysis of the binding ability of hybridoma antibodies to hTACI-ECD-Fc. [Figure 11B] This figure shows the ELISA analysis of the binding ability of hybridoma antibodies to hTACI-ECD-Fc. [Figure 12A] This figure shows a flow cytometry analysis of the binding ability of hybridoma antibodies to L-BCMA+ cells. [Figure 12B] This figure shows a flow cytometry analysis of the binding ability of hybridoma antibodies to L-BCMA+ cells. [Figure 13A] This figure shows a flow cytometry analysis of the binding ability of hybridoma antibodies to L cells. [Figure 13B] This figure shows a flow cytometry analysis of the binding ability of hybridoma antibodies to L cells. [Figure 14A] This figure shows the flow cytometry analysis of the competitive binding of soluble BCMA to mAb001. [Figure 14B] This figure shows the flow cytometry analysis of the competitive binding of soluble BCMA to mAb001. [Figure 14C] This figure shows the flow cytometry analysis of the competitive binding of soluble BCMA to mAb001. [Figure 14D] This figure shows the flow cytometry analysis of the competitive binding of soluble BCMA to mAb001. [Figure 14E] This figure shows the flow cytometry analysis of the competitive binding of soluble BCMA to mAb001. [Figure 14F] This figure shows the flow cytometry analysis of the competitive binding of soluble BCMA to mAb001. [Figure 15A] This figure shows the ELISA analysis of the binding activity of chimeric antibodies to hBCMA-ECD-Fc. [Figure 15B] This figure shows the ELISA analysis of the binding activity of chimeric antibodies to hBCMA-ECD-Fc. [Figure 16A] This figure shows flow cytometry analysis of the binding activity of chimeric antibodies to L-BCMA+ and L cells. [Figure 16B] This figure shows flow cytometry analysis of the binding activity of chimeric antibodies to L-BCMA+ and L cells. [Figure 17A] This figure shows the detection of transfection efficiency of engineered T cells targeting the human BCMA chimeric antigen receptor. The staining method for recombinant human BCMA protein Fc fragment identifies the expression level of the CAR gene-encoded protein on the cell membrane surface of CART-BCMA cells cultured for 7 days. [Figure 17B] This figure shows the detection of transfection efficiency of engineered T cells targeting the human BCMA chimeric antigen receptor. The staining method for recombinant human BCMA protein Fc fragment identifies the expression level of the CAR gene-encoded protein on the cell membrane surface of CART-BCMA cells cultured for 7 days. [Figure 18A] This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 18B] This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 18C] This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 18D] This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 18E] This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 18F] This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 18G] This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 18H]This figure shows the expression level of CD137 on the T cell membrane surface as detected by flow cytometry. [Figure 19A] This figure shows the secretion level of IFNγ in the culture supernatant detected by the ELISA method. [Figure 19B] This figure shows the secretion level of IFNγ in the culture supernatant detected by the ELISA method. [Figure 20A] This figure shows RTCA real-time label-free cell analysis to detect the killing effect of CART-BCMA on target cells. Human BCMA-positive A549-BCMA-1D6 tumor cell lineage, monkey BCMA-positive A549-BCMA-M tumor cell lineage, and BCMA-negative A549 tumor cell lineage were co-cultured in 200 μl of CBMG-RC-09a for 8 hours according to various effector / target ratios, and the killing rate was calculated. [Figure 20B] This figure shows RTCA real-time label-free cell analysis to detect the killing effect of CART-BCMA on target cells. Human BCMA-positive A549-BCMA-1D6 tumor cell lineage, monkey BCMA-positive A549-BCMA-M tumor cell lineage, and BCMA-negative A549 tumor cell lineage were co-cultured in 200 μl of CBMG-RC-09a for 8 hours according to various effector / target ratios, and the killing rate was calculated. [Figure 20C] This figure shows RTCA real-time label-free cell analysis to detect the killing effect of CART-BCMA on target cells. Human BCMA-positive A549-BCMA-1D6 tumor cell lineage, monkey BCMA-positive A549-BCMA-M tumor cell lineage, and BCMA-negative A549 tumor cell lineage were co-cultured in 200 μl of CBMG-RC-09a for 8 hours according to various effector / target ratios, and the killing rate was calculated. [Figure 20D]This figure shows RTCA real-time label-free cell analysis to detect the killing effect of CART-BCMA on target cells. Human BCMA-positive A549-BCMA-1D6 tumor cell lineage, monkey BCMA-positive A549-BCMA-M tumor cell lineage, and BCMA-negative A549 tumor cell lineage were co-cultured in 200 μl of CBMG-RC-09a for 8 hours according to various effector / target ratios, and the killing rate was calculated. [Figure 20E] This figure shows RTCA real-time label-free cell analysis to detect the killing effect of CART-BCMA on target cells. Human BCMA-positive A549-BCMA-1D6 tumor cell lineage, monkey BCMA-positive A549-BCMA-M tumor cell lineage, and BCMA-negative A549 tumor cell lineage were co-cultured in 200 μl of CBMG-RC-09a for 8 hours according to various effector / target ratios, and the killing rate was calculated. [Figure 20F] This figure shows RTCA real-time label-free cell analysis to detect the killing effect of CART-BCMA on target cells. Human BCMA-positive A549-BCMA-1D6 tumor cell lineage, monkey BCMA-positive A549-BCMA-M tumor cell lineage, and BCMA-negative A549 tumor cell lineage were co-cultured in 200 μl of CBMG-RC-09a for 8 hours according to various effector / target ratios, and the killing rate was calculated. [Modes for carrying out the invention]

[0117] This disclosure relates to anti-BCMA antibodies, antibody fragments (e.g., antigen-binding portions of antibodies), and chimeric antigen receptors that may be used in various therapeutic, preventive, diagnostic, and other applications.

[0118] Antibodies, or their antigen-binding moieties, can include, but are not limited to, humanized antibodies, human antibodies, monoclonal antibodies, chimeric antibodies, polyclonal antibodies, recombinantly expressed antibodies, and the aforementioned antigen-binding moieties. An example of an antibody antigen-binding moiety is the portion of the antibody that specifically binds to BCMA.

[0119] This disclosure provides a method for treating diseases such as cancer in a subject by administering an effective amount of the antibody or its antigen-binding portion, or immune cells containing the chimeric antigen receptor, to the subject.

[0120] The disclosure also includes methods for blocking the function of BCMA in mammals, which include administering a composition to a mammal containing the antibody or its antigen-binding portion, or immune cells containing the chimeric antigen receptor.

[0121] Another method of this disclosure relates to inhibiting the growth and / or differentiation of BCMA-expressing cells, including contacting cells with immune cells containing the antibody or antigen-binding fragment, or the chimeric antigen receptor.

[0122] Following extensive and thorough investigations and mass screenings, numerous anti-BCMA monoclonal antibodies were obtained. This disclosure further provides the construction of BCMA-targeting chimeric antigen receptors, as well as methods for preparing and identifying the activity of T cells expressing BCMA-targeting chimeric antigen receptors. Specifically, the inventors successfully screened nine hybridoma cell lines (6G10-1D7, 99B3G3, 105C10F1, 113B3F12, 109C5F3C1, 143D6F4, 151A9A4, 107B11E1D7, and 107A9A4D2), and the antibodies they secreted recognize the soluble extracellular domain of human BCMA (e.g., on the cell membrane surface). Antibodies secreted by 6G10-1D7, 99B3G3, 109C5F3C1, 143D6F4, 151A9A4, 107B11E1D7, and 107A9A4D2 can also cross-recognize the soluble extracellular domain of the cynomolgus monkey BCMA molecule. Anti-BCMA chimeric antigen receptors (CARs) were also constructed. These CARs exhibit varying responsiveness to BCMA-positive target cells and possess desired specific cytotoxicity against target cells.

[0123] Mice were immunized with a fusion protein (hBCMA-ECD-Fc) of the extracellular domain of human BCMA (Met1-Ala54) and the human IgG1 Fc fragment (Pro100-Lys330). Monoclonal antibodies against human BCMA were screened using hybridoma technology. The antibodies had to be able to cross-recognize cynomolgus monkey BCMA (cynoBCMA-ECD-Fc) and not recognize tumor necrosis factor receptor superfamily member 13B (TACI, which shares a common ligand with BCMA).

[0124] This disclosure relates to the light chain variable region (V L ) and heavy chain variable region (V H The present invention provides an anti-BCMA antibody or its antigen-binding moiety, which includes ).

[0125] This disclosure relates to the light chain variable region (V L ) and heavy chain variable region (V H The present invention provides a chimeric antigen receptor (CAR) that includes an anti-BCMA antigen-binding region.

[0126] In certain embodiments, the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) is approximately 80% to approximately 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, and less than or equal to the amino acid sequence shown in SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69. Examples include light chain variable regions containing amino acid sequences that are identical by all or approximately 83%, at least or approximately 84%, at least or approximately 85%, at least or approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%.

[0127] In certain embodiments, the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) is approximately 80% to approximately 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, and less than or equal to the amino acid sequence shown in SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 70. Examples include heavy chain variable regions containing amino acid sequences that are identical by all or approximately 83%, at least or approximately 84%, at least or approximately 85%, at least or approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%.

[0128] In certain embodiments, the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) is the light chain variable region (V L ) and heavy chain variable region (V H ) are listed, and here, V L and V H(i) SEQ ID NOs. 53 and 54 respectively, (ii) SEQ ID NOs. 55 and 56 respectively, (iii) SEQ ID NOs. 57 and 58 respectively, (vi) SEQ ID NOs. 59 and 60 respectively, (v) SEQ ID NOs. 61 and 62 respectively, (vi) SEQ ID NOs. 63 and 64 respectively, (vii) SEQ ID NOs. 65 and 66 respectively, (viii) SEQ ID NOs. 67 and 68 respectively, or (ix) SEQ ID NOs. 69 and 70 respectively, and approximately 80% to approximately 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, less Contains / has amino acid sequences that are identical by or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, at least or approximately 83%, at least or approximately 84%, at least or approximately 85%, at least or approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%.

[0129] The light chain variable region of the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) is approximately 80% to approximately 100%, at least or approximately 70%, of the amino acid sequences shown in (i) SEQ ID NOs. 76, 77, and 78, respectively; (ii) SEQ ID NOs. 82, 83, and 84, respectively; (iii) SEQ ID NOs. 88, 89, and 90, respectively; (iv) SEQ ID NOs. 94, 95, and 96, respectively; (v) SEQ ID NOs. 100, 101, and 102, respectively; (vi) SEQ ID NOs. 106, 107, and 108, respectively; (vii) SEQ ID NOs. 112, 113, and 114, respectively; (viii) SEQ ID NOs. 118, 119, and 120, respectively; or (ix) SEQ ID NOs. 124, 125, and 126, respectively. It may include one, two, or three complementary determination regions (CDRs) that are identical by or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, at least or approximately 83%, at least or approximately 84%, at least or approximately 85%, at least or approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%.

[0130] The heavy chain variable region of the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) is approximately 80% to approximately 100%, at least or at least 70%, less than the amino acid sequence shown in (i) SEQ ID NOs. 79, 80, and 81, respectively; (ii) SEQ ID NOs. 85, 86, and 87, respectively; (iii) SEQ ID NOs. 91, 92, and 93, respectively; (iv) SEQ ID NOs. 97, 98, and 99, respectively; (v) SEQ ID NOs. 103, 104, and 105, respectively; (vi) SEQ ID NOs. 109, 110, and 111, respectively; (vii) SEQ ID NOs. 115, 116, and 117, respectively; (viii) SEQ ID NOs. 121, 122, and 123, respectively; or (ix) SEQ ID NOs. 127, 128, and 129, respectively. It may include one, two, or three complementary determination regions (CDRs) that are identical by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or approximately 100%.

[0131] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) comprises three CDRs (CDR1, CDR2, and CDR3) that are identical to the amino acid sequences shown in (i) SEQ ID NOs. 76, 77, and 78, respectively; (ii) SEQ ID NOs. 82, 83, and 84, respectively; (iii) SEQ ID NOs. 88, 89, and 90, respectively; (iv) SEQ ID NOs. 94, 95, and 96, respectively; (v) SEQ ID NOs. 100, 101, and 102, respectively; (vi) SEQ ID NOs. 106, 107, and 108, respectively; (vii) SEQ ID NOs. 112, 113, and 114, respectively; (viii) SEQ ID NOs. 118, 119, and 120, respectively; or (ix) SEQ ID NOs. 124, 125, and 126, respectively. The heavy chain variable regions of the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) include three CDRs (CDR1, CDR2, and CDR3) which are identical to the amino acid sequences shown in (i) SEQ ID NOs. 79, 80, and 81, respectively; (ii) SEQ ID NOs. 85, 86, and 87, respectively; (iii) SEQ ID NOs. 91, 92, and 93, respectively; (iv) SEQ ID NOs. 97, 98, and 99, respectively; (v) SEQ ID NOs. 103, 104, and 105, respectively; (vi) SEQ ID NOs. 109, 110, and 111, respectively; (vii) SEQ ID NOs. 115, 116, and 117, respectively; (viii) SEQ ID NOs. 121, 122, and 123, respectively; or (ix) SEQ ID NOs. 127, 128, and 129, respectively.

[0132] In certain embodiments, the anti-BCMA antigen-binding region (or anti-BCMA antibody or its antigen-binding portion) is a light chain variable region (V L ) and heavy chain variable region (V H ) including, here, (i)V LThis includes CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively. H These are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, and at least It includes three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, having amino acid sequences that are identical by or approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100%, (ii)V LThis includes CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively. H These are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, and at least It includes three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, having amino acid sequences that are identical by or approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100%, (iii)V LThis includes CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively. H These are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, and at least It includes three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, having amino acid sequences that are identical by or approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100%, (iv)V LThis includes CDR1, CDR2, and CDR3 of three CDRs having amino acid sequences that are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. H These are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, and at least It includes three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, having amino acid sequences that are identical by or approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100%, (v)V LThese are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, and at least approximately 86% of the amino acid sequences shown in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively. It comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are identical by at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%, and V H These are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, and less than approximately 80% of the amino acid sequences shown in SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. It includes three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences that are identical by at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or approximately 100%, respectively. (vi)V LThese are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, and at least approximately 86% of the amino acid sequences shown in SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively. It comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are identical by at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%, and V H These are approximately 80% to approximately 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, at least or approximately 83%, at least or approximately 84%, at least or approximately 85%, at least or approximately 86%, and less than It includes three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences that are identical by at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or approximately 100%, respectively. (vii)V LThese are approximately 80% to 100%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, and at least 86% of the amino acid sequences shown in SEQ ID NO: 112, 113, and 114, respectively. It comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are identical by at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%, and V H These are approximately 80% to approximately 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, at least or approximately 83%, at least or approximately 84%, at least or approximately 85%, at least or approximately 86%, and less than It includes three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences that are identical by at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or approximately 100%, respectively. (viii)V LThese are approximately 80% to approximately 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, at least or approximately 83%, at least or approximately 84%, at least or approximately 85%, and at least or approximately 86% of the amino acid sequences shown in SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively. It comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are identical by at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%, and V H These are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, and at least It includes CDR1, CDR2, and CDR3, three complementarity-determining regions (CDRs) having amino acid sequences that are identical by or approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100%, or (ix)V LThese are the amino acid sequences shown in SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126, respectively, and are approximately 80% to 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, at least or approximately 83%, at least or approximately 84%, at least or approximately 85%, and at least or approximately 86%. It comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are identical by at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%, and V H These are approximately 80% to approximately 100%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 99%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, and less than approximately 80% of the amino acid sequences shown in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively. It includes three complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, which have amino acid sequences that are identical by at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or approximately 100%.

[0133] In certain embodiments, CDR, V H , and / or V L These have sequence mutations. For example, CDRs and Vs have one, two, three, four, five, six, seven, or eight of the total residues substituted or deleted, or less than 20%, less than 30%, or less than 40% of the total residues. H , or V L This can be present in antibodies (or their antigen-binding moieties) that bind to BCMA, or in CARs.

[0134] Furthermore, antibodies or their antigen-binding moieties, or CARs, in which specific amino acids are substituted, deleted, or added are also within the scope of this disclosure. These changes do not substantially affect the biological properties of the peptide, such as binding activity. For example, an antibody or its antigen-binding moiety, or CAR, may have amino acid substitutions in its framework region that improve binding to the antigen. In another example, a select few acceptor framework residues may be replaced with corresponding donor amino acids. The donor framework may be a mature or germline human antibody framework sequence or consensus sequence. Instructions on methods for inducing phenotypically silent amino acid substitutions can be found in: *Science*, Vol. 247, pp. 1306-1310 (1990) by Bowie et al.; *Science*, Vol. 244, pp. 1081-1085 (1989) by Cunningham et al.; *Current Protocols in Molecular Biology*, edited by Ausbel, published by John Wiley and Sons, Inc. (1994); and *Molecular Cloning: A Laboratory Manual* by T. Maniatis, E.F. Fritsch, and J. Sambrook. This manual is provided in the Cold Spring Harbor Laboratory (Cold Spring Harbor, New York) (1989), Methods in Molecular Biology, Vol. 243: pp. 307-331 (1994) by Pearson, and Science, Vol. 256: pp. 1443-1445 (1992) by Gonnet et al.

[0135] The peptide may be a functionally active variant of an antibody or its antigen-binding moiety or CAR disclosed herein, for example, in which less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of amino acid residues are substituted or deleted, but is not limited to these, while essentially maintaining the same immunological properties, including binding to BCMA.

[0136] Antibodies or their antigen-binding moieties, or CARs, may also include variants, analogs, orthologues, homologs, and derivatives of peptides exhibiting biological activity, such as BCMA binding. Peptides may include peptides comprising one or more analogs, substituted links, and other modifications known in the art of amino acids (including, for example, amino acids not found in nature, amino acids found only in unrelated biological systems, and modified amino acids of mammalian origin).

[0137] Antibodies or their antigen-binding moieties can be derivatized or linked to other functional molecules. For example, an antibody or its antigen-binding moiety can be functionally linked (by chemical coupling, genetic fusion, non-covalent interactions, etc.) to one or more other molecular entities, such as another antibody, a detectable activator, an immunosuppressant, a cytotoxic agent, a pharmaceutical product, a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or polyhistidine tag), an amino acid linker, a signal sequence, an immunogenic carrier, or a ligand useful for protein purification, such as glutathione-S-transferase, a histidine tag, and Staphylococcus protein A. Examples of cytotoxic agents include radioisotopes, chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, and fragments thereof. Such cytotoxic agents can be coupled to the antibody or its antigen-binding moiety of this disclosure using standard procedures and used, for example, to treat patients who are suitable for antibody therapy.

[0138] A single type of derivatized protein is produced by crosslinking two or more proteins (of the same or different types). Suitable crosslinking agents include heterobifunctional crosslinking agents having two distinct reactive groups isolated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimidyl) or homobifunctional crosslinking agents (e.g., disuccinimidyl suberate). Useful detectable active ingredients that can derivatize (or label) proteins include fluorescent active ingredients, various enzymes, prosthetic groups, luminescent substances, bioluminescent substances, and radioactive substances. Non-limiting exemplary fluorescent detectable active ingredients include fluorescein, fluorescein isothiocyanate, rhodamine, and phycoerythrin. Proteins or antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, and glucose oxidase. Proteins can also be derivatized with prosthetic groups (e.g., streptavidin / biotin and avidin / biotin).

[0139] Antibodies may be or are not limited to full length, but may include Fab, F(ab')2, Fab', F(ab)', Fv, single-chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragments (e.g., Nature, Vol. 341, pp. 544-546, 1989) by Ward et al., isolated CDRs, diabodies, triabodies, tetrabodies, linear antibodies, antibody fragments (or multiple fragments) having antigen-binding moieties containing single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single-chain antibodies produced by linking antibody fragments using recombinant methods or synthetic linkers are also included in this disclosure. Science, 1988, Vol. 242: pp. 423-426, by Bird et al.; Proceedings of the National Academy of Sciences, USA, 1988, Vol. 85: pp. 5879-5883, by Houston et al.

[0140] The antibody or its antigen-binding portion may include at least one constant domain, for example, (a) an IgG constant domain, (b) an IgA constant domain, etc.

[0141] Any antibody isotype, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE, is included in this disclosure. The antibody or its antigen-binding moiety may be a mammalian (e.g., mouse, human) antibody or its antigen-binding moiety. The antibody light chain may be kappa-type or lambda-type.

[0142] The antibodies or antigen-binding moieties of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific or bispecific antibodies or fragments thereof may be specific to different epitopes of one target polypeptide (e.g., BCMA) or may contain antigen-binding domains specific to two or more target polypeptides (e.g., antigen-binding domains specific to BCMA and another antigen). In one embodiment, a multispecific antibody or its antigen-binding moiety comprises at least two different variable domains, where each variable domain can specifically bind to a different antigen or a different epitope on the same antigen. See Journal of Immunology, Vol. 147, pp. 60-69, by Tutt et al. (1991) and Trends in Biotechnol., Vol. 22, pp. 238-244, by Kufer et al. (2004). This antibody can be conjugated to another functional molecule, such as another peptide or protein, or co-expressed with them. For example, an antibody or a fragment thereof can be functionally conjugated to one or more other molecular entities, such as another antibody or antibody fragment (e.g., by chemical coupling, genetic fusion, non-covalent association, or other methods) to produce a bispecific or multispecific antibody having a second binding specificity. For example, this disclosure includes a bispecific antibody in which one arm of the immunoglobulin is specific to BCMA and the other arm of the immunoglobulin is specific to a second therapeutic target or conjugated to a therapeutic portion described herein.

[0143] The antibodies or antigen-binding moieties described herein are useful as affinity purifiers. In this method, the antibody or a fragment thereof is immobilized on a solid phase such as a protein A resin using a method known in the art.

[0144] This anti-BCMA antibody or its antigen-binding moiety is also useful in diagnostic assays that detect and / or quantify BCMA proteins, for example, in detecting BCMA expression in specific cells, tissues, or serum.

[0145] The antibodies or antigen-binding moieties described herein may be used in any known assay method, including competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, for example, Monoclonal Antibodies: A Manual of Techniques by Zola, pp. 147-158 (CRC Publishing, 1987).

[0146] antibody The terms “antibody” or “immunoglobulin” can refer to a heterotetrameric glycoprotein of approximately 150,000 daltons with identical structural features, containing two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by disulfide covalent bonds, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes differs. Each heavy and light chain also has intrachain disulfide bonds arranged at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the constant region of the light chain opposite the first constant region of the heavy chain, and the variable region of the light chain opposite the variable region of the heavy chain. Certain amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0147] The term "variable" can mean that the sequence of a particular portion of the variable region of an antibody varies, thereby shaping the binding and specificity of different specific antibodies to its specific antigen. Nevertheless, variability is not uniformly distributed throughout the variable region of the entire antibody, but is concentrated in three fragments of the complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portion of the variable region is called the framework region (FR). The natural heavy and light chain variable regions each contain four FRs, which are generally in a β-folded structure and linked by three CDRs, forming an adapter ring. In some cases, a partial β-folded structure may also be formed. The CDRs in each chain are closely linked to each other by the FRs and, together with the CDRs of another chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). While the constant regions are not directly involved in antibody binding to antigens, they exhibit various effector functions, such as being involved in antibody-dependent cytotoxicity.

[0148] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct categories (called κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be classified into different types according to the amino acid sequence of their heavy chain constant region. There are mainly five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of different types of immunoglobulins are well known to those skilled in the art.

[0149] Generally, the antigen-binding properties of an antibody can be explained by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs), which divide the fragment into four spaced framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a cyclic structure, and their spatial structures are close together by the β-folds formed by the FRs between the CDRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of antibodies of the same type, the amino acids that make up the FRs or CDRs can be determined.

[0150] This disclosure includes not only complete antibodies, but also immunologically active antibody fragments or fusion proteins formed by antibodies and other sequences. Therefore, this disclosure also includes antibody fragments, derivatives, and analogs.

[0151] In this disclosure, antibodies include mouse antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric monoclonal antibodies and humanized monoclonal antibodies, contain human and non-human portions, can be obtained by standard DNA recombination techniques, and are all useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as chimeric antibodies having a variable region of a mouse-derived monoclonal antibody and a constant region of a human-derived immunoglobulin (see, for example, U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety). Humanized antibodies may refer to antibody molecules derived from non-human species that include one or more complementarity-determining regions (CDRs) derived from non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety). These chimeric monoclonal antibodies and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0152] In this disclosure, the antibody may be monospecific, bispecific, triplicate, or multispecific.

[0153] In this disclosure, antibodies also include their conserved variants, meaning that up to 10, 8, 5, or 3 amino acids, compared to the amino acid sequence of the antibody provided herein, are substituted with amino acids having similar properties to form a polypeptide. For example, conserved variant polypeptides can be generated by amino acid substitutions as shown in Table A.

[0154] [Table 6]

[0155] Anti-BCMA antibody This disclosure provides an antibody against BCMA having high specificity and high affinity, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence in the VH region and the light chain comprises an amino acid sequence in the VL region.

[0156] In certain embodiments, the antibody is encoded by the nucleotide sequence shown below. H Region and V L Includes area: [Table 7]

[0157] In certain embodiments, the antibody is a mouse antibody.

[0158] In certain embodiments, the antibody comprises a heavy chain and a light chain having the following amino acid sequences: [Table 8]

[0159] In certain embodiments, the antibody is a chimeric antibody.

[0160] In certain embodiments, the antibody has the following amino acid sequence V H Region and V L Includes area: [Table 9]

[0161] The CDR sequences of antibodies CP01 to CP09 are shown below.

[0162] [Table 10] TIFF0007883955000011.tif252166TIFF0007883955000012.tif128163

[0163] In certain embodiments, the antibody is a single-chain antibody.

[0164] In certain embodiments, any of the above-described amino acid sequences may optionally include derivative sequences in which at least one amino acid is added, deleted, modified, and / or substituted, thereby maintaining the binding affinity of BCMA.

[0165] In certain embodiments, a sequence formed by the addition, deletion, modification, and / or substitution of at least one amino acid sequence may have at least 80%, at least 85%, at least 90%, or at least 95% homology to the amino acid sequence. The number of added, deleted, modified, and / or substituted amino acid residues may be 1 to 7, 1 to 5, 1 to 3, or 1 to 2.

[0166] The antibodies provided by this disclosure may be double-chain or single-chain antibodies and may be selected from animal-derived antibodies, chimeric antibodies and humanized antibodies, human-animal chimeric antibodies, or fully humanized antibodies.

[0167] The antibody derivatives provided by this disclosure may be single-chain antibodies and / or antibody fragments, e.g., Fab, Fab', (Fab')2, or other antibody derivatives known in the art, and one or more antibodies of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes.

[0168] In particular, the animal could be a mammal such as a mouse.

[0169] The antibodies provided in this disclosure may be chimeric antibodies, humanized antibodies, or CDR-grafted and / or CDR-modified antibodies targeting human BCMA.

[0170] Specifically, in the embodiments of this application, four hybridoma cell fusions were performed. A total of 14 hybridoma cells (6G10-1D7, 99B3G3, 102A12H6, 107A11F1, 107A9A4, 107B11E1, 100H2D12C6, 105C10F1, 113B3F12, 109C5F3C1, 97B8G8D12, 143D6F4, 151A9A4, and 152D8E8) were selected for antibody production in all 13 of the 14 clones (except 97B8G8D12). Except for 152D8E8 (CP09), all of the remaining 12 hybridoma antibodies were able to bind to the soluble extracellular domain of the human BCMA molecule. With the exception of 105C10F1(CP03), 152D8E8(CP09), and 113B3F12, all of the remaining 10 hybridoma antibodies were able to cross-recognize the soluble extracellular domain of the cynomolgus monkey BCMA molecule. With the exception of 102A12H6 and 152D8E8(CP09), all of the remaining 11 hybridoma antibodies were able to recognize the extracellular domain of the human BCMA molecule on the cell surface.

[0171] The antibody variable regions of a total of 17 hybridoma cell lines were sequenced. Twelve hybridoma cell lines were monoclonal according to sequencing: 6G10-1D7, 99B3G3, 102A12H6, 100H2D12C6, 105C10F1, 113B3F12, 109C5F3C1, 97B8G8D12, 143D6F4, 151A9A4, 152D8E8, and 107B11E1D7. Of these, 99B3G3, 100H2D12C6, and 97B8G8D12 had the same sequence in their antibody variable regions. Five hybridoma cell lines were found to be polyclonal upon sequencing: 07A11F1, 107A9A4, 107B11E1, 107A9A4D2, and 107A11F1B7. Of these, 107A11F1, 107A9A4, and 107B11E1 each had four heavy chains and one light chain, while 107A9A4D2 and 107A11F1B7 each had one heavy chain and two light chains and possessed the same sequence. Five monoclonal hybridoma cell lines (6G10-1D7, 99B3G3, 105C10F1, 113B3F12, and 107B11E1D7) and one polyclonal hybridoma cell line (107A9A4D2) were selected to express chimeric antibodies. Of these, 107A9A4D2VH was expressed paired with 107A9A4D2VL-1 and 107A9A4D2VL-2, respectively. ELISA and flow cytometry detection revealed that all chimeric antibodies against hybridomas 6G10-1D7, 99B3G3, 105C10F1, and 107B11E1D7 bound to the soluble extracellular domain of human BCMA molecules on the cell surface. Chimeric antibodies formed by pairing the heavy chain 107A9A4D2VH and light chain 107A9A4D2VL-2 of hybridoma 107A9A4D2 all bound to the soluble extracellular domain of human BCMA molecules on the cell surface. Chimeric antibodies formed by pairing 107A9A4D2VH and 107A9A4D2VL-1 did not bind to the soluble extracellular domain of human BCMA molecules on the cell surface.

[0172] Antibody preparation The DNA molecule sequences of the antibodies or fragments provided by this disclosure can be obtained by conventional techniques such as PCR amplification or genome library screening. Furthermore, single-chain antibodies can be formed by fusing the coding sequences of the light and heavy chains together.

[0173] Once the relevant sequence is obtained, it can be obtained in large batches by recombination. Generally, the relevant sequence is obtained by cloning the relevant sequence into a vector, then introducing the relevant sequence into cells, and then isolating the relevant sequence from the grown host cells using conventional methods.

[0174] Furthermore, especially when the fragment length is short, related sequences can be synthesized using artificial synthesis methods. Generally, fragments with long sequences can be obtained by first synthesizing multiple small fragments and then concatenating those small fragments together.

[0175] Currently, the DNA sequences encoding the antibodies (or their fragments or derivatives) provided by this disclosure can be obtained entirely by chemical synthesis. These DNA sequences can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of this disclosure by chemical synthesis.

[0176] This disclosure also relates to vectors comprising the aforementioned appropriate DNA sequences and appropriate promoters or regulatory sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0177] The host cell may be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Examples of animal cells include (but are not limited to) CHO-S cells and HEK-293 cells.

[0178] Typically, the obtained host cells are cultured and transformed under conditions suitable for the expression of the antibodies provided by the present disclosure. Next, conventional immunoglobulin purification steps such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ionization exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, as well as other conventional isolation methods and purification methods well-known to those skilled in the art, are used for purification to obtain the antibodies provided by the present disclosure.

[0179] The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of a monoclonal antibody can be determined by the Scatchard analysis of Munson et al., Analytical Biochemistry, Vol. 107: p. 220 (1980).

[0180] The antibodies provided by the present disclosure can be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, by using the physical, chemical, and other properties of the recombinant protein, the recombinant protein can be isolated and purified by various isolation methods. These methods are well-known to those skilled in the art. Examples of these methods include, but are not limited to, conventional refolding treatments, treatment with protein precipitants (salting-out method), centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0181] Antibody-drug conjugate (ADC) The present disclosure further provides an antibody-drug conjugate (ADC) based on the antibodies provided by the present disclosure.

[0182] Typically, an ADC comprises an antibody and an effector molecule, and the antibody is conjugated to the effector molecule, for example, chemically conjugated. The effector molecule can be a drug having therapeutic activity. Further, the effector molecule can be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0183] The antibodies and effector molecules provided by the present disclosure can be conjugated by a coupling agent. Examples of coupling agents include one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. A non-selective coupling agent refers to a compound that forms a covalent bond with an effector molecule and an antibody, for example, glutaraldehyde. The coupling agent using a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (for example, cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0184] Some residues on the antibody (for example, Cys or Lys) can be used to link a variety of functional groups including imaging reagents (for example, chromogenic groups and fluorescent groups), diagnostic reagents (for example, MRI contrast agents and radioisotopes), stabilizers (for example, glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. A functional agent (for example, a drug, a detection reagent, and a stabilizer) can be conjugated (linked by a covalent bond) to the antibody. The functional agent can be linked to the antibody directly or indirectly by a linker.

[0185] Antibodies can be conjugated with drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in the intracellular environment, for example, by degradation at a target site, thus releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, such as peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucosidases. Examples of peptidyl linkers include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., hydrazone linkers, which are hydrolyzed when the pH is below 5.5) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by a protease.

[0186] Before linking the antibody, the linker may have an active reactive group that can react with several amino acid residues, where linking is achieved via the active reactive group. Examples of sulfhydryl-specific active reactive groups include maleimide compounds, halogenated amides (e.g., iodized, brominated, or chlorinated), halogenated esters (e.g., iodized, brominated, or chlorinated), halogenated methyl ketones (e.g., iodized, brominated, or chlorinated), benzyl halides (e.g., iodized, brominated, or chlorinated), vinyl sulfones, and pyridyl disulfides, mercury derivatives such as 3,6-di-(mercury-methyl)dioxane whose counterion is an acetate ion, chloride ion, or nitrate ion, and poly(methylenedimethyl sulfide ether) thiosulfonates. An example of a linker is maleimide linked to an antibody by thiosuccinimide.

[0187] The drug can be any drug that is cytotoxic, inhibits cell growth, or is immunosuppressive. In one implementation, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino group, carboxyl group, sulfhydryl group, hydroxyl group, or keto group that can form a bond with the linker. When the drug is directly linked to the linker, the drug has a reactive active group before being linked to the antibody.

[0188] Useful drug categories include, for example, antitubulin drugs, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibitors, and vinca alkaloids. Particularly useful examples of cytotoxic drugs include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatin, camptothecin, duocalmycin, etoposide, meitansin, and meitansinoid compounds (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolidine[1,4]benzodiazepines (PBD), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0189] ADCs can be formed in a simple process using a drug-linker. In other implementations, ADCs can be formed in a two-step or multi-step process using a bifunctional linker compound. For example, in the first step, a cysteine ​​residue reacts with the reactive portion of the linker, and in the next step, the functional group on the linker reacts with the drug, thereby forming the ADC.

[0190] Generally, functional groups on the linker are selected to facilitate specific reactions with appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently bonded to the linker via 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides), isocyanates and isothiocyanates (suitable for reactions with amines and alcohols), and activated esters such as N-hydroxysuccinimide esters (suitable for reactions with amines and alcohols). These linking strategies, and others described in "Bioconjugate Techniques," 2nd edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that, in the case of a selective reaction between a drug portion and a linker, each element of a complementary pair can be used in both the linker and the drug, provided that the reactive functional groups in the complementary pair are selected.

[0191] This disclosure further provides a method for preparing an antibody-drug conjugate (ADC), which may further comprise conjugating an antibody to a drug-linker compound under conditions sufficient to form an ADC.

[0192] In some forms of implementation, the methods provided by this disclosure include conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these forms of implementation, the methods provided by this disclosure further include conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody by the linker.

[0193] In some implementation forms, antibody-drug conjugates (ADCs) have the following molecular formula: [ka] As shown in the formula (wherein Ab is the antibody, LU is the linker, D is the drug, and the subscript p is a value selected from 1 to 8).

[0194] Chimeric antigen receptor (CAR) The chimeric antigen receptors (CARs) provided herein may comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain may comprise a target-specific binding element (also known as an antigen-binding domain). The intracellular domain may comprise a costimulatory signaling region and a ζ chain moiety. The costimulatory signaling region may refer to a portion of the intracellular domain containing a costimulatory molecule. The costimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than the antigen receptor or its ligand.

[0195] Linkers can be incorporated between the extracellular and transmembrane domains of a CAR, or between the cytoplasmic and transmembrane domains of a CAR. The term "linker" can refer to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain. Linkers may contain 0 to 300 amino acids, 2 to 100 amino acids, or 3 to 50 amino acids.

[0196] In certain embodiments, the extracellular domain of the CAR includes an antigen-binding domain that targets BCMA. When the CAR is expressed in T cells, antigen recognition can occur based on its antigen-binding specificity. When T cells bind to the relevant antigen, the T cells can influence the tumor cells to prevent growth or induce tumor cell death, thereby potentially reducing or eliminating the tumor burden in the patient.

[0197] The antigen-binding domain can be fused with one or more intracellular domains from the co-stimulatory molecule and the ζ chain. In one embodiment, the antigen-binding domain is fused with an intracellular domain formed by a combination of the 4-1BB signaling domain and the CD3ζ signaling domain.

[0198] The terms “antigen-binding domain” and “single-chain antibody fragment” may refer to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment that possesses antigen-binding activity. An Fv antibody contains the VH and VL regions of the antibody, but does not contain a constant region, and has the smallest antibody fragment of all antigen-binding sites. Generally, an Fv antibody may further contain a polypeptide linker between the VH and VL domains to form the structure required for antigen binding. The antigen-binding domain is generally an scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 that of a complete antibody. A single-chain antibody may be an amino acid chain sequence encoded by a nucleotide chain. In certain embodiments, the scFv specifically recognizes BCMA.

[0199] For hinge regions and transmembrane regions (transmembrane domains), CARs may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain that naturally associates with one of the domains within the CAR is used. In some examples, modifications may be made by selecting or substituting amino acids in the transmembrane domain to prevent such a domain from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interaction with other elements of the receptor complex.

[0200] Examples of intracellular domains of CAR include the 4-1BB signaling domain and the CD3ζ signaling domain.

[0201] Based on the light and heavy chains of 9 antibodies obtained through screening in the present disclosure, 18 chimeric antigen receptors (CARs) were constructed. These CARs have various reactivities against BCMA-positive target cells and have desirable specific killing ability against target cells. Specifically, the above-mentioned 9 antibodies have a V H region and a V L region, including:

Table 11

[0202] In certain embodiments, the gene structure of the CAR may include a leader sequence (signal peptide), an antigen recognition sequence, a linker region, a transmembrane region, a co-stimulatory factor signal region, and a CD3ζ signal transduction region, and the linking sequences are as follows: [CD8 LS]-[VL-linker-VH]-[hinge-CD8TM]-[4-1BB]-[CD3ζ], or [CD8 LS]-[VH-linker-VL]-[hinge-CD8TM]-[4-1BB]-[CD3ζ]

[0203] The present disclosure identified the correlation between the expression time and expression intensity of different CAR structures on the cell membrane surface after virus infection, and identified the difference in the ease of expression of different CAR structure proteins. This finding points out the difference between the expression level of CAR protein on the membrane surface and the persistence of the in vivo activity of CART under the same infection conditions of different CAR structures.

[0204] Vector The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as by screening a library from cells expressing the gene, obtaining the gene from a vector known to contain the gene, or using standard techniques for directly isolating the gene from cells and tissues containing the gene. Optionally, the target gene can be synthesized and generated.

[0205] This disclosure also provides vectors into which the expression cassettes provided herein are inserted. Retroviral vectors, such as lentiviruses, are suitable carriers for achieving long-term gene transfer because they enable long-term and stable integration of the transgene and transfer of the transgene in daughter cells. Lentiviral vectors are advantageous over vectors derived from oncogenic retroviruses, such as mouse leukemia virus, because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

[0206] The expression cassettes or nucleic acid sequences in this disclosure can be operably ligated to a promoter and generally incorporated into expression vectors. These vectors are suitable for eukaryotic cell replication and incorporation. Typical cloning vectors include a promoter that can be used to regulate the expression of transcription and translation termination factors, initial sequences, and the expected nucleic acid sequence.

[0207] The expression construct may also utilize standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art; see, for example, U.S. Patent No. 5,399,346, U.S. Patent No. 5,580,859, and U.S. Patent No. 5,589,466. In certain embodiments, this disclosure provides gene therapy vectors.

[0208] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors targeted include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0209] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al.'s *Molecular Cloning: A Laboratory Manual* (2001, Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, a suitable restriction enzyme site, and one or more selectable markers (e.g., International Publication No. 01 / 96584, International Publication No. 01 / 29058, and U.S. Patent No. 6,326,193).

[0210] Many virus-based systems have been developed to introduce genes into mammalian cells. For example, retroviruses provide a suitable platform for gene delivery systems. Using techniques known in the art, selected genes can be inserted into vectors and packaged into retroviral particles. Recombinant viruses can then be isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art. Adenovirus vectors are used in some implementations. Many adenovirus vectors are known in the art. Retroviral vectors are used in one implementation.

[0211] Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Generally, these are located 30-110 bp upstream of the initiation site, although many promoters have recently been shown to also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible to maintain promoter function even if these elements invert or move relative to one another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased by as much as 50 bp before activity begins to decrease. Depending on the promoter, individual elements appear to be able to initiate transcription either cooperatively or independently.

[0212] One example of a suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably ligated to the promoter sequence. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). Nevertheless, other constitutive promoter sequences may also be used, but are not limited to the Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Rous sarcoma virus promoter, and, but are not limited to, human gene promoters such as actin promoters, myosin promoters, heme promoters, and creatine kinase promoters. Inducible promoters are also considered part of this disclosure. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably ligated to the inducible promoter when such expression is expected, or turn off such expression when such expression is not expected. Examples of inductive promoters, though not limited to them, include the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0213] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may contain either or both a selectable marker gene or a reporter gene to identify and select expressing cells from a population of cells being investigated that have been transfected or infected with a viral vector. In other embodiments, the selectable marker may be supported on a single fragment of DNA and used in a co-transfection procedure. Both sides of the selectable marker and reporter gene may have appropriate regulatory sequences to ensure expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0214] Reporter genes can be used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are those that are not present in the recipient organism or tissue, or are expressed by the recipient organism or tissue, and whose expression is clearly indicated by several easily detectable characteristics, such as enzymatic activity. Reporter gene expression is determined at an appropriate time after the DNA has been introduced into recipient cells. Suitable reporter genes include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al. (2000), FEBS Letters, Vol. 479: pp. 79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with at least five adjacent regions exhibiting the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be ligated to a reporter gene and used to evaluate the reagent's ability to regulate promoter-driven transcription.

[0215] Methods for introducing genes into cells and expressing those genes in cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0216] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Calcium phosphate transfection is a method for introducing polynucleotides into host cells.

[0217] Biological methods for introducing target polynucleotides into host cells include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent No. 5,350,674 and U.S. Patent No. 5,585,362.

[0218] Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery carrier in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0219] When using nonviral delivery systems, an exemplary delivery tool is liposomes. Lipid preparations can be used to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can be associated with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both liposomes and oligonucleotides, captured within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, housed in micelles, or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vectors associated with a composition are not limited to any specific structure in solution. For example, they may exist as micelles in a bilayer structure or have a "broken-down" structure. They may also simply be dispersed in solution and may form aggregates of heterogeneous size or shape. Lipids are fatty substances that occur naturally or can be synthesized. Examples of lipids include naturally occurring lipid droplets in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, as well as their derivatives.

[0220] In certain embodiments, the vector is a lentiviral vector.

[0221] Therapeutic use of CAR T This disclosure includes therapeutic uses of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of this disclosure. Transduced T cells can target BCMA, a tumor cell marker, synergistically activate T cells, trigger a T cell immune response, and thereby significantly improve the efficiency of T cell killing against tumor cells.

[0222] Accordingly, the Disclosure further provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, comprising the step of administering the CAR-T cells of the Disclosure to a mammal.

[0223] In one implementation form, this disclosure includes a cell therapy in which the patient's own T cells (or xenodont cells) are isolated, activated and genetically modified to generate CAR-T cells, and then injected into the same patient. Thus, the likelihood of developing graft-versus-host disease is extremely low, and the antigen is recognized by the T cells in a non-MHC-restricted manner. Furthermore, a single CAR-T cell can treat any cancer that expresses that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, resulting in long-lasting effects that can lead to sustained tumor control.

[0224] In one implementation, the CAR-T cells provided by this disclosure can undergo stable T cell proliferation in vivo over a continuously extendable period. Furthermore, the CAR-mediated immune response may be part of an adoptive immunotherapy process in which CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, CAR-T cells against BCMA will elicit a specific immune response against cells expressing BCMA.

[0225] The data disclosed herein specifically disclose a lentiviral vector comprising an anti-BCMA scFv, a hinge, a transmembrane region, and 4-1BB and CD3ζ signaling domains, but this disclosure should be interpreted as including any number of variations in each of the components of the construct.

[0226] Cancers that can be treated include non-angiogenic or substantially non-angiogenic tumors, as well as angiogenic tumors. Cancers may include non-solid tumors (such as hematological malignancies like leukemia and lymphoma) or solid tumors. The types of cancers that can be treated with the CARs, antibodies, or their antigen-binding moieties provided herein include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as benign and malignant tumors such as some leukemia or lymphoid malignancies, sarcomas, carcinomas, and melanomas, and also include adult tumors / cancers and pediatric tumors / cancers.

[0227] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include acute leukemia (acute lymphoblastic leukemia, acute myeloblastic leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic erythrocytic leukemia, etc.), leukemia including chronic leukemia (chronic myeloblastic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (painless and high-grade types), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0228] The term "solid tumor" generally refers to an abnormal mass of tissue that does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (sarcoma, carcinoma, lymphoma, etc.). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0229] CAR-modified T cells can also be used as a type of vaccine for ex-vivo immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0230] In ex vivo immunization, at least one of the following steps is performed in vitro: i) cell amplification, ii) introduction of CAR-encoding nucleic acids into cells, and / or iii) cell cryopreservation, after which the cells are administered to mammals.

[0231] Ex vivo procedures are well known in the art and will be discussed in more detail below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transfected or transfected in vitro) with a vector expressing the CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to obtain a therapeutic effect. The mammalian recipient may be human, and the CAR-modified cells may be autologous to the recipient. Optionally, the cells may be allogeneic, syngeneic, or heterologous to the recipient.

[0232] In addition to the use of cell-based vaccines for ex vivo immunization, this disclosure also provides compositions and methods for in vivo immunization that induce an immune response to an antigen in a patient.

[0233] This disclosure provides a method for treating a tumor, comprising administering a therapeutically effective dose of CAR-modified T cells provided herein to a subject in need thereof.

[0234] The CAR-modified T cells provided by the present disclosure can be administered alone or in combination with diluents and / or other components or cell populations such as IL-2, IL-17 or other cytokines as a pharmaceutical composition. Briefly, the pharmaceutical composition provided by the present disclosure can include the target cell population described herein in combination with one or more pharmaceutically or physiologically acceptable vectors, diluents, or excipients. Such compositions can include buffer solutions such as neutral buffered saline and sulfate buffer solutions, carbohydrates such as glucose, mannose, sucrose, dextran, and mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The composition can be suitable for intravenous administration.

[0235] The pharmaceutical composition provided by the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administrations will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but appropriate dosages can be determined by clinical trials.

[0236] When an "immunologically effective dose", "anti-tumor effective dose", "tumor-suppressive effective dose", or "therapeutic dose" is indicated, the exact dosage of the composition to be administered can be determined by a physician considering age, body weight, tumor size, degree of infection or metastasis, and individual differences in the disease in the patient (subject). The pharmaceutical composition containing the T cells described herein can be administered at a dosage of 10 4 cells to 10 9 cells per kg of body weight, or at a dosage of 10 5 cells to 10 6It can generally be shown that T cells can be administered in doses of a certain number of cells (including all integers within these ranges). T cell compositions can also be administered multiple times in these doses. Cells can be administered using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New England Journal of Medicine, Vol. 319: p. 1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the art in the medical field by monitoring the patient's signs of the disease and thus adjusting the treatment.

[0237] The administration of the composition to the subject may be carried out in any suitable manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intralymphatically, intraspinally, or intramuscularly, or by intravenous injection (iv), or intraperitoneally. In one implementation, the T cell composition provided herein is administered to a patient by intradermal or subcutaneous injection. In certain embodiments, the T cell composition is administered by intravenous injection (iv). The T cell composition may be injected directly into a tumor, lymph node, or site of infection.

[0238] In certain embodiments, cells activated and proliferated using the methods described herein or other methods known in the art to proliferate T cells to therapeutic levels are administered to a patient in combination with any number of relevant therapeutic forms (e.g., before, simultaneously, or after). Therapeutic forms include, but are not limited to, treatment with reagents, such as antiviral agents, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab for MS patients, or elphazumab monoclonal therapy for psoriasis patients, or other therapeutics for PML patients. In a further implementation, the T cells provided by this disclosure may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate esters, and FK506, antibodies, or other immunotherapeutic agents. In certain embodiments, the cell compositions provided by this disclosure are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), and cyclophosphamide. For example, a subject may receive standard treatment of high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an injection of immune cells proliferated according to this disclosure. In certain embodiments, the proliferated cells are administered before or after surgery.

[0239] The therapeutic doses administered to patients will vary widely depending on the exact nature of the condition being treated and the individual receiving treatment. The dose ratios for human administration can be implemented according to accepted practices in the relevant field. Generally, 1 × 10⁻⁶ 6 pieces~1×10 10 Individual modified T cells (e.g., CAR-T20 cells) can be administered to the patient by intravenous reinfusion at each treatment or treatment stage.

[0240] CART Pharmacy The present disclosure provides CAR-T cells, and a pharmaceutically acceptable carrier, diluent, or excipient. In one mode of implementation, the preparation is a liquid preparation. In one embodiment, the preparation is an injection. In one embodiment, the concentration of CAR-T cells in the preparation is 1×10 3 cells to 1×10 8 cells / ml, or 1×10 4 cells to 1×10 7 cells / ml.

[0241] In certain embodiments, the preparation may include a buffer solution such as neutral buffered saline and sulfate buffered saline, carbohydrates such as glucose, mannose, sucrose, dextran, and mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (such as aluminum hydroxide), and preservatives. The preparation may be suitable for intravenous administration.

[0242] Pharmaceutical composition Furthermore, the present disclosure provides a composition. In certain embodiments, the composition is a pharmaceutical composition comprising the above-described antibody or its active fragment, its fusion protein, its ADC, or the corresponding CAR-T cells, and a pharmaceutically acceptable carrier. Generally, these substances can be prepared in a non-toxic and inert pharmaceutically acceptable aqueous carrier medium having a pH of about 5-8, or about 6-8, although the pH value can vary widely depending on the nature of the substance being prepared and the disease being treated. The prepared pharmaceutical composition can be administered by conventional routes including (but not limited to) intratumoral administration, intraperitoneal administration, intravenous administration, or topical administration.

[0243] The antibodies described in the present disclosure can also be used in cell therapy for intracellular expression of nucleotide sequences. For example, the antibodies are used in chimeric antigen receptor T cell immunotherapy (CAR-T).

[0244] The pharmaceutical compositions provided by this disclosure can be used directly for binding to TF protein molecules and can therefore be used for the prevention and treatment of tumors and other diseases. Furthermore, other therapeutic agents can be used simultaneously.

[0245] The pharmaceutical compositions provided by this disclosure comprise a safe and effective dose (e.g., 0.001% to 99% by weight, 0.01% to 90% by weight, or 0.1% to 80% by weight) of the monoclonal antibody (or its conjugate) provided by this disclosure, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to) saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Pharmaceutical preparations should be adapted to the administration method. The pharmaceutical compositions provided by this disclosure may be prepared, for example, in the form of injections from saline or aqueous solutions containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions, such as injections or solutions, should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective dose, e.g., approximately 1 mg to approximately 5 mg per kg of body weight per day. Furthermore, the polypeptides provided by this disclosure may also be used in combination with other therapeutic agents.

[0246] The use of a pharmaceutical composition involves administering a safe and effective dose of an immunoconjugate to a mammal, where a safe and effective dose is generally at least about 10 mg per kg of body weight and, in most cases, not exceeding about 50 mg per kg of body weight. For example, the dose ranges from about 10 mg per kg of body weight to about 20 mg per kg of body weight. Naturally, the specific dose should also take into account factors such as the route of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0247] Detection purpose and kit The antibodies or conjugates provided by this disclosure may be used for detection purposes, for example, to detect a sample and thereby provide diagnostic information.

[0248] Specimens (samples) include cell samples, tissue samples, and biopsy samples. The term "biopsy" can include all types of biopsies known to those skilled in the art. Therefore, biopsies can include, for example, tumor resection samples and tissue samples prepared by endoscopic methods or by fine-needle or needle biopsy of organs.

[0249] Examples of samples include fixed or preserved cell or tissue samples.

[0250] Furthermore, the Disclosure provides a kit comprising the antibody (or fragment thereof) or CAR of the Disclosure. In one embodiment, the kit further includes a container, an operating manual, a buffer, and the like. In one embodiment, the antibody can be mounted on a test plate.

[0251] Furthermore, this disclosure provides the use of antibodies. For example, antibodies are used to prepare diagnostic preparations or drugs for the prevention and / or treatment of BCMA-positive cancers / tumors.

[0252] The antibodies provided in this disclosure can recognize the soluble extracellular domain of human BCMA molecules on the cell membrane surface. Some antibodies can also cross-recognize the soluble extracellular domain of cynomolgus monkey BCMA molecules.

[0253] The CAR-T cells provided in this disclosure do not recognize tumor necrosis factor receptor superfamily member 13B.

[0254] The CAR-T cells provided in this disclosure possess desirable specific cytotoxicity against BCMA-positive target cells.

[0255] The following examples of specific ways in which this disclosure is implemented are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way.

[0256] General materials and methods The materials and methods used in the examples are as follows:

[0257] 1. Sources of cells and mice K562-BCMA + :K562-BCMA-B22D8 (Supplied by Cellular Biomedicine Group) K562 (supplied by ATCC, Cellular Biomedicine Group) L-BCMA + :L-BCMA-1E6-A4 (Supplied by Cellular Biomedicine Group) L cells (mouse fibroblasts, ATCC: catalog number CRL-2648 (trademark), lot 63903687, supplied by Cellular Biomedicine Group, Inc.) Sp2 / 0-Ag14 cells (supplied by ATCC, ChemPartner) Balb / c mouse (Shanghai Slac) SJL Mouse (Shanghai Slack Company)

[0258] 2. Main Reagents [Table 12]

[0259] 3. Main experimental equipment [Table 13]

[0260] 4. Experimental Design 4.1 Immunization of mice and detection of serum titers Five Balb / c mice and five SJL mice were divided into two groups and immunized with hBCMA-ECD-Fc according to Table 3. As shown in Table 4, at least two booster immunizations should be performed after the first immunization (for the third booster immunization, use K562-BCMA).+ Cells were used. After immunization, mouse serum titers were detected using ELISA and FACS. Hybridoma cell fusion screening was performed after serum titers met the requirements (ELISA reached a dilution of 1:10000, or FACS reached a dilution of 1:1000).

[0261] [Table 14]

[0262] [Table 15]

[0263] 4.2 Screening of Hybridoma Cell Lines 4.2.1 Fusion of Hybridoma Cells FACS detection was used to identify mice with the highest serum titer (one each from Balb / c and SJL), and hybridoma cells were obtained by fusing lymphocytes and Sp2 / O-Ag14 cells from the spleen and lymph nodes using electrofusion.

[0264] 4.2.2 Monoclonal screening of hybridoma cells After erythrocyte lysis, the fused cells from each mouse were placed on a 96-well plate at a rate of 2.5 × 10⁶ per plate. 6 Individual cells were smeared onto a total of 20 plates. After 10 days, a primary screening was performed, and the binding ability of the culture medium supernatant to the hBCMA-ECD-Fc protein was detected by ELISA. Clones with positive test results were transferred to 24-well plates and cultured for growth.

[0265] For cells after proliferation culture, ELISA was used to detect the binding ability of the culture supernatant to hBCMA-ECD-Fc and cynoBCMA-ECD-Fc, and FACS was used to detect the L-BCMA binding ability of the culture supernatant. + Cellular binding ability was detected (secondary screening). Ultimately, L-BCMA +We selected clones that had strong cell binding and performed subclonal screening.

[0266] 4.2.3 Subclonal screening of hybridoma cells The monoclonal cells obtained in 4.2.2 were smeared onto two 96-well plates, and the ELISA and FACS screening processes described in 4.2.2 were repeated to finally obtain subclonal cell lines. Target clones were transferred to culture bottles for proliferation, a seed bank was established, and 4 to 6 units of cells from each line were cryopreserved (0.5 to 1 × 10⁶). 7 (Individual cell / unit).

[0267] 4.3 Production and Purification of Hybridoma Antibodies Monoclonal cells that bind to both human BCMA and cynomolgus monkey BCMA were selected, and small batches were produced for trial production. The cells were cultured in 250 ml to 500 ml cell culture shaking flasks, and the produced antibodies were purified using a protein A affinity column. These were then sequentially processed to remove endotoxins, yielding 3 mg to 5 mg of antibody, which was then characterized.

[0268] 4.4 Identification of purified hybridoma antibodies Identifying antibodies involves the following three points: a) Analysis of binding specificity to hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and hTACI-ECD-Fc (ELISA) b) L cells and L-BCMA + Analysis of cell binding ability (FACS) c) Analysis of competitive binding to soluble BCMA (FACS).

[0269] 4.5 Sequencing of antibody variable regions RNA is extracted from target clone hybridoma cells, the RNA is reverse transcribed into cDNA, the antibody VH / VL gene fragment is amplified by PCR, and the gene sequence of the antibody variable region of the target clone is obtained by sequencing.

[0270] 4.6 Identification of Chimeric Antibody Expression The VH region of the antibody obtained from sequencing is cloned into an IgG1,κ recombinant antibody heavy chain expression vector, and the VL region is cloned into an IgG1,κ recombinant antibody light chain expression vector to construct an expression plasmid for a chimeric antibody. HEK293T cells are transfected using these two plasmids. After culturing for 3 days, the culture supernatant is collected. hBCMA-ECD-Fc (ELISA) and L-BCMA + To determine the binding ability of the supernatant to cells (FACS).

[0271] Example 1: Immunization of mice and detection of serum titer Mice were immunized. Three booster immunizations and four fusions were performed. As shown in Table 5, the first two booster immunizations used hBCMA-ECD-Fc to immunize the mice, followed by fusions with F0109 and F0227. The third booster immunization used K562-BCMA to immunize the mice. + We used the cells and then fused F0508 and F0614.

[0272] [Table 16]

[0273] Figures 1A-1B show the results of ELISA analysis of mouse serum titers after the first booster immunization. Following the first booster immunization using hBCMA-ECD-Fc, ELISA revealed that the serum of 10 mice at a 1:10k dilution all possessed binding ability to hBCMA-ECD-Fc and cynoBCMA-ECD-Fc. Of these, the titer of SJL number 5066 was relatively low, while the remaining 9 mice showed similar titers.

[0274] The FACS detection results are shown in Figure 2. Except for JL number 5066, all remaining mouse serums at a 1:1000 dilution showed K562-BCMA + It possessed a strong binding ability to [the substance].

[0275] Figures 3A-3B show the results of ELISA analysis of mouse serum titers after a second booster immunization. Following a second booster immunization using hBCMA-ECD-Fc, ELISA detected that the serum of 10 mice at a 1:10k dilution all possessed the desired binding ability to hBCMA-ECD-Fc and cynoBCMA-ECD-Fc and had similar titers.

[0276] The FACS detection results are shown in Figure 4. All serum samples from 10 mice at a 1:1000 dilution showed K562-BCMA. + It bound to [the target molecule]. In the two mouse groups, Balb / c number 5062 and SJL number 5067 showed the highest titers.

[0277] Mouse Balb / c number 5062 died before fusion and after immunization, so mice Balb / c number 5065 and SJL number 5067 were selected for the first fusion F0109. Mouse SJL number 5070 died before fusion and after immunization, so mice Balb / c number 5063 and SJL number 5068 were selected for the second fusion F0227 (no positive clones were obtained from this fusion for screening).

[0278] Only one clone was generated in F0109, and no positive clones were generated in F0227, therefore, K562-BCMA + A third booster immunization was performed using cells. Mouse serum (TB2-2) was collected before immunization, and mouse serum (TB3) was collected one week after immunization.

[0279] The results of ELISA analysis of mouse titers after the third booster immunization are shown in Figures 5A-5B. All four mouse serum samples, diluted 1:10k, showed desirable binding affinity to hBCMA-ECD-Fc and cynoBCMA-ECD-Fc both before and after immunization, and exhibited similar titers.

[0280] The FACS detection results are shown in Figure 6. All four mice's serum samples at a 1:1000 dilution showed L-BCMA. + The molecules bound to the cells. In the two mouse groups, Balb / c number 5064 and SJL number 5069 showed the highest titers.

[0281] Following the third booster immunization, Balb / c number 5064 and SJL number 5069 were selected for the third fusion F0508, and Balb / c number 5061 and SJL number 5066 were selected for the fourth fusion F0614.

[0282] Example 2: Screening of hybridoma cell lines 2.1 Screening of fused F0109 hybridoma cells Cells after electrofusion were smeared onto 40 96-well plates. Of these, Balb / c number 5065 was smeared onto plates 1-20, and SJL number 5067 was smeared onto plates 21-40. Primary screening: ELISA detected the binding ability of the culture medium supernatant to hBCMA-ECD-Fc. The negative control group was NC-Fc, with a set threshold of D value > 0.8, while OD450(hBCMA ECD-Fc)-OD450(hFc) > 0.5, yielding a total of 68 clones. After proliferation culture, secondary screening was performed, and 6G10 of Balb / c number 5065 was found to be hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and K562-BCMA + It exhibits strong binding affinity to cells, and SJL number 5067, 34D1, is K562-BCMA + The clones showed good binding affinity to the cells. These two clones were selected as parent clones, and subclonal screening was performed.

[0283] Derived from Balb / c number 5065, the monoclonal 6G10-1D7 and 6G10-1H2, as well as the hybrid clones 6G10-1B11 and 6G10-1D3 obtained from subclonal screening, are hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and K562-BCMA + It has a strong binding affinity to and the subclonal 34D1-2H2 derived from SJL number 5067 is hBCMA-ECD-Fc and K562-BCMA + It exhibited strong binding affinity to [the target substance]. Five clones were selected and cryopreserved. Clone 6G10-1D7 was selected, and antibody production was carried out in small batches.

[0284] 2.2 Screening of fused F0227 hybridoma cells Subclonal screening of fused F0227 revealed hBCMA-ECD-Fc and L-BCMA + No positive clones capable of binding to cells were obtained.

[0285] 2.3 Screening of fused F0508 hybridoma cells Cells after electrocellular fusion were smeared onto 40 96-well plates. Of these, Balb / c number 5064 was smeared onto plates 81-100, and SJL number 5069 was smeared onto plates 101-120. Primary screening: ELISA detected the binding affinity of the culture medium supernatant to hBCMA-ECD-Fc. The negative control group was NC-Fc, the set threshold was D value ≥ 2, and a total of 79 clones were obtained. After growth culture, secondary screening was performed, and all 19 clones obtained (109C5, 107A11, 107B11, 116H6, 102G2, 102A12, 111F11, 107A9, 111D5, 105F9, 114C6, 97B8, 97C4, 104H4, 115G7, 116A9, 100H2, 99B3, and 110A11) were found to be hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and L-BCMA. +The cells showed strong binding affinity, and all four resulting clones (120D6, 120A10, 105C10, and 113B3) were hBCMA-ECD-Fc and L-BCMA. + These 23 clones showed strong binding affinity to cells, and subclonal screening was performed using them as parent clones, as shown in Figures 7A-7D.

[0286] Each parental clone was smeared onto a 96-well plate, and subclonal screening was performed. In the primary screening, the subclonals of all six clones—105F9, 111F11, 120D6, 104H4, 115G7, and 110A11—were negative. 120A10 had a subclonal that was negative in ELISA, but all other clones were negative. Screening of 120A10 before subcloning showed no cross-reactivity with cynomolgus monkey BCMA, so this clone was not subjected to secondary screening. The 96-well plates smeared with the above clones were cultured for a further two days. Subsequently, ELISA screening was performed again, and the screening results were still negative. Therefore, subclonal screening of these seven parental clones was discontinued.

[0287] The remaining 16 parent clones were subjected to secondary screening. The five subclones obtained from the screening—99B3G3, 102A12H6, 107A11F1, 107A9A4, and 107B11E1—were all hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and L-BCMA. + It exhibited strong binding affinity to cells but did not bind to NC-Fc. Both subclones, 105C10F1 and 113B3F12, were hBCMA-ECD-Fc and L-BCMA. + It exhibited strong binding affinity to [subc], but did not bind to cynoBCMA-ECD-Fc or NC-Fc. The seven subclones mentioned above were selected, and antibodies were produced in small batches.

[0288] Furthermore, four clones, 97B8G8, 100H2D12, 109C5F3, and 114C6C8, were not monoclonal upon microscopic examination and were subjected to a second subcloning. The results of the second primary subclonal screening are as follows: 114C6C8 did not produce positive subclones and was therefore no longer retained. Clones 97B8G8D12, 97B8G8E6, 97B8G8F7, 97B8G8G2, 100H2D12B10, 100H2D12C6, 100H2D12D5, 100H2D12H3, 109C5F3C1, 109C5F3D2, 109C5F3D4, and 109C5F3H3 were selected and subjected to a secondary screening. These results indicate that both obtained clones, 100H2D12C6 and 109C5F3C1, were hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and L-BCMA. + This indicates that it has strong binding affinity to cells but did not bind to NC-Fc. The generated clone 97B8G8D12 has strong binding affinity to hBCMA-ECD-Fc and cynoBCMA-ECD-Fc, and L-BCMA + The cells exhibited weak binding affinity. Three clones were selected as described above, and antibody production was carried out in small batches.

[0289] Most subclones derived from clones 116A9, 111D5, and 116H6 exhibited strong binding affinity to NC-hFc; therefore, FACS was used to screen all subclones from these three clones. All subclones from 116A9 were negative. The subclone from 116H6C10 was positive but exhibited strong binding affinity to NC-hFc. Ten subclones were selected from 111D5 and subjected to secondary screening; all subclones exhibited strong binding affinity to NC-hFc. Consequently, clones 116A9, 111D5, and 116H6, as well as their subclones, were no longer retained.

[0290] Clones 97C4F2, 102G2B5, 102G2D1, and 102G2F3 are L-BCMA + Because they did not bind to the cells, these were no longer retained either.

[0291] Sequencing results showed that 107A11F1, 107B11E1, and 107A9A4 each possessed four heavy chains and one light chain, and these three clones were subjected to a second subclonal screening. The results showed that the subclones 107A11F1, 107A9A4D2, 107A11F1B7, and 107B11E1D7, were hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and L-BCMA + This indicates that the cells exhibited strong binding affinity. Three of these clones were selected and sequenced, and subclones 107A9A4B2, 107A9A4D8, 107A9A4E9, 107A11F1C5, 107A11F1E6, 107A11F1E7, 107B11E1A8, 107B11E1B10, and 107B11E1C11 were cryopreserved.

[0292] 2.4 Screening of fused F0614 hybridoma cells Cells after electrofusion were smeared onto 40 96-well plates. Of these, Balb / c number 5061 was smeared onto plates 121-140, and SJL number 5066 was smeared onto plates 141-160. ELISA was used for primary screening to detect the binding affinity of the culture medium supernatant to hBCMA-ECD-Fc. The set threshold was D value ≥ 0.8. A total of 33 clones were obtained. After proliferation culture, secondary screening was performed. All five obtained clones (151A9, 156E11, 149H4, 143D6, and 154B8) were hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and L-BCMA. + It showed binding affinity to NC-Fc but did not bind to NC-Fc. One obtained clone (152D8) was found to have binding affinity to hBCMA-ECD-Fc and L-BCMA +It exhibited strong binding affinity to NC-Fc but did not bind to NC-Fc. These six clones were selected as parent clones, and subclonal screening was performed.

[0293] Each parental clone was smeared onto a 96-well plate and subclonal screening was performed. Since all three subclones of clones 156E11, 149H4, and 154B8 were negative, these three parental clones were not subjected to secondary screening.

[0294] Subclones were selected from the remaining three parental clones and secondary screening was performed. The results showed that all three subclones, 143D6F4, 151A9A4, and 152D8E8, were hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and L-BCMA. + This indicates that it has a strong binding affinity to NC-Fc and did not bind to NC-Fc. Three subclones were selected, and antibody production was carried out in small batches. 143D6D8 and 151A9F1 were cryopreserved.

[0295] 2.5 Summary of Hybridoma Cell Screening Results As shown in Figures 7A-7D, four hybridoma cell fusions were performed. Following subclonal screening, 14 hybridoma cell lines (6G10-1D7, 99B3G3, 102A12H6, 107A11F1, 107A9A4, 107B11E1, 100H2D12C6, 105C10F1, 113B3F12, 109C5F3C1, 97B8G8D12, 143D6F4, 151A9A4, and 152D8E8) were selected for small-batch hybridoma antibody production and purification, as well as sequencing of the antibody variable region.

[0296] Sequencing results showed that the antibody variable region sequences of 100H2D12C6 and 97B8G8D12 were identical to those of 99B3G3. 107A11F1, 107A9A4, and 107B11E1 were not monoclonal. These three hybridoma cells were subjected to a second subcloning. 107A9A4D2, 107A11F1B7, and 107B11E1D7 were selected and their antibody variable regions were sequenced. The antibody variable regions of 107A9A4D2 and 107A11F1B7 had the same sequence, each containing one heavy chain and two light chains. The screening data for the hybridoma supernatants described above are shown in Table 6.

[0297] [Table 17]

[0298] Example 3: Production and purification of hybridoma antibodies A total of 14 hybridoma cells were selected, and antibody production was performed in small batches. Of these, cell 97B8G8D12 did not produce antibodies. All of the remaining 13 clones successfully produced the corresponding antibodies, as shown in Table 7. The SDS-PAGE results, as shown in Figure 8, indicate that pure antibodies were obtained from purification.

[0299] [Table 18]

[0300] Example 4 Identification of purified hybridoma antibodies 1.0 μg / ml of hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and hTACI-ECD-Fc were smeared onto plates, and the specificity of the hybridoma antibodies was detected by ELISA.

[0301] The ELISA analysis results are shown in Figures 9A to 11B. The purified hybridoma antibody mAb001 (6G10-1D7) was able to bind to the extracellular domain of human BCMA, but had a weak binding affinity to the extracellular domain of cynomolgus monkey BCMA. mAb001 (6G10-1D7) bound to the extracellular domain of cynomolgus monkey BCMA only at high concentrations. mAb013 (152D8E) did not bind to the extracellular domains of human BCMA or cynomolgus monkey BCMA. mAb004 (105C10F1) and mAb008 (13B3F12) were able to bind to the extracellular domain of human BCMA, but could not recognize the extracellular domain of cynomolgus monkey BCMA. All other purified hybridoma antibodies were able to bind to the extracellular domains of human BCMA and cynomolgus monkey BCMA. None of the hybridoma antibodies recognized hTACI-ECD-Fc. The EC50 values ​​for each antibody's binding to hBCMA-ECD-Fc, cynoBCMA-ECD-Fc, and hTACI-ECD-Fc are shown in Table 8.

[0302] [Table 19]

[0303] The flow cytometry detection results are shown in Figures 12A to 13B. With the exception of mAb003 and mAb013, all of the remaining 11 hybridoma antibodies are L-BCMA. + It exhibited desirable binding affinity to cells and did not show nonspecific binding to L cells.

[0304] The results of the competitive binding of hybridoma antibodies to soluble BCMA are shown in Figures 14A-14F. When the concentration of hBCMA-ECD-Fc exceeds 240 ng / ml, K562-BCMA + The binding of cells to mAb001 (final concentration EC80: 1 μg / ml) could be competitively inhibited, where the IC50 was 731 ng / ml and the corresponding molar ratio was 1.7:1. K562-BCMA +If the BCMA concentration in the culture medium supernatant exceeds 120 ng / ml, K562-BCMA + The binding of cells to mAb001 (final concentration EC80: 1 μg / ml) was competitively inhibited, with an IC50 of 466 ng / ml and a corresponding molar ratio of 10.4:1.

[0305] Example 5: Sequencing of antibody variable regions A total of 17 hybridoma cell lines were subjected to sequencing of the antibody variable region. The sequencing results showed that 12 hybridoma cell lines—6G10-1D7(CP01), 99B3G3(CP02), 102A12H6, 100H2D12C6, 105C10F1(CP03), 113B3F12, 109C5F3C1(CP06), 97B8G8D12, 143D6F4(CP07), 151A9A4(CP08), 152D8E8(CP09), and 107B11E1D7(CP05)—were monoclonal. 99B3G3(CP02), 100H2D12C6, and 97B8G8D12 had the same antibody variable region sequence. Five hybridoma cell lines—107A11F1, 107A9A4, 107B11E1, 107A9A4D2, and 107A11F1B7(CP04)—were polyclonal. 107A11F1, 107A9A4, and 107B11E1 each had four heavy chains and one light chain. 107A9A4D2 and 107A11F1B7(CP04) each had one heavy chain and two light chains with the same sequence.

[0306] Table 9 shows the sequence of the antibody variable region.

[0307] [Table 20]

[0308] Example 6 Expression and identification of chimeric antibodies V of the antibody obtained from sequencing H The region was cloned into an IgG1,κ recombinant antibody heavy chain expression vector, V LThe region was cloned into an IgG1,κ recombinant antibody light chain expression vector to construct an expression plasmid for the chimeric antibody. Five monoclonal hybridoma cell lines (6G10-1D7, 99B3G3, 105C10F1, 113B3F12, and 107B11E1D7) and one polyclonal hybridoma cell line (107A9A4D2) were selected to express the chimeric antibody. Of these, 107A9A4D2VH was expressed paired with 107A9A4D2VL-1 and 107A9A4D2VL-2, respectively. The heavy and light chain sequences of the recombinantly expressed chimeric antibody are as follows (underlined portion is the variable region sequence of the antibody):

[0309] 1. Hybridoma 6G10-1D7 >6G10-1D7VH heavy chain (SEQ ID NO: 39, V H (Underlined) MEFGLSWLFLVAILKGVQC EVQLQQSGPELVKPGASMKISCKASDYSFTDYIMTWVKQSHGKNLEWIGLINPYNGGTTYNQKFKDKATFTVDKSSTTAYMDLLSLTSEDSAVYYCARRGITTDYYTMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >6G10-1D7VL light chain (Sequence ID 40): MDMRVPAQLLGLLLLWFPGSRC DIVMTQSQRFMSTSVGDRVSITCKASQSVGTAVAWYQQTPGQFPKLLIYSTSNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSTYPLTFGSGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0310] 2. Hybridoma 99B3G3 >99B3G3VH Heavy Chain (Sequence No. 41, V H (Underlined) MEFGLSWLFLVAILKGVQC EVQLQQSGPVLVKPGASVKMSCKASGYTFTDLYMNWVKQSHGKSLEWIGVINPYNGGTSYNQKFKAKATLTVDKSSITAYMELNSLTSEDSAVYYCARGDSIYVMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >99B3G3VL Light chain (Sequence number 42, V L (Underlined) MDMRVPAQLLGLLLLWFPGSRC DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSIQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTFTLTISSVQAEDLADYFCQQHYSSPLTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0311] 3. Hybridoma 105C10F1 >105C10F1VH Heavy Chain (Sequence ID 43, V H (Underlined) MEFGLSWLFLVAILKGVQC EVKLLQSGGGLVQPGGSLKLSCAASGIDFSRYWMSWVRRAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDAAKNTLYLQMSKVRSEDTALYYCATLYYDYDGDYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >105C10F1VL Light chain (Sequence number 44, V L (Underlined) MDMRVPAQLLGLLLLWFPGSRC DIVMTPSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQHYNSYPFTFGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0312] 4. Hybridoma 113B3F12 >113B3F12VH Heavy Chain (Sequence No. 45, V H (Underlined) MEFGLSWLFLVAILKGVQC EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGGTDYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARRRESYGTSYQGAYFDSWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >113B3F12VL Light chain (Sequence number 46, V L (Underlined) MDMRVPAQLLGLLLLWFPGSRC DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLSISDLEQEDIATYFCQQVITLPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0313] 5. Hybridoma 107B11E1D7 >107B11E1D7VH Heavy Chain (Sequence No. 47, V H (Underlined) MEFGLSWLFLVAILKGVQC EVQLQQSGPVLVKPGASVKMSCKASGYTFTDLYMNWLKQSHGKRLEWIGVINPYNGGTSYNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARGDSIYVMDYWGQGTSFTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >107B11E1D7VL Light chain (Sequence number 48, V L (Underlined) MDMRVPAQLLGLLLLWFPGSRC ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSTSPKLWIYDTSKLSSGVPGRFSGSGSGKSYSLTISSMEAEDVATYYCFQGSGYPLFTFGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0314] 6. Hybridoma 107A9A4D2 >107A9A4D2VH Heavy Chain (Sequence No. 49, V H (Underlined) MEFGLSWLFLVAILKGVQC EVQLQQSGPVLVKPGASVKMSCKASGYTFTDLYMNWLKQSHGKRLEWIGVINPYNGGTSYNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARGDSIYVMDYWGQGTSFTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >107A9A4D2VL-1 Light chain (Sequence ID 50, V L (Underlined) MDMRVPAQLLGLLLLWFPGSRC ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSTSPKLWIYDTSKLSSGVPGRFSGSGSGKSYSLTISSMEAEDVATYYCFQGSGYPLFTFGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >107A9A4D2VL-2 Light Chain (Sequence ID 51, V L (Underlined) MDMRVPAQLLGLLLLWFPGSRC DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSIQKNYLAWYQQKPGQSPKLLIYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0315] Example 7: Identification of Chimeric Antibody Expression The results of ELISA analysis for the identification of chimeric antibody expression are shown in Figures 15A-15B. The results of flow cytometry detection are shown in Figures 16A-16B. Chimeric antibodies of hybridomas 6G10-1D7, 99B3G3, 105C10F1, and 107B11E1D7 bound to the soluble extracellular domain of human BCMA molecules on the cell surface. All chimeric antibodies formed by pairing the heavy chain 107A9A4D2VH and light chain 107A9A4D2VL-2 of hybridoma 107A9A4D2 bound to the soluble extracellular domain of human BCMA molecules on the cell surface. Chimeric antibodies formed by pairing 107A9A4D2VH and 107A9A4D2VL-1 did not bind to the soluble extracellular domain of human BCMA molecules on the cell surface. See Table 10 for detailed data.

[0316] [Table 21]

[0317] Example 8 Construction of a lentiviral expression vector containing a CAR structure Using the light and heavy chains of the nine antibodies obtained from the screening described above, shown in Table 11, 18 chimeric antigen receptors (CARs) were constructed. The structure of a CAR includes a signal peptide (leader sequence), an antigen-binding region, a linker region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ), and the hinge region is as follows: [CD8 LS]-[VL-Linker-VH]-[Hinge-CD8TM]-[4-1BB]-[CD3ζ], or, [CD8 LS]-[VH-Linker-VL]-[Hinge-CD8TM]-[4-1BB]-[CD3ζ]

[0318] [Table 22]

[0319] Based on the CAR sequences constructed above, expression vectors were constructed by full-length DNA synthesis and cloning. The selected expression vector was a pWPT lentiviral vector, and the cloning sites were BamHI and SalI. The specific sequences of each CAR are as follows:

[0320] (1) The leader sequence (signal peptide) was the leader sequence of the CD8 antigen: MALPVTALLLPLALLLHAARP (Sequence ID 52)

[0321] (2) Single-chain variable region light chain (VL) sequence derived from BCMA-CP01 antibody: DIVMTQSQRFMSTSVGDRVSITCKASQSVGTAVAWYQQTPGQFPKLLIYSTSNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSTYPLTFGSGTKLELK (Sequence ID 53)

[0322] (3) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP01 antibody: EVQLQQSGPELVKPGASMKISCKASDYSFTDYIMTWVKQSHGKNLEWIGLINPYNGGTTYNQKFKDKATFTVDKSSTTAYMDLLSLTSEDSAVYYCARRGITTDYYTMDYWGQGTSVTVSS(Sequence ID 54)

[0323] (4) Single-chain variable region light chain (VL) sequence derived from BCMA-CP02 antibody: DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSIQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSSPLTFGAGTKLELK(Sequence ID 55)

[0324] (5) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP02 antibody: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDLYMNWVKQSHGKSLEWIGVINPYNGGTSYNQKFKAKATLTVDKSSITAYMELNSLTSEDSAVYYCARGDSIYVMDYWGQGTSVTVSS(Sequence ID 56)

[0325] (6) Single-chain variable region light chain (VL) sequence derived from BCMA-CP03 antibody: DIVMTPSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQHYNSYPFTFGSGTKLEIK (Sequence ID 57)

[0326] (7) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP03 antibody: EVKLLQSGGGLVQPGGSLKLSCAASGIDFSRYWMSWVRRAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDAAKNTLYLQMSKVRSEDTALYYCATLYYDYDGDYAMDYWGQGTSVTVSS(Sequence ID 58)

[0327] (8) Single-chain variable region light chain (VL) sequence derived from BCMA-CP04 antibody: DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSIQKNYLAWYQQKPGQSPKLLIYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELK(Sequence ID 59)

[0328] (9) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP04 antibody: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDLYMNWLKQSHGKRLEWIGVINPYNGGTSYNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARGDSIYVMDYWGQGTSFTVSS(Sequence ID 60)

[0329] (10) Single-chain variable region light chain (VL) sequence derived from BCMA-CP05 antibody: ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSTSPKLWIYDTSKLSSGVPGRFSGSGSGKSYSLTISSMEAEDVATYYCFQGSGYPLFTFGSGTKLEIK (Sequence ID 61)

[0330] (11) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP05 antibody: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDLYMNWLKQSHGKRLEWIGVINPYNGGTSYNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARGDSIYVMDYWGQGTSFTVSS(Sequence ID 62)

[0331] (12) Single-chain variable region light chain (VL) sequence derived from BCMA-CP06 antibody: DIVMTQSPSSLALSVGQKVTMSCKSSQSLLDNSNQKHYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYTAPLTFGAGTKLALK(Sequence ID 63)

[0332] (13) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP06 antibody: EVQLQQSGPVLVKPGASVKMSCKVSGYTFTDYYMNWVKQSHGKSLEWIGVITPYNGANRYNQKFKGKATLTVDKSSSTAYMEVSSLTSEDSAVYYCARGDSIYVMDYWGQGTSVIVSS(Sequence ID 64)

[0333] (14) Single-chain variable region light chain (VL) sequence derived from BCMA-CP07 antibody: DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSIQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGGGTKLELK (Sequence ID 65)

[0334] (15) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP07 antibody: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYSLNWVKQSHGKSLEWIGVVNPYNGGTSHNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARPDSIYVMDYWGQGTSVTVSS(Sequence ID 66)

[0335] (16) Single-chain variable region light chain (VL) sequence derived from BCMA-CP08 antibody: DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSNIQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELK (Sequence ID 67)

[0336] (17) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP08 antibody: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYSLNWVKQSHGKSLEWIGVVNPYNGGTTYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARPDSIYVMDSWGQGTSVTVSS(Sequence ID 68)

[0337] (18) Single-chain variable region light chain (VL) sequence derived from BCMA-CP09 antibody: DIKMTQSPSSMYVSLGERVTITCKASQDINRNLSWFQQKPGKSPKTLIYRANRLVDGVPLRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPRTFGGGTKLEIK(Sequence ID 69)

[0338] (19) Single-chain variable region heavy chain (VH) sequence derived from BCMA-CP09 antibody: QVTLKESGPGILQSSQTLSLTCSFSGFSLNTSGMGVNWIRQSSGKDLEWLAHIYWNDDKRYNPSLKSRLTISKDTSRNQVFLRITSVDATDTATYFCCRSRLSFDYWGHGTTLTVSS (Sequence ID 70)

[0339] (20) The linker sequences between the heavy and light chains in the single-chain variable region of BCMA-CP01 / R, BCMA-CP02 / R, BCMA-CP03 / R, BCMA-CP04 / R, BCMA-CP05 / R, BCMA-CP06 / R, BCMA-CP07 / R, BCMA-CP08 / R, and BCMA-CP09 / R are as follows: GGGGSGGGGSGGGGS (Sequence No. 71)

[0340] (21) Arrangement of hinge regions (and linker regions): FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (Sequence ID 72)

[0341] (22) Sequence of the transmembrane domain, which is the CD8 (CD8™) transmembrane domain: IYIWAPLAGTCGVLLLSLVITLYC (Sequence ID 73)

[0342] (23) Sequence of intracellular signaling motifs from 4-1BB in the co-stimulatory region: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence ID 74)

[0343] (24) Sequence of the immunoreceptor tyrosine-dependent activation motif (ITAM) from the TCR complex in the cytoplasmic signaling domain of CD3ζ: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 75)

[0344] Example 9 Preparation of CAR-T cells (1) Venous blood was collected from healthy individuals and separated by density gradient centrifugation to obtain mononuclear cells (PBMCs). (2) On day 0, PBMCs were inoculated into cell culture flasks that had been pre-coated with CD3 monoclonal antibody (OKT3) at a final concentration of 5 μg / mL and retronectin (purchased from Takara Bio) at a final concentration of 10 μg / mL. The culture medium was CBMG-RC-09a cell medium containing 1% human albumin. Recombinant human interleukin-2 (CBMG-RC-05b) at a final concentration of 1000 U / mL was added. The culture medium was incubated in a CO2 incubator at 37°C and 5% saturated humidity. (3) On day 1, the supernatant of the cultured PBMCs was slowly removed and fresh CBMG-RC-09a cell medium containing 1% human albumin was added. Recombinant human interleukin 2 (CBMG-RC-05b) was added to the medium to a final concentration of 1000 U / mL. The cells were kept cultured in a CO2 incubator at 37°C and 5% saturated humidity. (4) On day 3, fresh culture medium, concentrated and purified CAR-BCMA lentivirus, protamine sulfate (12 μg / ml), and CBMG-RC-05b at a final concentration of 1000 U / mL were added. After transduction for 12 hours in a 37°C, 5% CO2 incubator, the culture medium was discarded, fresh medium was added, and incubation was continued in a 37°C, 5% CO2 incubator. (5) From day 6, CART-BCMA cells were used for the desired activity test.

[0345] Example 10: Detection of the integration rate of CAR genes into the T cell genome and the expression level of proteins encoded by CAR genes on the membrane surface. In Example 9, 0.5 × 10⁶ cells were cultured for 7 days. 6Using individual CART-BCMA cells, the Fc fragment of recombinant human BCMA protein was stained, and then the expression level of CAR-BCMA protein on the T cell membrane surface was analyzed using a flow cytometer.

[0346] The results are shown in Figures 17A-17B. Except for three CAR-T lentiviruses, BCMA-CP01, BCMA-CP09, and BCMA-CP09R, which did not exhibit transfection rates, all 15 other BCMA-CP CAR-T cells showed relatively high T-cell surface expression of CAR after 7 days of transduction. Cells 6C and 8C served as positive controls.

[0347] Example 11: Detection of in vitro activation ability of CART-BCMA In Example 9, CART-BCMA cells cultured for 7 days were used to detect the proteins CD137 and IFNγ, which are indicators of cell activation levels. 5 Each CART-BCMA cell was cultured for 18 hours in 200 μl of CBMG-RC-09a medium in a 1:1 ratio with either human BCMA-positive A549-BCMA-1D6, MM.1S, and RPMI8226 tumor cell lineages, monkey BCMA-positive A549-BCMA-M and BCMA-negative A549 tumor cell lineages, or tumor cells without these additives. Next, the expression level of CD137 on the T cell membrane surface was detected by flow cytometry, and the secretion level of IFNγ in the culture supernatant was detected by ELISA.

[0348] The results are shown in Figures 18A-18H and 19A-19B. BCMA-CP01R, BCMA-CP03, and BCMA-CP03R showed high-specificity IFN-γ release and CD137 activation-specific upregulation expression only in A549-BCMA-1D6 cells. BCMA-CP02 / R, BCMA-CP04 / R, and BCMA-CP05 / R showed high-specificity IFN-γ release and CD137 activation-specific upregulation expression in both A549-BCMA-1D6 and A549-BCMA-M cells.

[0349] Example 12: Detection of cytotoxicity of CART-BCMA cells against target cells RTCA (Real-time Cell Analysis) was performed to detect the cytotoxicity of CART cells (BCMA-CP01 / R~BCMA-CP05 / R) cultured for 14 days in Example 9 and CART cells (BCMA-CP06 / R~BCMA-CP09 / R) cultured for 12 days. These cells were then co-cultured for 8 hours in 200 μl of CBMG-RC-09a medium with BCMA-negative cells (A549), human BCMA-positive autoconstructed cells A549-BCMA-1D6, or monkey BCMA-positive autoconstructed cells A549-BCMA-M in the ratios shown in Figure 4. The cytotoxicity of the CART cells against each target cell was then analyzed.

[0350] The results are shown in Figures 20A-20F. BCMA-CP01R, BCMA-CP03, and BCMA-CP03R exhibited strong cytotoxicity against A549-BCMA-1D6 cells. BCMA-CP02 / R, BCMA-CP04 / R, and BCMA-CP05 / R exhibited strong cytotoxicity against A549-BCMA-1D6 and A549-BCMA-M cells, but did not have a significant killing effect against negative target cells A549. The untransduced control group (NT) also did not have a significant killing effect against target cells.

[0351] The scope of the present invention is not limited to those specifically shown and described above. Those skilled in the art will recognize that there are appropriate choices for the described examples of materials, components, structures, and dimensions. Numerous references, including patents and various publications, are cited and discussed in the detailed description of the present invention. Such citations and discussions of references are provided solely to clarify the detailed description of the present invention, and do not constitute prior art to the inventions described herein. All references cited and discussed herein are incorporated herein by reference in their entirety. Those skilled in the art will be able to recall variations, modifications, and other implementations of those described herein without departing from the spirit and scope of the present invention. While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the present invention. The matters shown in the above detailed description and accompanying drawings are presented as examples and not as limitations.

Claims

1. Light chain variable region (V Ｌ ) and heavy chain variable region (V Ｈ ) including, (i) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively, and V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively. (ii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively, and V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively. (iii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively. (iv) the V Ｌ comprises three CDRs, CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively, and the V Ｈ comprises CDR1, CDR2, and CDR3 of three complementarity determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively, or (v) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. (vi) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively. (vii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively, or (viiii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively, and the V Ｈ This comprises an anti-BCMA antibody or its antigen-binding moiety, including CDR1, CDR2, and CDR3 of three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively.

2. The aforementioned V Ｌ and V Ｈ The antibody or antigen-binding moiety according to claim 1, wherein the antibody or antigen-binding moiety has the amino acid sequences shown in (i) SEQ ID NO: 53 and SEQ ID NO: 54, respectively, (ii) SEQ ID NO: 55 and SEQ ID NO: 56, respectively, (iii) SEQ ID NO: 57 and SEQ ID NO: 58, respectively, (iv) SEQ ID NO: 59 and SEQ ID NO: 60, respectively, (v) SEQ ID NO: 61 and SEQ ID NO: 62, respectively, (vi) SEQ ID NO: 63 and SEQ ID NO: 64, respectively, (vii) SEQ ID NO: 65 and SEQ ID NO: 66, respectively, or (viiii) SEQ ID NO: 67 and SEQ ID NO: 68, respectively.

3. The antibody or its antigen-binding portion according to claim 1 or 2, wherein the antibody or its antigen-binding portion is selected from the group consisting of (a) a whole immunoglobulin molecule, (b) scFv, (c) a Fab fragment, (d) F(ab')2, and (e) a disulfide-bonded Fv.

4. An antibody conjugate comprising an antibody or its antigen-binding portion according to claim 1 or 2, linked to a conjugate portion selected from the group consisting of a detectable marker, drug, toxin, cytokine, radionuclide, enzyme, targeting portion, and combinations thereof.

5. A composition comprising an antibody or its antigen-binding moiety according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

6. A nucleic acid encoding an antibody or its antigen-binding portion according to any one of claims 1 to 3.

7. Light chain variable region (V Ｌ ) and heavy chain variable region (V Ｈ It includes an anti-BCMA antigen binding region, (i) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively, and V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively. (ii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively, and V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively. (iii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively. (iv) Said V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively. (v) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. (vi) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively. (vii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively, and the V Ｈ This includes CDR1, CDR2, and CDR3, which are three complementarity-determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively, or (viiii) The above V Ｌ This includes three CDRs, CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively, and the V Ｈ This is a chimeric antigen receptor (CAR) comprising three complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, each having the amino acid sequences shown in SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively.

8. The aforementioned V Ｌ and V Ｈ The CAR according to claim 7, wherein (i) the amino acid sequences shown in SEQ ID NO: 53 and SEQ ID NO: 54, respectively, (ii) SEQ ID NO: 55 and SEQ ID NO: 56, respectively, (iii) SEQ ID NO: 57 and SEQ ID NO: 58, respectively, (iv) SEQ ID NO: 59 and SEQ ID NO: 60, respectively, (v) SEQ ID NO: 61 and SEQ ID NO: 62, respectively, (vi) SEQ ID NO: 63 and SEQ ID NO: 64, respectively, (vii) SEQ ID NO: 65 and SEQ ID NO: 66, respectively, or (viiii) SEQ ID NO: 67 and SEQ ID NO: 68, respectively.

9. The CAR according to claim 7 or 8, wherein the anti-BCMA antigen-binding region is a single-chain variable fragment (scFv) that specifically binds to BCMA.

10. The CAR according to any one of claims 7 to 9 further includes one or more of the following: (a) signal peptide, (b) Hinge region, the hinge region includes the hinge regions of Ig4, CD8, CD28, CD137, or a combination thereof. (c) Transmembrane domains, the transmembrane domains comprising the transmembrane domains of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof. (d) Co-stimulatory regions, the co-stimulatory regions include the co-stimulatory regions of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof, and (e) Cytoplasmic signaling domain, the cytoplasmic signaling domain includes the cytoplasmic signaling domain of CD3ζ.

11. An immune cell expressing the CAR according to any one of claims 7 to 10, wherein the immune cell is a T cell, a natural killer (NK) cell, a natural killer T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a stem cell, a macrophage, or a dendritic cell.

12. A nucleic acid encoding a CAR according to any one of claims 7 to 10.

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