Bispecific chimeric antigen receptors targeting BCMA-CD19 and their applications

Abstract

The present invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its use. The bispecific chimeric antigen receptor comprises an extracellular antigen recognition domain, the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain, the anti-BCMA extracellular antigen recognition domain comprises a BCMA VH and a BCMA VL, the amino acid sequences of the BCMA VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the amino acid sequences of the BCMA VL complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

Description

[Technical Field]

[0001] This application relates to the field of biomedicine, and more specifically to a bispecific chimeric antigen receptor targeting BCMA-CD19 and its applications. [Background technology]

[0002] Multiple myeloma, defined as the malignant proliferation of plasma cells in the bone marrow, accounts for 1% of all cancer types and is the second most common hematological malignancy. Multiple studies have shown that multiple myeloma is more common in people over 60 years of age, and its incidence has been steadily increasing in recent years. For most patients, multiple myeloma is incurable and eventually progresses to relapsed / refractory multiple myeloma. The survival time for patients with relapsed / refractory multiple myeloma who do not respond to existing multiple myeloma treatments (such as immunomodulators, proteasome inhibitors, and antibody drugs) is only about 13 months.

[0003] B-cell maturation antigen (BCMA) is a transmembrane glycoprotein belonging to the tumor necrosis factor receptor family. BCMA is highly expressed in multiple myeloma cells but not in most other cells. Malignant tumor plasma cells typically express higher levels of BCMA than normal plasma cells, and BCMA upregulation promotes the proliferation of multiple myeloma cancer cells, while downregulation of its expression can inhibit their proliferation.

[0004] Furthermore, since multiple myeloma, like B-cell tumors, generally does not express the CD19 molecule, CD19 is generally not a target for multiple myeloma treatment. However, some literature suggests that mild drug-resistant and relapsed multiple myeloma clones also express CD19. + Phenotypes may be used This suggests that...

[0005] Chimeric antigen receptors (CARs) are core components of CAR cell therapies and may include a target region (e.g., a region that binds to tumor-associated antigens (TAAs)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T cell immunotherapy is considered one of the most promising methods for overcoming tumors. CAR-T cells are T cells that express CAR proteins using genetic modification methods. Such CAR proteins have the ability to recognize untreated proteins on the membrane surface independently of antigen presentation, thus leading to T cell activation and functional effects. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In 2021, Bristol-Myers Squibb and Bluebird Bio jointly announced that the U.S. Food and Drug Administration (FDA) had approved a CAR-T cell therapy targeting BCMA (bb2121). This CAR-T cell therapy is used in adult patients with relapsed or refractory multiple myeloma after fourth-line treatment (including immunomodulatory agents, proteasome inhibitors, and antibody therapy). This is the world's first CAR-T cell therapy targeting BCMA. Developing more effective cell therapies targeting BCMA is of practical importance. [Effects of the Invention]

[0007] This application provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its applications. The inventors constructed multiple expression vectors for a bispecific chimeric antigen receptor targeting BCMA-CD19 and prepared bispecific CAR-T cells targeting BCMA-CD19. The inventors also confirmed that BCMA-CD19 bispecific CAR-T cells have good antitumor function at the cellular level. [Means for solving the problem]

[0008] It comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the extracellular antigen recognition domain includes an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain. A bispecific chimeric antigen receptor targeting BCMA-CD19, wherein the anti-BCMA extracellular antigen recognition domain comprises BCMA VH and BCMA VL, the amino acid sequences of the BCMA VH complementarity-determining regions CDR1, CDR2, and CDR3 comprise the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of the BCMA VL complementarity-determining regions CDR1, CDR2, and CDR3 comprise the amino acid sequences represented by SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0009] In one embodiment of the bispecific chimeric antigen receptor described above, the anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, the amino acid sequences of the CD19 VH complementarity-determining regions CDR1, CDR2, and CDR3 each comprise the amino acid sequences represented by SEQ ID NOs. 7, SEQ ID NOs. 8, and SEQ ID NOs. 9, respectively, and the amino acid sequences of the CD19 VL complementarity-determining regions CDR1, CDR2, and CDR3 each comprise the amino acid sequences represented by SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, respectively.

[0010] In one embodiment of the bispecific chimeric antigen receptor described above, the BCMA VH sequence includes the amino acid sequence represented by SEQ ID NO: 13, and the BCMA VL sequence includes the amino acid sequence represented by SEQ ID NO: 14.

[0011] In one embodiment of the bispecific chimeric antigen receptor described above, the BCMA VH sequence includes the amino acid sequence represented by SEQ ID NO: 15, and the BCMA VL sequence includes the amino acid sequence represented by SEQ ID NO: 16.

[0012] In one embodiment of the bispecific chimeric antigen receptor described above, the CD19 VH sequence includes the amino acid sequence represented by SEQ ID NO: 17, and the CD19 VL sequence includes the amino acid sequence represented by SEQ ID NO: 18.

[0013] In certain embodiments of the above-mentioned bispecific chimeric antigen receptor, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one structure selected from the group consisting of CD19 VL sequence - first linker sequence - CD19 VH sequence - second linker sequence - BCMA VL sequence - third linker sequence - BCMA VH sequence, BCMA VL sequence - fourth linker sequence - BCMA VH sequence - fifth linker sequence - CD19 VL sequence - sixth linker sequence - CD19 VH sequence, BCMA VL sequence - seventh linker sequence - CD19 VL sequence - eighth linker sequence - CD19 VH sequence - ninth linker sequence - BCMA VH sequence, and CD19 VL sequence - tenth linker sequence - BCMA VL sequence - eleventh linker sequence - BCMA VH sequence - twelfth linker sequence - CD19 VH sequence.

[0014]

[0015] In certain embodiments of the above-mentioned bispecific chimeric antigen receptor, the first linker sequence, the second linker sequence, the third linker sequence, the fourth linker sequence, the fifth linker sequence, the sixth linker sequence, the seventh linker sequence, the eighth linker sequence, the ninth linker sequence, the tenth linker sequence, the eleventh linker sequence, and the twelfth linker sequence are each independently selected from one or more of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0016] In certain embodiments of the above-mentioned bispecific chimeric antigen receptor, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises the amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 20.

[0017] In certain embodiments of the above-mentioned bispecific chimeric antigen receptor, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. Optionally, the amino acids of the hinge region are derived from CD8α. Further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence represented by SEQ ID NO: 21.

[0018] In certain embodiments of the above-mentioned bispecific chimeric antigen receptor, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70. Optionally, the amino acids of the transmembrane region are derived from CD8α. Further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence represented by SEQ ID NO: 22.

[0019] In certain embodiments of the above-mentioned bispecific chimeric antigen receptor, the intracellular domain comprises an intracellular signaling region. Optionally, the intracellular domain further comprises a co-stimulatory signaling region.

[0020] In certain embodiments of the above-mentioned bispecific chimeric antigen receptor, the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk. Optionally, the intracellular signaling region is derived from CD3ζ. Further optionally, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence represented by SEQ ID NO: 23.

[0021] In one embodiment of the bispecific chimeric antigen receptor described above, the co-stimulatory signaling region is derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88, optionally the co-stimulatory signaling region is derived from CD28 or 4-1BB, and optionally the amino acid sequence of the co-stimulatory signaling region includes the amino acid sequence represented by SEQ ID NO: 24.

[0022] In one embodiment of the bispecific chimeric antigen receptor described above, the bispecific chimeric antigen receptor further comprises a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor, optionally the guide peptide is derived from CD8α, and optionally the amino acid sequence of the guide peptide comprises the amino acid sequence represented by SEQ ID NO: 25.

[0023] In one embodiment of the bispecific chimeric antigen receptor described above, the bispecific chimeric antigen receptor includes the amino acid sequence represented by SEQ ID NO: 28 or SEQ ID NO: 29.

[0024] This application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor described above.

[0025] In one embodiment of the isolated nucleic acid molecule described above, the nucleotide sequence encoding the bispecific chimeric antigen receptor described above is 1) A nucleotide sequence represented by SEQ ID NO: 37, which encodes the BCMA VH amino acid sequence represented by SEQ ID NO: 13, and a nucleotide sequence represented by SEQ ID NO: 38, which encodes the BCMA VL amino acid sequence represented by SEQ ID NO: 14, and / or 2) It includes a nucleotide sequence represented by SEQ ID NO: 39 that encodes the CD19 VH amino acid sequence represented by SEQ ID NO: 17, and a nucleotide sequence represented by SEQ ID NO: 40 that encodes the CD19 VL amino acid sequence represented by SEQ ID NO: 18.

[0026] This application also provides a vector containing the isolated nucleic acid molecule described above.

[0027] In one embodiment of the above vector, the vector is an expression vector; in another embodiment, the vector is a viral vector; and in yet another embodiment, the vector is a lentiviral vector.

[0028] This application also provides engineered immunoeffector cells comprising the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0029] In one embodiment of the manipulated immune effector cells described above, the manipulated immune effector cells are selected from one or more of the following: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0030] In one embodiment of the manipulated immune effector cells described above, the manipulated immune effector cells are T lymphocytes, and optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

[0031] This application also provides a pharmaceutical composition comprising the above-described manipulated immunoeffector cells and a pharmaceutically acceptable adjuvant.

[0032] In one embodiment of the above pharmaceutical composition, the pharmaceutically acceptable adjuvant includes a protective agent.

[0033] In one embodiment of the above pharmaceutical composition, the pharmaceutically acceptable adjuvant comprises a cell cryopreservation solution.

[0034] In one embodiment, the above pharmaceutical composition is administered by intravenous injection.

[0035] This application also provides the use of the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, the above-mentioned vector, or the above-mentioned engineered immune effector cell in the preparation of a pharmaceutical used for the treatment of a disease or condition associated with BCMA expression.

[0036] In one embodiment of the use described above, the disease or condition associated with the expression of the BCMA is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or recurrent multiple myeloma.

[0037] In one embodiment of the above use, the disease or condition associated with the expression of BCMA is an autoimmune disease.

[0038] In the embodiments described above, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0039] This application also provides a method for treating a disease or condition associated with BCMA expression, comprising the step of administering an effective amount of the above-described engineered immunoeffector cells or the above-described pharmaceutical composition to a subject who needs to be treated for a disease or condition associated with BCMA expression.

[0040] In one embodiment of the above method, the disease or condition associated with the expression of BCMA is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or recurrent multiple myeloma.

[0041] In one embodiment of the above method, the disease or condition associated with the expression of BCMA may be an autoimmune disease.

[0042] In one embodiment of the above method, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0043] In one embodiment of the above method, the method of administration is intravenous injection.

[0044] In one embodiment of the above method, the administration method involves administering an effective amount of the above-described manipulated immune effector cells or the above-described pharmaceutical composition to the subject by a single injection.

[0045] In one embodiment of the above method, the effective amount of the manipulated immunoeffector cells or the pharmaceutical composition is 1 × 10 5 ~1 × 10 7 It is cells / kg.

[0046] This application also provides the above-described manipulated immune effector cells or the above-described pharmaceutical composition for use in the treatment of diseases or conditions related to BCMA expression.

[0047] In one embodiment of the manipulated immune effector cells or the pharmaceutical composition described above, the disease or condition associated with the expression of the BCMA is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or relapsed multiple myeloma.

[0048] In some embodiments of the manipulated immune effector cells or the pharmaceutical composition described above, the disease or condition associated with the expression of BCMA may be an autoimmune disease.

[0049] In one embodiment of the manipulated immune effector cells or the pharmaceutical composition described above, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia. [Brief explanation of the drawing]

[0050] [Figure 1] Figure 1 shows schematic structural diagrams of various BCMA-CD19 bispecific CARs, CD19 CARs, and BCMA CARs in Example 1 of this application. [Figure 2A] Figure 2A shows the CAR expression of two untreated CARs (i.e., CD19-2A-BCMA and BCMA-2A-CD19) linked by the self-cleaving polypeptide T2A on the surface of CAR-T cells (6 days after infection) in Example 2 of this application. The left panel of Figure 2A is the CD19-2A-BCMA group, the center panel is the BCMA-2A-CD19 group, and the right panel is the UTD group (T cells without CAR transduction). [Figure 2B] Figures 2B to 2E show CAR expression on the surface of four bispecific CAR-T cells (i.e., Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, and Loop BCMA-CD19 cells) (6 days after infection) in Example 2 of this application. Figure 2B shows the Tan CD19-BCMA group, Figure 2C shows the Tan BCMA-CD19 group, Figure 2D shows the Loop CD19-BCMA group, and Figure 2E shows the Loop BCMA-CD19 cell group. [Figure 3]Figures 3A to 3C show the cytokine release of various BCMA-CD19 bispecific CARs, CD19 CARs, and BCMA CARs after activation by positive target cells in Example 4 of this application. Figure 3A shows the release of IL-2 in each group, Figure 3B shows the release of IFN-γ in each group, and Figure 3C shows the release of TNF-α in each group. In particular, Figures 3A, 3B, and 3C each contain two dotted boxes. In the first dotted box in Figures 3A, 3B, and 3C, the five bars, from left to right, represent cytokine release from BCMA cells after activation by UTD cells, TanCD19-BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells, and K562-BCMA cells. In the second dotted box in Figures 3A, 3B, and 3C, the five bars, from left to right, represent cytokine release from CD19 cells after activation by UTD cells, TanCD19-BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells, and K562-CD19 cells. [Figure 4] Figures 4A to 4C show the cell-killing effects of various BCMA-CD19 bispecific CARs, CD19 CARs, and BCMA CAR cells on different target cells in Example 5 of this application. Figure 4A shows the NALM6 group, which are CD19+BCMA target cells; Figure 4B shows the MM.1S group, which are BCMA+CD19 target cells; and Figure 4C shows the NALM6-KO CD19 group, which are negative target cell controls in which BCMA is not expressed but CD19 is knocked out. [Figure 5] Figures 5A to 5D show the sustained proliferation of CAR-T cells in each group after multiple antigen stimulations in Example 6 of this application. Figure 5A shows the proliferation of CD3+ cells stimulated with MM.1S cells, Figure 5B shows the proliferation of CD3+ cells stimulated with NALM6 cells, Figure 5C shows the proliferation of CAR+ cells stimulated with MM.1S cells, and Figure 5D shows the proliferation of CAR+ cells stimulated with NALM6 cells. [Modes for carrying out the invention]

[0051] Embodiments of the present invention will be described using the following specific examples. Those familiar with this art will readily understand other advantages and effects of the present invention from the disclosure herein.

[0052] This application is further described below. Unless otherwise specified, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are widely used terms and routine procedures in the corresponding fields. For a better understanding of this invention, definitions and explanations of the relevant terms are provided below.

[0053] In this application, “chimeric antigen receptor (CAR)” is a core component of CAR cell therapy and may include an extracellular antigen-recognition domain (e.g., a portion that binds to tumor-associated antigens (TAA), a hinge region, a transmembrane region, and an intracellular domain). CAR-T (chimeric antigen receptor T) cell immunotherapy is considered one of the most promising methods for overcoming tumors. CAR-T cells are T cells that express CAR proteins using genetic modification methods. Such CAR proteins have the ability to recognize untreated proteins on the membrane surface independently of antigen presentation, thus leading to T cell activation and functional effects.

[0054] In this application, “extracellular antigen recognition domain” refers to the antigen recognition domain (ARD). The reason why CAR cell therapy products (such as CAR-T cells) can specifically recognize and / or bind to target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. To date, antigen recognition domains have been derived from antibody single-chain variable fragments (abbreviated as scFv), receptor-ligand interactions, TCR mimics, or variable lymphocyte receptors (VLRs). To date, the most common source is the antibody scFv segment. The scFv contains an antibody heavy chain variable region (VH region) and an antibody light chain variable region (VL region), which are linked by peptide chains such as GSTSGSGKPGSGEGSTKG, which are linker sequences consisting of 18 amino acids. scFv antibodies targeting two or more targets include VH and VL regions that target different targets, and these different regions are linked directly or indirectly via ligation sequences. The arrangement can be in one of the following forms: target1 VL-target1 VH-target2 VL-target2 VH, target2 VL-target2 VH-target1 VL-target1 VH, target1 VL-target2 VL-target2 VH-target1 VH, or target2 VL-target1 VL-target1 VH-target2 VH, where "-" indicates linkage via a ligation sequence.

[0055] In this application, “specific recognition and / or binding” refers to the recognition and / or binding between a CAR and a specific target, where the CAR binding to this target is performed with higher affinity, binding activity, ease, and / or longer duration than CAR binding to other targets.

[0056] In this application, the “hinge region” refers to the linker segment between the extracellular antigen recognition domain and the transmembrane domain. This region enables the CAR to recognize an antigen by providing a range of activity to the antigen recognition domain. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. Furthermore, typical hinge regions also contain several residues that are involved in CAR dimerization and contribute to enhanced antigen sensitivity.

[0057] In this application, “transmembrane region” refers to the transmembrane domain that connects the intracellular and extracellular components of a CAR structure. Different transmembrane domains may affect CAR expression and stability to some extent, but they do not directly participate in signal transduction and instead improve downstream signal transduction through their interactions. The transmembrane region may originate from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0058] In this application, the “intracellular domain” includes an intracellular signaling region and may further include a co-stimulatory signaling region.

[0059] In this application, “intracellular signaling region” refers to the activation of at least one normal effector function of an immune effector cell that is a factor in CAR expression. The intracellular signaling region may be derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0060] In this application, the “co-stimulatory signaling region” is present because, in addition to stimulation by antigen-specific signals, many immune effector cells require co-stimulation to promote the proliferation, differentiation, and survival of activated cells, as well as effector function. In some examples, the CAR may further include one or more co-stimulatory signaling regions, which may be derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0061] In this application, “isolated” generally means obtained from nature by artificial means. When an “isolated” substance or component exists in nature, it may be that the natural environment in which it is located has been altered, or the substance has been separated from that natural environment, or both. For example, if an unisolated polynucleotide or polypeptide exists in a living animal, a highly purified version of that polynucleotide or polypeptide isolated from this natural state is called an isolated polynucleotide or polypeptide. “Isolated” does not exclude substances obtained artificially or synthetically from their natural state by artificial means, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0062] In this application, "guide peptide" refers to a short peptide preceding the extracellular antigen recognition domain (e.g., scFv sequence), whose function is to guide recombinant proteins synthesized intracellularly to be transported outside the cell. Commonly used guide peptides include human CD8α signaling peptide or human GM-CSF receptor α signaling peptide.

[0063] In this application, one of the key factors determining the therapeutic effect of CAR immune cells is the selection of tumor target antigens. In this application, "BCMA" refers to B cell maturation antigens, which are members of the tumor necrosis factor receptor superfamily. Human BCMA is expressed almost exclusively on plasma cells and multiple myeloma cells. BCMA may be a suitable tumor antigen target for immunotherapies against multiple myeloma. However, because the specific antigens on the surface of multiple myeloma cells are heterogeneous, the selection of its antigen target is not always singular. By selecting the appropriate target, the antitumor activity of CAR-T cells can be optimized. The "CD19" molecule is currently a major target for treating hematological malignancies derived from B lymphocytes and is also attracting attention in CAR-T cell therapy research. Most malignant tumor cells derived from B cells express the CD19 molecule on their surface. Multiple myeloma, like B cell line tumors, generally does not express the CD19 molecule. Therefore, CD19 is not typically used as a target for the treatment of multiple myeloma. However, some literature studies suggest that even small amounts of drug-resistant and relapsing multiple myeloma clones may also contain CD19 + This suggests that phenotypes are involved. When using this dual-target CAR-T product, as long as one tumor antigen target is recognized, it is possible to activate CAR-T cells and prevent leakage of the tumor antigen. Compared to preparing CAR immune cells that target different targets separately and using them together, dual-target CAR immune cells have the advantages of 1. requiring fewer immune cells, being easier to prepare, and saving costs, and 2. from an administration standpoint, the safety and operability of administering one product is far superior to that of administering two products.

[0064] In this application, “linker sequence” generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links any structure / region of the chimeric antigen receptor of the present invention. The linker sequence may consist of different amino acid residues (e.g., glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linker sequences may be used if it is desirable that two adjacent domains not spatially interfere with each other.

[0065] In this application, “isolated nucleic acid molecule” generally refers to an isolated form of a nucleotide, deoxyribonucleotide, or ribonucleotide of any length that can be isolated from the natural environment or synthetic analogues.

[0066] In this application, when performing CAR gene transfer / transfection and target gene expression, the gene transfer / transfection methods mainly include viral and non-viral methods. For example, these include methods using gamma retroviral vectors, lentiviral vectors, adenovirus-related viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transfer.

[0067] A "vector" generally refers to a nucleic acid delivery carrier into which a protein-coding polynucleotide can be inserted, enabling protein expression. A vector can be transformed, transduced, or transfected into a host cell so that its genetic material elements can be expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain various elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may also contain an origin of replication. The vector may also contain components that assist in cell entry, such as viral particles, liposomes, or protein coatings, but are not limited to these. “Transposons” refer to discontinuous DNA segments that have the ability to move between chromosomal regions and carry genetic information, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects. In some examples, mRNA can also be transduced into T cells by electrotransduction.

[0068] In this application, "immune effector cells" generally refers to cells involved in the immune response, such as cells that promote immune effector responses. Immune effector cells can be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0069] In this application, “pharmaceutical composition” generally refers to a pharmaceutical composition suitable for administration to a patient, which may include the immunoeffector cells described in this application and may further include one or more pharmaceutically acceptable adjuvants, such as carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives. In some examples, the pharmaceutically acceptable adjuvants include protective agents such as cell cryopreservation solutions. In some examples, the pharmaceutical composition of this application is a cell suspension or cryopreserved cells thereof.

[0070] In this application, “Subject” generally refers to human or non-human animals, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cattle, sheep, or monkeys.

[0071] In this application, "includes" generally refers to the inclusion of explicitly specified features, but not to the exclusion of other elements.

[0072] In this application, "about" generally refers to a range of variation above or below a specified value that is acceptable to a person skilled in the art, for example, a variation within ±0.5% to 10% above or below 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0073] Chimeric antigen receptors, nucleic acids, vectors, immune effector cells, pharmaceutical compositions In one embodiment, the present application comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain. The present invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19, wherein the anti-BCMA extracellular antigen recognition domain comprises BCMA VH and BCMA VL, the amino acid sequences of the BCMA VH complementarity-determining regions CDR1, CDR2, and CDR3 comprise the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of the BCMA VL complementarity-determining regions CDR1, CDR2, and CDR3 comprise the amino acid sequences represented by SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0074] In antibodies, common rules for splitting CDRs include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying websites that implement these rules, by inputting VH and VL sequences and selecting the corresponding rule, CDR sequences based on different rules can be obtained. In this application, the IMGT rule is used to split the CDR. However, those skilled in the art should understand that the scope of protection of this application includes combinations of CDR sequences obtained by analysis using different rules.

[0075] In some examples, the anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19 VL, the amino acid sequences of the CD19 VH complementarity-determining regions CDR1, CDR2, and CDR3 include the amino acid sequences represented by SEQ ID NOs. 7, SEQ ID NOs. 8, and SEQ ID NOs. 9, respectively, and the amino acid sequences of the CD19 VL complementarity-determining regions CDR1, CDR2, and CDR3 include the amino acid sequences represented by SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, respectively.

[0076] In some examples, the BCMA VH sequence includes the amino acid sequence represented by SEQ ID NO: 13, and the BCMA VL sequence includes the amino acid sequence represented by SEQ ID NO: 14.

[0077] In some examples, the BCMA VH sequence includes the amino acid sequence represented by SEQ ID NO: 15, and the BCMA VL sequence includes the amino acid sequence represented by SEQ ID NO: 16.

[0078] In some examples, the CD19 VH sequence includes the amino acid sequence represented by SEQ ID NO: 17, and the CD19 VL sequence includes the amino acid sequence represented by SEQ ID NO: 18.

[0079] In some cases, the application also includes substitutions, deletions, additions and / or insertions of one or more amino acids in the amino acid sequence of any one of the above-described bispecific chimeric antigen receptors, and activity equivalent to that of any one of the above-described chimeric antigen receptors. Those skilled in the art will know that amino acids in the FR region of the VH and VL sequences can be substituted so that the CDR region of the modified antibody retains a suitable antigen-binding site during the humanization process. Accordingly, the application will certainly include different amino acid sequences obtained by humanizing the FR region of the VH and VL sequences based on the above-described CDR. Furthermore, those skilled in the art will know that, if necessary, 1%, 2%, 3%, or 10% or less of the amino acid sequence in the CDR may be substituted, deleted, added and / or inserted so that the CDR region of the modified antibody retains a suitable antigen-binding site during the humanization process, and these are also included in the application.

[0080] In some examples, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes one structure selected from the group consisting of CD19 VL sequence-1st linker sequence-CD19 VH sequence-2nd linker sequence-BCMA VL sequence-3rd linker sequence-BCMA VH sequence, BCMA VL sequence-4th linker sequence-BCMA VH sequence-5th linker sequence-CD19 VL sequence-6th linker sequence-CD19 VH sequence, BCMA VL sequence-7th linker sequence-CD19 VL sequence-8th linker sequence-CD19 VH sequence-9th linker sequence-BCMA VH sequence, and CD19 VL sequence-10th linker sequence-BCMA VL sequence-11th linker sequence-BCMA VH sequence-12th linker sequence-CD19 VH sequence.

[0081] In some examples, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes one structure selected from the group consisting of BCMA VL sequence-7th linker sequence-CD19 VL sequence-8th linker sequence-CD19 VH sequence-9th linker sequence-BCMA VH sequence and CD19 VL sequence-10th linker sequence-BCMA VL sequence-11th linker sequence-BCMA VH sequence-12th linker sequence-CD19 VH sequence.

[0082] In some examples, the first linker sequence, the second linker sequence, the third linker sequence, the fourth linker sequence, the fifth linker sequence, the sixth linker sequence, the seventh linker sequence, the eighth linker sequence, the ninth linker sequence, the tenth linker sequence, the eleventh linker sequence, and the twelfth linker sequence are each independently selected from one or more of sequence numbers 34, 35, and 36.

[0083] In some cases, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes the amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 20.

[0084] In some examples, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α, optionally the amino acid sequence of the hinge region is derived from CD8α, and further optionally the amino acid sequence of the hinge region includes the amino acid sequence represented by SEQ ID NO: 21.

[0085] In some examples, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70, and optionally the amino acid sequence of the transmembrane region is derived from CD8α, and further optionally the amino acid sequence of the transmembrane region includes the amino acid sequence represented by SEQ ID NO: 22.

[0086] In some examples, the intracellular domain includes an intracellular signaling region, optionally further including a co-stimulatory signaling region, and further optionally the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk, and further optionally the intracellular signaling region is derived from CD3ζ, for example the amino acid sequence of the intracellular signaling region includes the amino acid sequence represented by SEQ ID NO: 23.

[0087] In some examples, the co-stimulatory signaling region is derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88, optionally the co-stimulatory signaling region is derived from CD28 or 4-1BB, and further optionally the amino acid sequence of the co-stimulatory signaling region includes the amino acid sequence represented by SEQ ID NO: 24.

[0088] In some examples, the bispecific chimeric antigen receptor further comprises a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor, optionally the guide peptide being derived from CD8α, and further optionally the amino acid sequence of the guide peptide comprising the amino acid sequence represented by SEQ ID NO: 25.

[0089] In some examples, the bispecific chimeric antigen receptor includes the amino acid sequence represented by SEQ ID NO: 28 or SEQ ID NO: 29.

[0090] In another embodiment, the application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor described above.

[0091] In some examples, the nucleotide sequence encoding the bispecific chimeric antigen receptor is 1) A nucleotide sequence represented by SEQ ID NO: 37, which encodes the BCMA VH amino acid sequence represented by SEQ ID NO: 13, and a nucleotide sequence represented by SEQ ID NO: 38, which encodes the BCMA VL amino acid sequence represented by SEQ ID NO: 14, and / or 2) It includes a nucleotide sequence represented by SEQ ID NO: 39 that encodes the CD19 VH amino acid sequence represented by SEQ ID NO: 17, and a nucleotide sequence represented by SEQ ID NO: 40 that encodes the CD19 VL amino acid sequence represented by SEQ ID NO: 18.

[0092] In another aspect, the application also provides vectors comprising the isolated nucleic acid molecules described above. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, and animal viruses. Examples of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0093] In some examples, the vector is an expression vector, optionally a viral vector, and further optionally a lentiviral vector.

[0094] In another embodiment, the application also provides engineered immune effector cells comprising the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0095] In some examples, the manipulated immune effector cells are selected from one or more of the following: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0096] In some cases, the manipulated immune effector cells are T lymphocytes, and optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

[0097] In some cases, the chimeric antigen receptor described in this application may or may be expressed on the surface of engineered immune effector cells.

[0098] In another embodiment, the application also provides a pharmaceutical composition comprising the above-described manipulated immunoeffector cells and a pharmaceutically acceptable adjuvant, the pharmaceutically acceptable adjuvant comprising one or more of a carrier, a protective agent, a stabilizer, and a diluent.

[0099] In some cases, the pharmaceutically acceptable adjuvant includes a protective agent such as a cell cryopreservation solution.

[0100] In some examples, the pharmaceutical composition is a cell suspension or cryopreserved cells thereof.

[0101] In some cases, the pharmaceutical composition is administered by intravenous injection.

[0102] Preparation method In another aspect, the application also provides a method for preparing engineered immunoeffector cells, comprising the step of transducing the vector described in the application into immunoeffector cells.

[0103] In some examples, the manipulated immune effector cells are selected from one or more of the following: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0104] In some cases, the manipulated immune effector cells are T lymphocytes, and optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

[0105] use In another aspect, the application also provides the use of the bispecific chimeric antigen receptor, isolated nucleic acid molecule, vector and / or engineered immunoeffector cell described herein in the preparation of a pharmaceutical for use in the treatment of a disease or condition associated with BCMA expression.

[0106] In some cases, the disease or condition associated with the expression of BCMA is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or recurrent multiple myeloma.

[0107] In some cases, the disease or condition associated with the expression of BCMA may be an autoimmune disease.

[0108] In some cases, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0109] In another aspect, the present application also provides a method for treating a disease or condition associated with the expression of BCMA, comprising administering to a subject in need of treatment for a disease or condition associated with the expression of BCMA an effective amount of the chimeric antigen receptor, isolated nucleic acid molecule, vector and / or engineered immune effector cell described in the present application.

[0110] In some examples, the disease or condition associated with the expression of BCMA is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or relapsed multiple myeloma.

[0111] In some examples, the disease or condition associated with the expression of BCMA can be an autoimmune disease.

[0112] In some examples, the autoimmune disease can be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis and autoimmune hemolytic anemia.

[0113] In some examples, the administration can be carried out by different methods such as intravenous administration, intratumoral administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, topical administration or intradermal administration. For example, the administration method can be administration to the subject by intravenous injection. In some examples, an effective amount of the engineered immune effector cells or pharmaceutical composition can be administered to the subject once or divided within a certain period, for example, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0114] In some examples, the dosage can be varied according to different indications, and the dosage can also be varied according to the severity of the patient's condition. The dosage can be 1×10 5 CAR positive T cells / kg to 1×10 7 CAR positive T cells / kg, for example, 1×10 5 CAR positive T cells / kg to 1×10 6 CAR positive T cells / kg, 1×10 6CAR positive T cells / kg~1×10 7 CAR positive T cells / kg, 0.5×10 6 CAR positive T cells / kg, 0.6×10 6 CAR positive T cells / kg, 0.7×10 6 CAR positive T cells / kg, 0.8×10 6 CAR positive T cells / kg, 0.9×10 6 CAR positive T cells / kg, 1.0×10 6 CAR positive T cells / kg, 1.1×10 6 CAR positive T cells / kg, 1.2×10 6 CAR positive T cells / kg, 1.3×10 6 CAR positive T cells / kg, 1.4×10 6 CAR positive T cells / kg, 1.5×10 6 CAR positive T cells / kg, 1.6×10 6 CAR positive T cells / kg, 1.7×10 6 CAR positive T cells / kg, 1.8×10 6 CAR positive T cells / kg, 1.9×10 6 CAR positive T cells / kg, 2.0×10 6 CAR-positive T cells / kg is also acceptable.

[0115] In some cases, subjects may include humans and non-human animals. For example, subjects may include, but are not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

[0116] In another aspect, the application also provides chimeric antigen receptors, isolated nucleic acid molecules, vectors, and / or engineered immune effector cells for use in treating diseases or conditions associated with BCMA expression.

[0117] In some cases, the disease or condition associated with the expression of BCMA may include non-solid tumors, and optionally, the non-solid tumor is a hematological malignancy.

[0118] In some cases, the disease or condition associated with the expression of BCMA may include multiple myeloma.

[0119] In some cases, the multiple myeloma is relapsed or refractory multiple myeloma.

[0120] While we do not wish to be bound by any theory, the following examples are provided solely to illustrate the chimeric antigen receptor, engineered immune effector cells, preparation methods, and uses of this application, and are not intended to limit their scope. The examples do not include detailed descriptions of conventional methods such as methods for constructing vectors and plasmids, methods for inserting protein-coding genes into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous publications, including Sambrook, J., Fritsch, EF and Maniais, T (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press. [Examples]

[0121] Example 1: Acquisition of BCMA-CD19 bispecific CAR-T cells To obtain a superior bispecific CAR structure, a BCMA-specific humanized antibody (the amino acid sequence of its VH is represented by SEQ ID NO: 13, the nucleotide sequence of its VH is represented by SEQ ID NO: 37, the amino acid sequence of its VL is represented by SEQ ID NO: 14, the nucleotide sequence of its VL is represented by SEQ ID NO: 38, the amino acid sequences of its VH CDR1, CDR2, and CDR3 are represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of its VL CDR1, CDR2, and CDR3 are represented by SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) and a CD19-specific antibody (the amino acid sequence of its VH is represented by SEQ ID NO: 17, the nucleotide sequence of its VH is represented by SEQ ID NO: 39, the amino acid sequence of its VL is represented by SEQ ID NO: 18, the nucleotide sequence of its VL is represented by SEQ ID NO: 40, the amino acid sequences of its VH CDR1, CDR2, and CDR3 are represented by SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, and its VL The amino acid sequences of CDR1, CDR2, and CDR3 (represented by SEQ ID NOs. 10, 11, and 12, respectively) were obtained. Considering the order of the target BCMA and CD19, as well as the changes in the order of VH and VL in the extracellular antigen recognition domains of each target, there are multiple options for designing the structure of a BCMA-CD19 bispecific CAR. Screening of candidate BCMA-CD19 bispecific CAR structures on second-generation CAR structures was selected.

[0122] Six BCMA-CD19 bispecific CAR structures, as shown in Figure 1, were tested, with BCMA CAR structures and CD19 CAR structures used as controls. In each CAR structure, the following structures were used in this example: the CD8α guide chain (represented by SEQ ID NO: 25) was used as the signal peptide, the CD8α structure (represented by SEQ ID NO: 21) was used as the hinge region and (represented by SEQ ID NO: 22) as the transmembrane region, 4-1BB (represented by SEQ ID NO: 24) was used as the intracellular costimulatory signal, and CD3ζ (represented by SEQ ID NO: 23) was used as the T cell activation signal.

[0123] 1. Construction of a lentiviral vector Six BCMA-CD19 bispecific CAR structures, as well as BCMA CAR and CD19 CAR structures as controls, were artificially synthesized as shown in Figure 1. The amino acid sequences of the Tan CD19-BCMA structure, Tan BCMA-CD19 structure, Loop CD19-BCMA structure, Loop BCMA-CD19 structure, CD19-2A-BCMA structure, BCMA-2A-CD19 structure, CD19 CAR structure, and BCMA CAR structure are represented by SEQ ID NOs. 26, 27, 28, 29, 30, 31, 32, and 33, respectively. The following description is only a brief summary of the main distinguishing regions in the CAR structures and does not fully show the signal peptide, linker sequence, hinge region, transmembrane region, intracellular costimulatory signaling region, and T cell activation signaling region. The order of each structure is shown in Figure 1. SP represents the signal peptide region, Hinge represents the hinge region, TM represents the transmembrane region, and the linker sequence is located between VL and VH.

[0124] 1)Tan CD19-BCMA structure: CD19 VL-CD19 VH-BCMA VL-BCMA VH 2)Tan BCMA-CD19 structure: BCMA VL-BCMA VH-CD19 VL-CD19 VH 3)Loop CD19-BCMA structure: BCMA VL-CD19 VL-CD19 VH-BCMA VH 4)Loop BCMA-CD19 structure: CD19 VL-BCMA VL-BCMA VH-CD19 VH 5) CD19-2A-BCMA structure: CD19 CAR-T2A-BCMA CAR (Complete CD19 CAR structure and BCMA CAR structure were observed before and after the self-cleaving polypeptide T2A in this structure. The complete CAR structure refers to the same signal peptide, hinge region, transmembrane region, intracellular costimulatory signaling region, and T cell activation signaling region as the other groups.) 6) BCMA-2A-CD19 structure: BCMA CAR-T2A-CD19 CAR (Complete BCMA CAR structure and CD19 CAR structure were observed before and after the self-cleaving polypeptide T2A, respectively. The complete CAR structure refers to the same signal peptide, hinge region, transmembrane region, intracellular costimulatory signaling region, and T cell activation signaling region as the other groups.) 7)CD19CAR structure:CD19 VL-CD19 VH 8)BCMA CAR structure: BCMA VL-CD19 VH

[0125] The six BCMA-CD19 bispecific CAR structures, one CD19 CAR structure, and one BCMA CAR structure described above were each constructed in modified empty lentiviral vectors (manufacturer: SBI Corporation, catalog numbers: CD500-CD800, conventional resistance modifications were performed as described in Example 1 of International Publication No. 2021 / 121227) to obtain CAR expression vectors. The CAR expression vectors and three packaging plasmids were then transfected into 293T cells, and after recovery and purification, functional lentiviral vectors were obtained. The three packaging plasmids were PMD2.0G (purchased from Biovector Corporation, catalog number: Biovector 012259), pMDLg / pRRE (purchased from Biovector Corporation, catalog number: Biovector 012251), and pRSV-Rev (purchased from Biovector Corporation, catalog number: Biovector 012253).

[0126] 2. Preparation of six corresponding BCMA-CD19 bispecific CAR-T cells, one CD19 CAR-T cell, and one BCMA CAR-T cell by lentiviral transduction. The transduction experiment was carried out according to conventional methods known to those skilled in the art. The transduction process is briefly described below.

[0127] 1) Selection of T cells Peripheral blood mononuclear cells (PBMCs) were isolated from the target apheresis cells, and then T cells were selected from the PBMC cells.

[0128] 2) Activation of T cells The isolated T cells were resuspended in complete lymphocyte culture medium (X-VIVO15 medium + 5% FBS + 300 IU / ml IL-2 or X-VIVO15 medium + 5% FBS + 5 ng / ml IL-15 + 10 ng / ml IL-7) and the final concentration (1-2) × 10⁻¹⁰ 6 The cells were divided into cells / ml, and 5-10 μL of CD3 / CD28 stimulating magnetic beads were added. The mixture was thoroughly mixed and incubated in an incubator at 37°C under 5% CO2 culture conditions for at least 24 hours.

[0129] 3) Transduction of T cells by lentivirus Activated cultured T cells were removed, and polyblen at a final concentration of 8 μg / ml was added and thoroughly mixed. The lentiviral vector was slowly added at an MOI of 2. After thorough mixing, the mixture was placed in a centrifuge and centrifuged at 1500 rpm for 1.5 hours. Then, it was placed in an incubator and cultured at 37°C under 5% CO2 conditions for at least 24 hours.

[0130] 4) Proliferation and culture of transduced T cells The transduced cells are removed and the cell density is set to (0.5~1) × 10⁻¹⁶ for use in subsequent examples. 6 The cell count was monitored to maintain a constant level of cells / ml. T cells were infected with lentiviruses containing Tan CD19-BCMA structure, Tan BCMA-CD19 structure, Loop CD19-BCMA structure, Loop BCMA-CD19 structure, CD19-2A-BCMA structure, BCMA-2A-CD19 structure, CD19 CAR, and BCMA CAR. The resulting T cells were named Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, CD19-2A-BCMA cells, BCMA-2A-CD19 cells, CD19 cells, and BCMA cells, respectively. Next, the six bispecific CAR structures were screened at the cellular level to determine the characteristics of each CAR structure, and superior bispecific CAR structures were selected.

[0131] Example 2: Detection of CAR molecules expressed on the surface of BCMA-CD19 bispecific CAR-T cells CAR protein molecules expressed on the surface of the six bispecific CAR-T cells obtained in Example 1 were detected. The six BCMA-CD19 bispecific CAR-T cells and UTD cells (T cells without CAR transduction) obtained in Example 1 were stained with PE fluorescently labeled CD19 antigen (manufacturer: ACRO Biosystems, catalog number: CD9-HP2H3) and FITC fluorescently labeled BCMA antigen (manufacturer: ACRO Biosystems, catalog number: BCA-HF254), and the percentage of CAR molecules positive was detected and analyzed by flow cytometry. First, as shown in Figure 2A, it was found that in CD19-2A-BCMA cells and BCMA-2A-CD19 cells, the CAR molecule linked to the T2A was not sufficiently expressed 6 days after T cell infection, and therefore, the two CAR structures in CD19-2A-BCMA cells and BCMA-2A-CD19 cells were initially eliminated. As shown in Figures 2B, 2C, 2D, and 2E, 6 days after T cell infection, the CAR expression rates of the other four bispecific CAR-T cells—Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, and Loop BCMA-CD19 cells—were 42.98%, 66.71%, 53.46%, and 61.63%, respectively (based on results after BCMA antigen staining, the reasoning of which is described in detail in Example 3).

[0132] Example 3: Detection of CAR molecules on the surface of BCMA-CD19 bispecific CAR-T cells The inventors selected a staining method by attempting single staining (single staining means staining bispecific CAR-T cells with only FITC-fluorescently labeled BCMA antigen or PE-fluorescently labeled CD19 antigen) and co-staining (co-staining means staining bispecific CAR-T cells with both FITC-fluorescently labeled BCMA antigen and PE-fluorescently labeled CD19 antigen) on bispecific CAR-T cells obtained in Example 1 11 days after T cell infection. Since the extracellular components of BCMA and CD19 are constructed on a single scFv and are always co-expressed, theoretically, the difference in results should be within the margin of error whether bispecific CAR-T cells are stained by single staining or co-staining. However, as shown in Table 1 (especially the grayscale portion), it was found that the selection of different antigens for staining significantly affected the calculation of CAR+ cells from bispecific CAR-T cells.

[0133] [Table 1]

[0134] Taking the characteristics of the proportion of CAR+ cells in Tan BCMA-CD19 cells as an example, when BCMA antigen was used for single staining and BCMA antigen + CD19 antigen was used for co-staining, the proportion of CAR+ cells detected via BCMA antigen was all within the margin of error. However, when CD19 antigen was used for single staining and BCMA antigen + CD19 antigen was used for co-staining, the values ​​detected via CD19 antigen varied greatly beyond the margin of error, and the proportion of CAR+ cells detected via CD19 antigen was significantly lower than the proportion of CAR+ cells detected via BCMA antigen. On the other hand, when CD19 antigen was used for detection, a significant difference was also observed between the proportion of CAR+ cells detected by single staining and the proportion of CAR+ cells detected by co-staining. The above observations were not limited by the relative positions of the BCMA extracellular antigen recognition domain and the CD19 extracellular antigen recognition domain in the CAR structure. Compared to the CD19 antigen, the BCMA antigen is smaller, and it is thought that it can more easily overcome the steric hindrance problem in the bispecific CAR structure that binds to the CAR protein. Therefore, in subsequent experiments of this application, the positive rate of BCMA-CD19 bispecific CARs was characterized using data from detection via BCMA antigen. Of course, data from detection via BCMA antigen in co-staining methods could also characterize the positive rate of BCMA-CD19 bispecific CARs, but the single-staining method was simpler.

[0135] Example 4: Cytokine release experiment in BCMA-CD19 bispecific CAR-T cells Cytokine release experiment: The four bispecific CAR-T cells obtained in Example 1 (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, CD19 CAR-T cells, and UTD cells were co-cultured with target cells in X-VIVO15 medium at a 1:1 effector-target ratio for 24 hours. The concentrations of IL-2, IFN-γ, and TNF-α in the cell supernatant were then detected by ELISA. K562 is a binegative target cell for BCMA and CD19, K562-BCMA is a positive target cell that exogenously expresses BCMA but does not express CD19, and K562-CD19 is a positive target cell that exogenously expresses CD19 but does not express BCMA. K562-BCMA cells did not express the CD19 antigen and were therefore not used for the detection of CD19 CAR-T cells. Similarly, K562-CD19 cells did not express the BCMA antigen and were therefore not used to detect BCMA CAR-T cells (in Figures 3A, 3B, and 3C, the K562-BCMA group and the K562-CD19 group each have one bar missing).

[0136] The experimental results for the release of cytokines IL-2, IFN-γ, and TNF-α are shown in Figures 3A, 3B, and 3C, respectively. The arrows in Figures 3A, 3B, and 3C indicate CAR-T cells with low cytokine release levels. As can be seen from Figures 3A, 3B, and 3C, the levels of various cytokines released by bispecific CARs in the Loop CD19-BCMA and Loop BCMA-CD19 structures were generally higher compared to the Tan CD19-BCMA and Tan BCMA-CD19 structures. In particular, as can be seen from the cytokine release levels in the Tan CD19-BCMA and Tan BCMA-CD19 structures, when scFvs far from the cell membrane in the Tan CD19-BCMA and Tan BCMA-CD19 structures were stimulated by the antigen, the levels of cytokines released by CAR-T cells were relatively low.

[0137] Example 5: Cell death experiment in BCMA-CD19 bispecific CAR-T cells Cell death experiment: The four bispecific CAR-T cells obtained in Example 1 (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, CD19 CAR-T cells, and UTD cells were co-cultured with target cells in X-VIVO15 medium at different effector-target ratios (0:1, 1:1, 3:1, or 9:1) for 4 hours each. Subsequently, the rate of target cell death was detected by detecting the activity of luciferase stably expressed in the target cells. NALM6 (human acute lymphoblastic leukemia cells) were CD19+BCMA - The target cells, MM.1S (human multiple myeloma cells), are BCMA+CD19 - Target cells, NALM6 - KO CD19 was a negative target cell control in which CD19 was knocked out and BCMA was not expressed. The results of cell death are shown in Figures 4A to 4C. As shown in Figure 4A, Tan CD19-BCMA cells, TanBCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and CD19 CAR-T cells all showed good killing effects against NALM6, a positive target cell that endogenously expresses CD19, while BCMA CAR-T cells and UTD cells did not show killing effects against NALM6, a positive target cell that endogenously expresses CD19. As shown in Figure 4B, Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and BCMA CAR-T cells all exhibited good killing effects against BCMA-endogenously expressing positive target cells MM.1S, while CD19 CAR-T cells and UTD cells did not exhibit killing effects against BCMA-endogenously expressing positive target cells MM.1S. As shown in Figure 4C, all cells did not exhibit killing effects against negative target cell controls in which CD19 was knocked out and BCMA was not expressed.

[0138] Example 6: Sustained proliferation of BCMA-CD19 bispecific CAR-T cells Antigen stimulation can activate CAR-T cells and induce CAR-T cell proliferation, but sustained T cell activation leads to cell depletion. Depleted T cells have reduced proliferative capacity and effector function. Sustained proliferation of BCMA-CD19 bispecific CAR-T cells was verified by detecting CD3+ cell proliferation (i.e., T cell proliferation), CAR+ cell proliferation, and the proportion of CAR+ cells after multiple antigen stimulation experiments.

[0139] Prior to antigen stimulation, the CAR positivity rates of all four bispecific CAR-T cell groups (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, and CD19 CAR-T cells obtained in Example 1 were adjusted using UTD to a level consistent with the proportion of the CAR-T cell group with the lowest CAR positivity rate. The CAR positivity rates of Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and BCMA CAR-T cells were based on BCMA antigen detection data, and the CAR positivity rate of CD19 CAR-T cells was based on CD19 antigen detection data. In multiple antigen stimulation experiments, each group of CAR-T cells was co-cultured with positive target cells in 24-well plates at a 1:2 effector-target ratio using 2 ml of X-VIVO15 medium per well, with 3 wells repeated for each cell group. The positive target cells used were MM.1S and NALM6, respectively, which were equivalent to BCMA and CD19 as antigens for multiple stimulation to test the effect of multiple stimulations with different antigens on the sustained proliferation of BCMA-CD19 bispecific CAR-T cells. MM.1S cells did not express the CD19 antigen and were therefore not used to detect CD19 CAR-T cells. Similarly, NALM6 cells did not express the BCMA antigen and were therefore not used to detect BCMA CAR-T cells.

[0140] After co-culturing CAR-T cells with positive target cells for 3 days, 500 μL of cells were removed and stained with fluorescently labeled CD3 antibody (manufacturer: BioLegend, catalog number: 300312) and BCMA antigen and CD19 antigen (same as in Example 2) (Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and BCMA CAR-T cells were stained with BCMA antigen, and CD19 CAR-T cells were stained with CD19 antigen). Detection and analysis were performed by flow cytometry to show the percentage and number of CAR-positive cells in CD3-positive cells. The number of CAR-positive cells in CD3-positive cells can also be calculated based on volume multiplier conversion (CD3 is a marker that distinguishes whether a cell is a T cell or not). Then, according to the calculation results, a certain amount of CAR-T cells were removed from each group and added to the corresponding positive target cells in an effector-target ratio of 1:2 for new stimulation, and this was repeated for 3-4 stimulations.

[0141] After multiple antigen stimulation experiments, the results of CD3+ cell proliferation (i.e., T cell proliferation) stimulated with MM.1S and NALM6 cells are shown in Figures 5A and 5B, respectively, and the results of CAR+ cell proliferation stimulated with MM.1S and NALM6 cells are shown in Figures 5C and 5D, respectively. As shown in Figures 5A and 5C, after multiple antigen stimulation experiments, the number of CD3+ cells (T cell proliferation) and CAR+ cells in Tan BCMA-CD19 cells stimulated with MM.1S cells were significantly lower than those in other CAR-T cell groups. As shown in Figures 5B and 5D, after multiple antigen stimulation experiments, the number of CD3+ cells and CAR+ cells in Tan CD19-BCMA cells stimulated with NALM6 cells were significantly lower than those in other CAR-T cell groups. Comparing Figure 5A with Figure 5B and Figure 5C with Figure 5D, it can be seen that the proliferative capacity of CAR-T cells stimulated with MM.1S cells was stronger than that of CAR-T cells stimulated with NALM6 cells. From the above results, it was found that when scFv far from the cell membrane of Tan-structured BCMA-CD19 bispecific CAR-T cells was stimulated with the antigen, the sustained proliferative capacity of CAR-T cells was relatively poor. However, the sustained proliferative capacity of Loop-structured CAR-T cells was generally stronger than that of Tan-structured CAR-T cells. Cells exhibited sustained proliferative capacity when stimulated with both BCMA and CD19 antigens, and the proliferative capacity of CAR-T cells stimulated with the BCMA antigen was stronger.

[0142] In summary, in vitro pharmacodynamic studies demonstrated that the Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, and Loop BCMA-CD19 structures exhibited superior performance in terms of T cell surface expression, cytokine release, and in vitro cell death. Regarding the sustained proliferation of CAR-T cells, the Loop CD19-BCMA and Loop BCMA-CD19 structures were significantly superior to the Tan CD19-BCMA and Tan BCMA-CD19 structures, demonstrating better function. Furthermore, the Loop CD19-BCMA and Loop BCMA-CD19 structures functioned similarly to BCMA CAR-T and CD19 CAR-T in various experiments and tests, exhibiting complete functionality against dual targets.

[0143] Array description Sequence ID 1: BCMA VH CDR1 Sequence ID 2: BCMA VH CDR2 Sequence ID 3: BCMA VH CDR3 Sequence ID 4: BCMA VL CDR1 Sequence ID 5: BCMA VL CDR2 (ETS, Glu-Thr Ser) Sequence ID 6: BCMA VL CDR3 Sequence ID 7: CD19 VH CDR1 Sequence ID 8: CD19 VH CDR2 Sequence ID 9: CD19 VH CDR3 Sequence ID 10: CD19 VL CDR1 Sequence ID 11: CD19 VL CDR2(SAT, Ser Ala Thr) Sequence ID 12: CD19 VL CDR3 Sequence ID 13: BCMA VH sequence (humanized) Sequence ID 14: BCMA VL sequence (humanized) Sequence ID 15: BCMA VH sequence (rabbit origin) Sequence ID 16: BCMA VL sequence (rabbit origin) Sequence ID 17: CD19 VH sequence Sequence ID 18: CD19 VL sequence SEQ ID NO: 19: Amino acid sequence of scFv in the Loop CD19-BCMA structure SEQ ID NO: 20: Amino acid sequence of scFv in the Loop BCMA-CD19 structure Sequence ID 21: Amino acid sequence of the hinge region Sequence ID 22: Amino acid sequence of the transmembrane region Sequence ID 23: Amino acid sequence of the intracellular signaling region SEQ ID NO: 24: Amino acid sequence of the co-stimulatory signaling region Sequence ID 25: Amino acid sequence of the guide peptide Sequence ID 26: Amino acid sequence of the Tan CD19-BCMA structure Sequence ID 27: Amino acid sequence of the Tan BCMA-CD19 structure Sequence ID 28: Amino acid sequence of the Loop CD19-BCMA structure Sequence ID 29: Amino acid sequence of the Loop BCMA-CD19 structure Sequence ID 30: Amino acid sequence of the CD19-2A-BCMA structure Sequence ID 31: Amino acid sequence of the BCMA-2A-CD19 structure Sequence ID 32: Amino acid sequence of the CD19 CAR structure Sequence ID 33: Amino acid sequence of the BCMA CAR structure Sequence ID 34: Linker Sequence Sequence ID 35: Linker Sequence Sequence ID 36: Linker Sequence Sequence ID 37: Nucleotide sequence encoding the BCMA VH amino acid sequence represented by Sequence ID 13 Sequence ID 38: Nucleotide sequence encoding the BCMA VL amino acid sequence represented by Sequence ID 14 Sequence ID 39: Nucleotide sequence encoding the CD19 VH amino acid sequence represented by Sequence ID 17 Sequence ID 40: Nucleotide sequence encoding the CD19 VL amino acid sequence represented by Sequence ID 18

Claims

1. It includes an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. The extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain. The anti-BCMA extracellular antigen recognition domain comprises BCMA VH and BCMA VL, The amino acid sequences of the BCMA VH complementarity-determining regions CDR1, CDR2, and CDR3 each include the amino acid sequences represented by GFSLSTYH (SEQ ID NO: 1), ISSSGST (SEQ ID NO: 2), and ARDLDYVIDL (SEQ ID NO: 3), respectively. The amino acid sequences of the BCMA VL complementarity-determining regions CDR1, CDR2, and CDR3 each include the amino acid sequences represented by PSVYNNY (SEQ ID NO: 4), ETS, and AGTYVSGDRRA (SEQ ID NO: 6), A bispecific chimeric antigen receptor that targets BCMA-CD19.

2. The anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, The amino acid sequences of the CD19 VH complementarity-determining regions CDR1, CDR2, and CDR3 each include the amino acid sequences represented by GYAFSSYW (SEQ ID NO: 7), IYPGDGDT (SEQ ID NO: 8), and ARKTISSVVDFYFDY (SEQ ID NO: 9), respectively. The bispecific chimeric antigen receptor according to claim 1, wherein the amino acid sequences of the CD19 VL complementarity-determining regions CDR1, CDR2, and CDR3 each include the amino acid sequences represented by QNVGTN (SEQ ID NO: 10), SAT, and QQYNRYPYT (SEQ ID NO: 12).

3. The BCMA VH sequence includes the amino acid sequence represented by SEQ ID NO: 13, and the BCMA VL sequence includes the amino acid sequence represented by SEQ ID NO: 14, or The bispecific chimeric antigen receptor according to claim 1 or 2, wherein the BCMA VH sequence comprises the amino acid sequence represented by SEQ ID NO: 15, and the BCMA VL sequence comprises the amino acid sequence represented by SEQ ID NO:

16.

4. The bispecific chimeric antigen receptor according to claim 2, wherein the CD19 VH sequence comprises the amino acid sequence represented by SEQ ID NO: 17, and the CD19 VL sequence comprises the amino acid sequence represented by SEQ ID NO:

18.

5. The bispecific chimeric antigen receptor according to claim 1, wherein the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one structure selected from the group consisting of CD19 VL sequence - 1st linker sequence - CD19 VH sequence - 2nd linker sequence - BCMA VL sequence - 3rd linker sequence - BCMA VH sequence, BCMA VL sequence - 4th linker sequence - BCMA VH sequence - 5th linker sequence - CD19 VL sequence - 6th linker sequence - CD19 VH sequence, BCMA VL sequence - 7th linker sequence - CD19 VL sequence - 8th linker sequence - CD19 VH sequence - 9th linker sequence - BCMA VH sequence, and CD19 VL sequence - 10th linker sequence - BCMA VL sequence - 11th linker sequence - BCMA VH sequence - 12th linker sequence - CD19 VH sequence.

6. The bispecific chimeric antigen receptor according to claim 5, wherein the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises one structure selected from the group consisting of BCMA VL sequence - seventh linker sequence - CD19 VL sequence - eighth linker sequence - CD19 VH sequence - ninth linker sequence - BCMA VH sequence and CD19 VL sequence - tenth linker sequence - BCMA VL sequence - eleventh linker sequence - BCMA VH sequence - twelfth linker sequence - CD19 VH sequence.

7. The bispecific chimeric antigen receptor according to claim 6, wherein the first linker sequence, the second linker sequence, the third linker sequence, the fourth linker sequence, the fifth linker sequence, the sixth linker sequence, the seventh linker sequence, the eighth linker sequence, the ninth linker sequence, the tenth linker sequence, the eleventh linker sequence, and the twelfth linker sequence are each independently selected from one or more of SEQ ID NOs: 34, SEQ ID NOs: 35, and SEQ ID NOs:

36.

8. The bispecific chimeric antigen receptor according to claim 5, wherein the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises an amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO:

20.

9. The hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α, and / or The bispecific chimeric antigen receptor according to claim 1, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

10. The bispecific chimeric antigen receptor according to claim 9, wherein the amino acids of the hinge region are derived from CD8α, and / or the amino acids of the transmembrane region are derived from CD8α.

11. The amino acid sequence of the hinge region includes the amino acid sequence represented by SEQ ID NO: 21, and / or The bispecific chimeric antigen receptor according to claim 10, wherein the amino acid sequence of the transmembrane region includes the amino acid sequence represented by SEQ ID NO:

22.

12. The bispecific chimeric antigen receptor according to claim 1, wherein the intracellular domain includes an intracellular signaling region.

13. The bispecific chimeric antigen receptor according to claim 12, wherein the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

14. The bispecific chimeric antigen receptor according to claim 13, wherein the intracellular signaling region is derived from CD3ζ.

15. The bispecific chimeric antigen receptor according to claim 14, wherein the amino acid sequence of the intracellular signaling region includes the amino acid sequence represented by SEQ ID NO:

23.

16. The bispecific chimeric antigen receptor according to claim 1, wherein the intracellular domain further comprises a co-stimulatory signaling region.

17. The bispecific chimeric antigen receptor according to claim 16, wherein the costimulatory signaling region is derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

18. The bispecific chimeric antigen receptor according to claim 17, wherein the costimulatory signaling region is derived from CD28 or 4-1BB.

19. The bispecific chimeric antigen receptor according to claim 18, wherein the amino acid sequence of the costimulatory signaling region includes the amino acid sequence represented by SEQ ID NO:

24.

20. The bispecific chimeric antigen receptor according to claim 1, further comprising a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor.

21. The bispecific chimeric antigen receptor according to claim 20, wherein the guide peptide is derived from CD8α.

22. The bispecific chimeric antigen receptor according to claim 21, wherein the amino acid sequence of the guide peptide includes the amino acid sequence represented by SEQ ID NO:

25.

23. A bispecific chimeric antigen receptor according to claim 1, comprising the amino acid sequence represented by SEQ ID NO: 28 or SEQ ID NO:

29.

24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific chimeric antigen receptor as described in claim 1.

25. The nucleotide sequence encoding the bispecific chimeric antigen receptor is 1) A nucleotide sequence represented by SEQ ID NO: 37, which encodes the BCMA VH amino acid sequence represented by SEQ ID NO: 13, and a nucleotide sequence represented by SEQ ID NO: 38, which encodes the BCMA VL amino acid sequence represented by SEQ ID NO: 14, and / or 2) The isolated nucleic acid molecule according to claim 24, comprising a nucleotide sequence represented by SEQ ID NO: 39, which encodes the CD19 VH amino acid sequence represented by SEQ ID NO: 17, and a nucleotide sequence represented by SEQ ID NO: 40, which encodes the CD19 VL amino acid sequence represented by SEQ ID NO:

18.

26. A vector comprising an isolated nucleic acid molecule as described in claim 24.

27. The vector according to claim 26, wherein the vector is an expression vector.

28. The vector according to claim 27, wherein the vector is a viral vector.

29. The vector according to claim 28, wherein the vector is a lentiviral vector.

30. Engineered immune effector cells comprising a bispecific chimeric antigen receptor according to claim 1, an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor according to claim 1, or a vector comprising the isolated nucleic acid molecule.

31. The manipulated immune effector cells according to claim 30, wherein the manipulated immune effector cells are selected from one or more of the following: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-Ts), NK cells differentiated from induced pluripotent stem cells (iPSC-NKs), and embryonic stem cells.

32. The manipulated immune effector cell according to claim 31, wherein the manipulated immune effector cell is a T lymphocyte.

33. The manipulated immune effector cells according to claim 32, wherein the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

34. A pharmaceutical composition comprising the manipulated immunoeffector cells described in claim 30 and a pharmaceutically acceptable adjuvant.

35. The pharmaceutical composition according to claim 34, wherein the pharmaceutically acceptable adjuvant comprises a protective agent.

36. The pharmaceutical composition according to claim 35, wherein the pharmaceutically acceptable adjuvant comprises a cell cryopreservation solution.

37. The pharmaceutical composition according to claim 34, which is administered by intravenous injection.

38. The use of a bispecific chimeric antigen receptor according to claim 1, an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor according to claim 1, or a vector comprising the isolated nucleic acid molecule, or engineered immunoeffector cells in the preparation of a pharmaceutical for the treatment of a disease or condition related to BCMA expression; The manipulated immune effector cells include the bispecific chimeric antigen receptor described in claim 1, an isolated nucleic acid molecule containing a nucleotide sequence encoding the bispecific chimeric antigen receptor described in claim 1, or a vector containing the isolated nucleic acid molecule.

39. The use according to claim 38, wherein the disease or condition associated with the expression of BCMA is cancer.

40. The use according to claim 39, wherein the cancer is multiple myeloma.

41. The use according to claim 40, wherein the cancer is refractory or recurrent multiple myeloma.

42. The use according to claim 38, wherein the disease or condition associated with the expression of BCMA is an autoimmune disease.

43. The use according to claim 42, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

44. Engineered immune effector cells or pharmaceutical compositions comprising said engineered immune effector cells for use in the treatment of diseases or conditions related to BCMA expression; The manipulated immune effector cells include the bispecific chimeric antigen receptor described in claim 1, an isolated nucleic acid molecule containing a nucleotide sequence encoding the bispecific chimeric antigen receptor described in claim 1, or a vector containing the isolated nucleic acid molecule.

45. The manipulated immune effector cells or pharmaceutical composition according to claim 44, wherein the disease or condition associated with the expression of BCMA is cancer.

46. The manipulated immune effector cells or pharmaceutical composition according to claim 45, wherein the cancer is multiple myeloma.

47. The manipulated immune effector cells or pharmaceutical composition according to claim 46, wherein the cancer is refractory or recurrent multiple myeloma.

48. The manipulated immune effector cells or pharmaceutical composition according to claim 44, wherein the disease or condition associated with the expression of BCMA is an autoimmune disease.

49. The manipulated immune effector cells or pharmaceutical composition according to claim 48, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

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