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CHIMERIC ANTIGEN RECEPTOR CAR OR CAR CONSTRUCT TARGETING BCMA AND CD19 AND APPLICATION THEREOF

20230203178 · 2023-06-29

    Inventors

    Cpc classification

    International classification

    Abstract

    Provided are a chimeric antigen receptor (CAR) or CAR construct containing antibodies against BCMA and CD19, a nucleic acid molecule encoding the CAR or CAR construct, a modified immune cell, and a method for preparing the immune cell. The CAR or CAR construct and the modified immune cell are used for the prevention and/or treatment of B cell related conditions (e.g., B cell and plasma cell related malignant tumors or autoimmune diseases (such as systemic lupus erythematosus)), and can effectively avoid target escape and prevent the recurrence of multiple myeloma.

    Claims

    1. A bispecific antibody or antigen-binding fragment thereof targeting BCMA and CD19, wherein the bispecific antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof targeting BCMA and a second antibody or antigen-binding fragment thereof targeting CD19, said first antibody or antigen-binding fragment thereof targeting BCMA comprises a first heavy chain variable region (VH) and/or a first light chain variable region (VL), the first VH and/or the first VL form a BCMA-binding site, and said second antibody or antigen-binding fragment thereof targeting CD19 comprises a second heavy chain variable region (VH) and/or a second light chain variable region (VL), the second VH and/or the second VL form a CD19-binding site, wherein, the first VH comprises: a first VH CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a first VH CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a first VH CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; the first VL comprises: a first VL CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a first VL CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a first VL CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; preferably, the first VH comprises the sequence as set forth in SEQ ID NO: 1 or a variant thereof; the first VL comprises the sequence as set forth in SEQ ID NO: 2 or a variant thereof; wherein, the variant has a sequence identity of at least 70%, at least 80%, at least 85%, 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 100% as compared to the sequence from which it is derived, or has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

    2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the second VH of the second antibody or antigen-binding fragment thereof targeting CD19 comprises: a second VH CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a second VH CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a second VH CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; the second VL comprises: a second VL CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a second VL CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or 53 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a second VL CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; preferably, the second VH comprises the sequence as set forth in SEQ ID NO: 3 or 76 or a variant thereof; the second VL comprises the sequence as set forth in SEQ ID NO: 4 or 77 or a variant thereof; wherein, the variant has a sequence identity of at least 70%, at least 80%, at least 85%, 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 100% as compared to the sequence from which it is derived, or has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

    3. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the bispecific antibody or antigen-binding fragment thereof comprises the following domains in sequence from the N-terminal to the C-terminal: (1) the first VH, the first VL, the second VH, the second VL; (2) the second VH, the second VL, the first VH, the first VL; (3) the first VL, the first VH, the second VL, the second VH; (4) the second VL, the second VH, the first VL, the first VH; (5) the first VH, the first VL, the second VL, the second VH; (6) the second VH, the second VL, the first VL, the first VH; (7) the first VL, the first VH, the second VH, the second VL; (8) the second VL, the second VH, the first VH, the first VL; (9) the first VL, the second VL, the second VH, the first VH; (10) the second VL, the first VL, the first VH, the second VH; (11) the first VH, the second VL, the second VH, the first VL; (12) the second VH, the first VL, the first VH, the second VL; (13) the first VL, the second VH, the second VL, the first VH; (14) the second VL, the first VH, the first VL, the second VH; (15) the first VH, the second VH, the second VL, the first VL; or (16) the second VH, the first VH, the first VL, the second VL; in any one of items (1) to (16), any adjacent variable regions are independently connected by a linker; preferably, in any one of items (1) to (16), the linker between the adjacent variable regions can be the same or different; preferably, the first VH comprises the sequence as set forth in SEQ ID NO: 1 or a variant thereof; the first VL comprises the sequence as set forth in SEQ ID NO: 2 or a variant thereof; wherein the variant has a sequence identity of at least 70%, at least 80%, at least 85%, 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 100% as compared to the sequence from which it is derived, or has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence from which it is derived; preferably, the second VH comprises the sequence as set forth in SEQ ID NO: 3 or 76 or a variant thereof; the second VL comprises the sequence as set forth in SEQ ID NO: 4 or 77 or a variant thereof; wherein, the variant has a sequence identity of at least 70%, at least 80%, at least 85%, 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 100% as compared to the sequence from which it is derived, or has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared to the sequence from which it is derived.

    4. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the first antibody or antigen-binding fragment thereof targeting BCMA or the second antibody or antigen-binding fragment thereof targeting CD19 is each independently selected from the group consisting of camelid Ig, IgNAR, Fab fragment, Fab′ fragment, F(ab′).sub.2 fragment, F(ab′).sub.3 fragment, Fv, single chain antibody (e.g., scFv, di-scFv, (scFv).sub.2), minibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein (“dsFv”) and single domain antibody (sdAb, nanobody), chimeric antibody, humanized antibody, single domain antibody, bispecific antibody or multi specific antibody; preferably, the first antibody or antigen-binding fragment thereof targeting BCMA or the second antibody or antigen-binding fragment thereof targeting CD19 is an scFv; more preferably, the BCMA-targeting scFv comprises a first VL as set forth in SEQ ID NO: 2, a linker as set forth in SEQ ID NO: 17, 18, 19, 20 or 68, a first VH as set forth in SEQ ID NO: 1; the CD19-targeting scFv comprises a second VL as set forth in SEQ ID NO: 4 or 77, a linker as set forth in SEQ ID NO: 17, 18, 19, 20 or 68, and a second VH as set forth in SEQ ID NO: 3 or 76; more preferably, the BCMA-targeting scFv has a sequence as set forth in SEQ ID NO: 25 or 27, and the CD19-targeting scFv has a sequence as set forth in SEQ ID NO: 26 or 28.

    5. The bispecific antibody or antigen-binding fragment thereof according to claim 1, which further comprises a heavy chain constant region (CH) and a light chain constant region (CL); preferably, the heavy chain constant region is selected from the group consisting of IgG, IgM, IgE, IgD and IgA; preferably, the light chain constant region is selected from κ or λ.

    6. A chimeric antigen receptor (CAR) targeting BCMA and CD19, which comprises an antigen-binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen-binding domain comprises the bispecific antibody or antigen-binding fragment thereof according to claim 1.

    7. The chimeric antigen receptor (CAR) according to claim 6, wherein the chimeric antigen receptor (CAR) comprises the following domains in sequence from the N-terminal to the C-terminal: (1) the first VH, the first VL, the second VH, the second VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (2) the second VH, the second VL, the first VH, the first VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (3) the first VL, the first VH, the second VL, the second VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (4) the second VL, the second VH, the first VL, the first VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (5) the first VH, the first VL, the second VL, the second VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (6) the second VH, the second VL, the first VL, the first VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (7) the first VL, the first VH, the second VH, the second VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (8) the second VL, the second VH, the first VH, the first VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (9) the first VL, the second VL, the second VH, the first VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (10) the second VL, the first VL, the first VH, the second VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (11) the first VH, the second VL, the second VH, the first VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (12) the second VH, the first VL, the first VH, the second VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (13) the first VL, the second VH, the second VL, the first VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (14) the second VL, the first VH, the first VL, the second VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; (15) the first VH, the second VH, the second VL, the first VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; or (16) the second VH, the first VH, the first VL, the second VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; optionally, in any one of items (1) to (16), any adjacent variable regions are independently connected by a linker; preferably, the linker between any adjacent variable regions is each independently selected from: a polypeptide having a sequence as shown in (GGGGS)x1 or (EAAAK)x2 (x1 and x2 are independently selected from integers from 1 to 6) or a polypeptide containing a sequence as set forth in SEQ ID NO: 68; preferably, in any one of items (1) to (16), the linker between the adjacent variable regions may be the same or different.

    8. The chimeric antigen receptor (CAR) according to claim 6, wherein the chimeric antigen receptor (CAR) comprises the following in sequence from the N-terminal to the C-terminal: an antigen-binding domain comprising the first VL, the first VH, the second VH and the second VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; or an antigen-binding domain comprising the second VH, the second VL, the first VL and the first VH, the spacer domain, the transmembrane domain, and the intracellular signaling domain; or an antigen-binding domain comprising the second VH, the first VL, the first VH and the second VL, the spacer domain, the transmembrane domain, and the intracellular signaling domain; wherein, any adjacent variable regions are independently connected by a linker; preferably, the linker between any adjacent variable regions is independently selected from a polypeptide with a sequence as set forth in SEQ ID NO: 17, 18, 19, 20 or 68.

    9. The chimeric antigen receptor (CAR) according to claim 6, wherein, the first VH comprises: a first VH CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a first VH CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a first VH CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; the first VL comprises: a first VL CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a first VL CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a first VL CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; the second VH comprises: a second VH CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a second VH CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a second VH CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and, the second VL comprises: a second VL CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a second VL CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or 53 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a second VL CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; preferably, the substitution is a conservative substitution.

    10. (canceled)

    11. The chimeric antigen receptor (CAR) according to claim 6, wherein the antigen-binding domain comprises the following in sequence from the N-terminal to the C-terminal: (1) the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 18, the first VH as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 19, the second VH as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 18 and the second VL as set forth in SEQ ID NO: 4; (2) the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 18, the first VH as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 20, the second VH as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 18 and the second VL as set forth in SEQ ID NO: 4; (3) the second VH as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 18, the second VL as set forth in SEQ ID NO: 4, the linker as set forth in SEQ ID NO: 19, the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 18 and the first VH as set forth in SEQ ID NO: 1; (4) the second VH as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 18, the second VL as set forth in SEQ ID NO: 4, the linker as set forth in SEQ ID NO: 20, the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 18 and the first VH as set forth in SEQ ID NO: 1; (5) the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 17, the first VH as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 19, the second VH as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 17 and the second VL as set forth in SEQ ID NO: 4; (6) the second VH as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 17, the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 19, the first VH as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 17 and the second VL as set forth in SEQ ID NO: 4; (7) the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 18, the first VH as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 19, the second VH as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO:68 and the second VL as set forth in SEQ ID NO:4; or (8) the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 18, the first VH as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 19, the second VH as set forth in SEQ ID NO: 76, the linker as set forth in SEQ ID NO:68 and the second VL as set forth in SEQ ID NO:77.

    12.-15. (canceled)

    16. The chimeric antigen receptor (CAR) according to claim 6, wherein the chimeric antigen receptor comprises the signal peptide, the antigen-binding domain, the spacer domain, the transmembrane domain, the intracellular signaling domain in sequence from the N-terminal to the C-terminal; preferably, the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (e.g., a sequence as set forth in SEQ ID NO: 49); preferably, the spacer domain comprises a hinge region of CD8 (e.g., CD8α) or IgG4 (e.g., a sequence as set forth in SEQ ID NO: 21 or 70); preferably, the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8α) or CD28 (e.g., a sequence as set forth in SEQ ID NO: 22 or 72); preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3ζ (e.g., a sequence as set forth in SEQ ID NO: 24 or 74), the costimulatory signaling domain comprises an intracellular signaling domain of CD137 (e.g., a sequence as set forth in SEQ ID NO: 23); preferably, the chimeric antigen receptor has an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs: 37-42, 64, 66; (2) a sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, 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 100% as compared to the sequence as set forth in any one of SEQ ID NO: 37-42, 64, 66.

    17. A chimeric antigen receptor (CAR) construct targeting BCMA and CD19, the CAR construct comprising independently a first CAR and a second CAR, wherein the first CAR comprises a first antibody or antigen-binding fragment thereof targeting BCMA, a spacer domain, a transmembrane domain and an intracellular signaling domain; the second CAR comprises a second antibody or antigen-binding fragment thereof targeting CD19, a spacer domain, a transmembrane domain and an intracellular signaling domain; wherein the first antibody or antigen-binding fragment thereof comprises a first VH and/or a first VL which are defined in claim 1, and the second antibody or antigen-binding fragment thereof comprises a second VH and/or a second VL, wherein: the second VH comprises: a second VH CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a second VH CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a second VH CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; the second VL comprises: a second VL CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; a second VL CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or 53 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; and a second VL CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto; preferably, the first antibody or antigen-binding fragment thereof targeting BCMA or the second antibody or antigen-binding fragment thereof targeting CD19 is an scFv; more preferably, the BCMA-targeting scFv comprises the first VL as set forth in SEQ ID NO: 2, the linker as set forth in SEQ ID NO: 17, 18, 19, 20 or 68, the first VH as set forth in SEQ ID NO: 1; the CD19-targeting scFv comprises the second VL as set forth in SEQ ID NO: 4 or 77, the linker as set forth in SEQ ID NO: 17, 18, 19, 20 or 68, the second VH as set forth in SEQ ID NO: 3 or 76; more preferably, the BCMA-targeting scFv has a sequence as set forth in SEQ ID NO: 25 or 27, and the CD19-targeting scFv has a sequence as set forth in SEQ ID NO: 26 or 28; preferably, the spacer domain is located between the antigen-binding domain and the transmembrane domain, the spacer domain is selected from a hinge domain and/or CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4); preferably, the hinge domain comprises a hinge region of CD8α, IgG4, PD1, CD152 or CD154; more preferably, the hinge domain comprises a hinge region of CD8α or IgG4; preferably, the transmembrane domain is selected from one or more transmembrane regions selected from the group consisting of: α, β or ζ chain of T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD28, CD137, CD152, CD154 and PD1; preferably, the transmembrane domain is selected from one or more transmembrane regions selected from the group consisting of: CD8α, CD28, CD4, PD1, CD152 and CD154; preferably, the transmembrane domain comprises the transmembrane region of CD8α or CD28; preferably, the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain; preferably, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain; preferably, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM); preferably, the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b and CD66d; more preferably, the primary signaling domain comprises an intracellular signaling domain of CD3ζ; preferably, the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) and DAP10; more preferably, the costimulatory signaling domain is selected from an intracellular signaling domain of CD28 or an intracellular signaling domain of CD137 (4-1BB) or a combination thereof; preferably, the first CAR and the second CAR further comprise a signal peptide at their N-terminal; preferably, the signal peptide comprises a heavy chain signal peptide (e.g., heavy chain signal peptide of IgG1), a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8α signal peptide; more preferably, the signal peptide is selected from a CD8α signal peptide; preferably, the first CAR has an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in SEQ ID NO: 29; (2) a sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, 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 100% as compared to the amino acid sequence as set forth in SEQ ID NO: 29; preferably, the second CAR has an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in SEQ ID NO: 30; (2) a sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, 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 100% as compared to the amino acid sequence as set forth in SEQ ID NO: 30; more preferably, the CAR construct has an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in SEQ ID NO:51; (2) a sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, 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 100% as compared to the amino acid sequence as set forth in SEQ ID NO: 51.

    18. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the chimeric antigen receptor CAR according to claim 6, preferably, the isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (1) a nucleotide sequence as set forth in any one of SEQ ID NOs: 43-48, 65, 67; (2) a sequence having a sequence identity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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 100% as compared to the nucleotide sequence as set forth in SEQ ID NO: 43-48, 65, 67.

    19. A nucleic acid construct, which comprises a first nucleotide sequence and a second nucleotide sequence encoding the first CAR and the second CAR in the CAR construct according to claim 17 respectively; preferably, the first nucleotide sequence and second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A or T2A); preferably, the self-cleaving peptide is P2A (e.g., P2A of the sequence as set forth in SEQ ID NO: 50); preferably, the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) a nucleotide sequence as set forth in SEQ ID NO: 52; (2) a sequence having a sequence identity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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 100% as compared to the nucleotide sequence as set forth in SEQ ID NO: 52.

    20. A vector, which comprises an isolated nucleic acid molecule comprising a nucleotide sequence encoding the CAR of claim 6; preferably, the vector is selected from the group consisting of DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector or viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.

    21. A host cell, which comprises (i) an isolated nucleic acid molecule comprising a nucleotide sequence encoding the CAR of claim 6 or (ii) a vector comprising the isolated nucleic acid molecule; preferably, the host cell is selected from an immune cell (e.g., human immune cell); more preferably, the immune cell is selected from the group consisting of T lymphocyte, NK cell, monocyte, macrophage or dendritic cell and any combination thereof.

    22. A method for preparing a cell expressing a chimeric antigen receptor, comprising: (1) providing a host cell; (2) obtaining a host cell capable of expressing the chimeric antigen receptor; wherein step (2) comprises introducing (i) an isolated nucleic acid molecule comprising a nucleotide sequence encoding the CAR of claim 6 or (ii) a vector comprising the isolated nucleic acid molecule into the host cell of step (1); preferably, the host cell is selected from an immune cell (e.g., human immune cell); preferably, the immune cell is selected from the group consisting of T lymphocyte, NK cell, monocyte, macrophage or dendritic cell and any combination thereof; preferably, in step (1), the immune cell undergoes pretreatment, and the pretreatment comprises sorting, activation and/or proliferation of the immune cell; more preferably, the pretreatment comprises contacting the immune cell with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cell and inducing its proliferation, thereby generating a pretreated immune cell; preferably, in step (2), the nucleic acid molecule or the vector is introduced into the host cell by viral infection; preferably, in step (2), the nucleic acid molecule or the vector is introduced into the host cell by means of non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method; preferably, a step of expanding the host cell obtained in the step (2) is further comprised after step (2).

    23. An engineered immune cell, which expresses the chimeric antigen receptor targeting BCMA and CD19 of claim 6; optionally, the chimeric antigen receptor targeting BCMA and CD19 is expressed on the surface of the engineered immune cell; optionally, the engineered immune cell also expresses a CAR that is not specific for BCMA and CD19; preferably, the CAR that is not specific for BCMA and CD19 has specificity for a target selected from the group consisting of: CD20, CD22, CD33, CD123 and CD138; optionally, the immune cell further comprises knockout of one or more endogenous genes, wherein the endogenous genes encode TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or immune checkpoint protein such as PD-1; preferably, the immune cell is derived from T lymphocyte, NK cell, monocyte, macrophage or dendritic cell and any combination thereof; and/or the immune cell is obtained from a patient or a healthy donor.

    24. (canceled)

    25. An immune cell composition, comprising the engineered immune cell according to claim 23; optionally, the composition also comprises an unmodified and/or unsuccessfully engineered immune cell; preferably, the number of engineered immune cells accounts for 10% to 100%, more preferably 40% to 80%, of the total number of cells in the immune cell composition.

    26. A kit, wherein the kit comprises: (i) the bispecific antibody or antigen-binding fragment thereof according to claim 1, or (ii) a chimeric antigen receptor (CAR) comprising the bispecific antibody or antigen-binding fragment thereof, or (iii) a nucleic acid molecule comprising a nucleotide sequence encoding the CAR, or (iv) a vector comprising the nucleic acid molecule, or (v) a host cell comprising the nucleic acid molecule or vector; optionally, the kit is used to prepare a chimeric antigen receptor or CAR construct targeting BCMA and CD19, or to prepare a cell expressing the chimeric antigen receptor or CAR construct.

    27. (canceled)

    28. A pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and/or excipient and one of the following: (i) the bispecific antibody or antigen-binding fragment thereof of claim 1, or (ii) a chimeric antigen receptor (CAR) comprising the bispecific antibody or antigen-binding fragment thereof, or (iii) an isolated nucleic acid molecule comprising a nucleotide sequence encoding the CAR, or (iv) a vector comprising the isolated nucleic acid molecule, or (v) a host cell comprising the isolated nucleic acid molecule or vector, or (vi) an engineered immune cell expressing the CAR, or (vii) an immune cell composition comprising the engineered immune cell; preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; more preferably, the additional pharmaceutically active agent is selected from an additional antibody, a fusion protein or a drug (e.g., an anti-tumor drug, such as a drug used in radiotherapy or a chemotherapy drug).

    29. (canceled)

    30. A method for preventing and/or treating a B cell-related disease or condition in a subject (such as a human), the method comprising administering to a subject in need thereof an effective amount of an active agent selected from the following: (i) the bispecific antibody or antigen-binding fragment thereof according to claim 1, or (ii) a chimeric antigen receptor (CAR) comprising the bispecific antibody or antigen-binding fragment thereof, or (iii) an isolated nucleic acid molecule comprising a nucleotide sequence encoding the CAR, or (iv) a vector comprising the isolated nucleic acid molecule, or (v) a host cell comprising the isolated nucleic acid molecule or vector, or (vi) an engineered immune cell expressing the CAR, or (vii) an immune cell composition comprising the engineered immune cell, or (viii) a pharmaceutical composition comprising any one of (i)-(vii); preferably, the B cell-related disease or condition is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, B cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunomodulatory disorder, rheumatic arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immunocyte-associated amyloidosis or monoclonal gammopathy of undetermined significance, systemic lupus erythematosus; preferably, the B cell-related disease or condition is a B cell malignancy, such as multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL); preferably, the B cell-related disease or condition is a B cell- and plasma cell-related disease or condition, such as an autoimmune disease such as systemic lupus erythematosus; preferably, the active agent is administered in combination with one or more of the following: (i) an agent that increases the efficacy of a cell comprising a CAR nucleic acid or CAR polypeptide; (ii) an agent that ameliorates one or more side effects associated with administration of a cell comprising a CAR nucleic acid or CAR polypeptide; (iii) an additional agent for treating a disease associated with BCMA and CD19; (iv) a second therapy, which is selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

    31. A method for preventing and/or treating a B cell-related disease or condition in a subject (e.g., a human), the method comprising the following steps: (1) providing an immune cell required by the subject; (2) introducing the isolated nucleic acid molecule according to claim 18 into the immune cell of step (1) to obtain an immune cell expressing a chimeric antigen receptor; (3) administering the immune cell obtained in step (2) to the subject; optionally, in step (3), the total dose of the immune cells comprises 1 to 5×10.sup.7 or 1 to 5×10.sup.8 cells; preferably, in step (3), the total dose of the immune cells is administered to the subject in divided doses.

    32. The chimeric antigen receptor (CAR) according to claim 6, characterized by one or more of the following: (i) the transmembrane domain is selected from one or more transmembrane regions selected from the group consisting of: α, β or λ chain of T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD28, CD137, CD152, CD154 and PD1; preferably, the transmembrane domain is selected from one or more transmembrane regions selected from the group consisting of: CD8α, CD28, CD4, PD1, CD152 and CD154; preferably, the transmembrane domain comprises the transmembrane region of CD8α or CD28; (ii) the spacer domain is located between the antigen-binding domain and the transmembrane domain, the spacer domain is selected from a hinge domain and/or CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4); preferably, the hinge domain comprises a hinge region of CD8α, IgG4, PD1, CD152 or CD154; more preferably, the hinge domain comprises a hinge region of CD8α or IgG4; (iii) the chimeric antigen receptor (CAR) further comprises a signal peptide at its N-terminal; preferably, the signal peptide comprises a heavy chain signal peptide (e.g., heavy chain signal peptide of IgG1), a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8α signal peptide; more preferably, the signal peptide is selected from a CD8α signal peptide; (iv) the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain; preferably, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain; preferably, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM); preferably, the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b and CD66d; more preferably, the primary signaling domain comprises an intracellular signaling domain of CD3ζ; preferably, the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) and DAP10; more preferably, the costimulatory signaling domain is selected from an intracellular signaling domain of CD28 or an intracellular signaling domain of CD137 (4-1BB) or a combination thereof.

    33. A vector, which comprises a nucleic acid construct comprising a first nucleotide sequence and a second nucleotide sequence encoding the first CAR and the second CAR in the CAR construct of claim 17 respectively; preferably, the vector is selected from the group consisting of DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector or viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.

    34. A host cell, which comprises: (i) a nucleic acid construct comprising a first nucleotide sequence and a second nucleotide sequence encoding the first CAR and the second CAR in the CAR construct of claim 17 respectively; or (ii) a vector comprising the nucleic acid construct; preferably, the host cell is selected from an immune cell (e.g., human immune cell); more preferably, the immune cell is selected from the group consisting of T lymphocyte, NK cell, monocyte, macrophage or dendritic cell and any combination thereof.

    35. A method for preparing a cell expressing a CAR construct, comprising: (1) providing a host cell; (2) obtaining a host cell capable of expressing the CAR construct; wherein step (2) comprises introducing (i) a nucleic acid construct comprising a first nucleotide sequence and a second nucleotide sequence encoding the first CAR and the second CAR in the CAR construct of claim 17 respectively; or (ii) a vector comprising the nucleic acid construct into the host cell of step (1); preferably, the host cell is selected from an immune cell (e.g., human immune cell); preferably, the immune cell is selected from the group consisting of T lymphocyte, NK cell, monocyte, macrophage or dendritic cell and any combination thereof; preferably, in step (1), the immune cell undergoes pretreatment, and the pretreatment comprises sorting, activation and/or proliferation of the immune cell; more preferably, the pretreatment comprises contacting the immune cell with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cell and inducing its proliferation, thereby generating a pretreated immune cell; preferably, in step (2), the nucleic acid molecule or the vector is introduced into the host cell by viral infection; preferably, in step (2), the nucleic acid molecule or the vector is introduced into the host cell by means of non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method; preferably, a step of expanding the host cell obtained in the step (2) is further comprised after step (2).

    36. An engineered immune cell, which expresses the chimeric antigen receptor (CAR) construct of claim 17; optionally, the CAR construct targeting BCMA and CD19 is expressed on the surface of the engineered immune cell in the form of co-expression of independent BCMA-targeting chimeric antigen receptor and CD19-targeting chimeric antigen receptor; optionally, the engineered immune cell also expresses a CAR that is not specific for BCMA and CD19; preferably, the CAR that is not specific for BCMA and CD19 has specificity for a target selected from the group consisting of: CD20, CD22, CD33, CD123 and CD138; optionally, the immune cell further comprises knockout of one or more endogenous genes, wherein the endogenous genes encode TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or immune checkpoint protein such as PD-1; preferably, the immune cell is derived from T lymphocyte, NK cell, monocyte, macrophage or dendritic cell and any combination thereof; and/or the immune cell is obtained from a patient or a healthy donor.

    37. An immune cell composition, comprising the engineered immune cell according to claim 35; optionally, the composition also comprises an unmodified and/or unsuccessfully engineered immune cell; preferably, the number of engineered immune cells accounts for 10% to 100%, more preferably 40% to 80%, of the total number of cells in the immune cell composition.

    38. A kit, wherein the kit comprises (i) the CAR construct of claim 17, or (ii) a nucleic acid construct comprising a first nucleotide sequence and a second nucleotide sequence encoding the first CAR and the second CAR in the CAR construct respectively, or (iii) a vector comprising the nucleic acid construct, or (v) a host cell comprising the nucleic acid construct or vector; optionally, the kit is used to prepare a CAR construct targeting BCMA and CD19, or to prepare a cell expressing CAR construct.

    39. A pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and/or excipient and one of the following: (i) the CAR construct of claim 17, or (ii) a nucleic acid construct comprising a first nucleotide sequence and a second nucleotide sequence encoding the first CAR and the second CAR in the CAR construct respectively, or (iii) a vector comprising the nucleic acid construct, or (iv) a host cell comprising the nucleic acid construct or vector, or (v) an engineered immune cell expressing the CAR construct, or (vi) an immune cell composition comprising the engineered immune cell; preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; more preferably, the additional pharmaceutically active agent is selected from an additional antibody, a fusion protein or a drug (e.g., an anti-tumor drug, such as a drug used in radiotherapy or a chemotherapy drug).

    40. A method for preventing and/or treating a B cell-related disease or condition in a subject (such as a human), the method comprising administering to a subject in need thereof an effective amount of an active agent selected from the following: (i) the CAR construct of claim 17, or (ii) a nucleic acid construct comprising a first nucleotide sequence and a second nucleotide sequence encoding the first CAR and the second CAR in the CAR construct respectively, or (iii) a vector comprising the nucleic acid construct, or (iv) a host cell comprising the nucleic acid construct or vector, or (v) an engineered immune cell expressing the CAR construct, or (vi) an immune cell composition comprising the engineered immune cell, or (vii) a pharmaceutical composition comprising any one of (i)-(vi); preferably, the B cell-related disease or condition is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, B cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunomodulatory disorder, rheumatic arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immunocyte-associated amyloidosis or monoclonal gammopathy of undetermined significance, systemic lupus erythematosus; preferably, the B cell-related disease or condition is a B cell malignancy, such as multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL); preferably, the B cell-related disease or condition is a B cell- and plasma cell-related disease or condition, such as an autoimmune disease such as systemic lupus erythematosus; preferably, the active agent is administered in combination with one or more of the following: (i) an agent that increases the efficacy of a cell comprising a CAR nucleic acid or CAR polypeptide; (ii) an agent that ameliorates one or more side effects associated with administration of a cell comprising a CAR nucleic acid or CAR polypeptide; (iii) an additional agent for treating a disease associated with BCMA and CD19; (iv) a second therapy, which is selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

    41. A method for preventing and/or treating a B cell-related disease or condition in a subject (e.g., a human), the method comprising the following steps: (1) providing an immune cell required by the subject; (2) introducing the nucleic acid construct of claim 19 into the immune cell of step (1) to obtain an immune cell expressing a CAR construct; (3) administering the immune cell obtained in step (2) to the subject; optionally, in step (3), the total dose of the immune cells comprises 1 to 5×10.sup.7 or 1 to 5×10.sup.8 cells; preferably, in step (3), the total dose of the immune cells is administered to the subject in divided doses.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0263] FIG. 1 shows a schematic diagram of the binding of the constructed chimeric antigen receptor or CAR construct to tumor antigens. BCMA- and CD19-targeting chimeric antigen receptor is expressed on the surface of engineered immune cell; BCMA- and CD19-targeting CAR construct is presented on the surface of engineered immune cell in the form of co-expression of independent BCMA-targeting chimeric antigen receptor and CD19-targeting chimeric antigen receptor after cleavage via a cleavable linker.

    [0264] FIG. 2 shows a schematic structural diagram of the constructed nucleic acid molecule encoding the chimeric antigen receptor or CAR construct.

    [0265] FIG. 3A shows the results of IL-2 secretion levels of BCMA-positive target cells stimulated with TanCAR 01-06CAR-T, H-BCMA CAR-T and blank T.

    [0266] FIG. 3B shows the results of IL-2 secretion levels of BCMA-positive target cells stimulated with TanCAR 08, 10 and blank T.

    [0267] FIG. 4A shows the results of IL-2 secretion levels of CD19-positive target cells stimulated with TanCAR 01-06CAR-T, H-CD19 CAR-T and blank T.

    [0268] FIG. 4B shows the results of IL-2 secretion levels of CD19-positive target cells stimulated with TanCAR 08, 10 and blank T.

    [0269] FIG. 5A shows the results of IFN-γ secretion levels of BCMA-positive target cells stimulated with TanCAR 01˜06 CAR-T, H-BCMA CAR-T and blank T.

    [0270] FIG. 5B shows the results of IFN-γ secretion levels of BCMA-positive target cells stimulated with TanCAR 08, 10 and blank T.

    [0271] FIG. 6A shows the results of IFN-γ secretion levels of CD19-positive target cells stimulated with TanCAR 01˜06 CAR-T, H-CD19 CAR-T and blank T.

    [0272] FIG. 6B shows the results of IFN-γ secretion levels of CD19-positive target cells stimulated with TanCAR 08, 10 and blank T.

    [0273] FIG. 7A shows the results of in vivo inhibition of the growth of BCMA-positive target cells in solid tumor model mice by TanCAR 02/08/10 CAR-T, H-CD19 CAR-T, H-BCMA CAR-T, H-BCMA CAR-T, H-CD19 CAR-T and blank T.

    [0274] FIG. 7B shows the results of in vivo inhibition of the growth of CD19-positive target cells in solid tumor model mice by TanCAR 02/08/10 CAR-T, H-CD19 CAR-T, H-BCMA CAR-T, H-BCMA CAR-T, H-CD19 CAR-T and blank T.

    [0275] FIG. 8 shows the results of in vivo inhibition of the growth of BCMA- and CD19-positive target cells in hematological tumor model mice by TanCAR 02/08/10 CAR-T and blank T.

    SEQUENCE INFORMATION

    [0276] The information of partial sequences involved in the present invention is provided in Table 1 below.

    TABLE-US-00002 TABLE 1 Description of sequences SEQ ID NO Description 1 H-BCMA VH Humanized antibody BCMA heavy chain variable region 2 H-BCMA VL Humanized antibody BCMA light chain variable region 3 H-CD19 VH Humanized antibody CD19 heavy chain variable region 4 H-CD19 VL Humanized antibody CD19 light chain variable region 5 H-BCMA VH CDR1 6 H-BCMA VH CDR2 7 H-BCMA VH CDR3 8 H-BCMA VL CDR1 9 H-BCMA VL CDR2 10 H-BCMA VL CDR3 11 H-CD19 VH CDR1 12 H-CD19 VH CDR2 13 H-CD19 VH CDR3 14 H-CD19 VL CDR1 15 H-CD19 VL CDR2 16 H-CD19 VL CDR3 17 Linker1 amino acid sequence 18 Linker2 amino acid sequence 19 Linker3 amino acid sequence 20 Linker4 amino acid sequence 21 CD8α hinge region amino acid sequence 22 CD8TM amino acid sequence 23 4-1BB intracellular signaling domain amino acid sequence 24 CD3ζ intracellular signaling domain amino acid sequence-1 25 Anti-murine-BCMA scFv amino acid sequence 26 FMC63 (murine-CD19 antibody) scFv amino acid sequence 27 H-BCMA scFv amino acid sequence 28 H-CD19 scFv amino acid sequence 29 H-BCMA CAR amino acid sequence 30 H-CD19 CAR amino acid sequence 31 Murine-BCMA scFv encoding nucleotide sequence 32 FMC63 (murine-CD19 antibody) scFv encoding nucleotide sequence 33 H-BCMA scFv encoding nucleotide sequence 34 H-CD19 scFv encoding nucleotide sequence 35 H-BCMA CAR encoding nucleic acid sequence 36 H-CD19 CAR encoding nucleic acid sequence 37 TanCAR 01 full-length amino acid sequence 38 TanCAR 02 full-length amino acid sequence 39 TanCAR 03 full-length amino acid sequence 40 TanCAR 04 full-length amino acid sequence 41 TanCAR 05 full-length amino acid sequence 42 TanCAR 06 full-length amino acid sequence 43 TanCAR 01 encding nucleotide sequence 44 TanCAR 02 encoding nucleotide sequence 45 TanCAR 03 encoding nucleotide sequence 46 TanCAR 04 encoding nucleotide sequence 47 TanCAR 05 encoding nucleotide sequence 48 TanCAR 06 encoding nucleotide sequence 49 N-signal peptide amino acid sequence 50 P2A amino acid sequence 51 BiCAR amino acid sequence 52 BiCAR encoding nucleotide sequence 53 Murine-CD19 VL CDR2 54 Linker1 encoding nucleotide sequence 55 Linker2 encoding nucleotide sequence 56 Linker3 encoding nucleotide sequence 57 Linker4 encoding nucleotide sequence 58 N-signal peptide encoding nucleotide sequence 59 H-BCMA VH encoding nucleotide sequence 60 H-BCMA VL encoding nucleotide sequence 61 H-CD19 VH encoding nucleotide sequence 62 H-CD19 VL encoding nucleotide sequence 63 SFFV encoding nucleotide sequence 64 TanCAR08 amino acid sequence 65 TanCAR08 encoding nucleotide sequence 66 TanCAR10 amino acid sequence 67 TanCAR10 encoding nucleotide sequence 68 Linker 5 amino acid sequence 69 Linker5 encoding nucleotide sequence 70 IgG4Hinge amino acid sequence 71 IgG4Hinge encoding nucleotide sequence 72 CD28TM amino acid sequence 73 CD28TM encoding nucleotide sequence 74 CD3ζ intracellular signaling domain amino acid sequence-2 75 CD3ζ intracellular signaling domain encoding nucleotide sequence-2 76 Murine-CD19 VH 77 Murine-CD19 VL 78 Murine-CD19 VH encoding nucleotide sequence 79 Murine-CD19 VL encoding nucleotide sequence 80 FMC63 CAR full-length amino acid sequence 81 FMC63 CAR encoding nucleotide sequence

    EXAMPLES

    [0277] The present invention will now be further described with reference to the following examples, which are intended to illustrate the present invention rather than to limit it.

    [0278] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Refined Laboratory Guide for Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used according to the conditions recommended by the product manufacturers. Those skilled in the art appreciate that the examples describe the present invention by way of example only, and are not intended to limit the scope covered by the present invention.

    Example 1: Generation of Humanized BCMA or CD19 Monoclonal Antibody

    [0279] 1.1 Generation of Humanized BCMA Monoclonal Antibody

    [0280] 1.1.1 Generation of Anti-BCMA Murine Antibody

    [0281] 1) Antigen Preparation

    [0282] The CDS sequences (NM_001192.2) of human BCMA were retrieved from the NCBI data, full gene synthesized, and cloned into vectors; CHO-K1 (ATCC) was resuscitated and cultured to logarithmic growth phase. The cells were infected with lentivirus to overexpress BCMA, and a CHO-K1-BCMA recombinant cell line with high expression of BCMA was constructed. The K562(ATCC)-BCMA cell line with high expression of BCMA was obtained by a similar method.

    [0283] 2) Immunization

    [0284] The CHO-K1-BCMA cell line expressing the target protein was resuscitated, 5 to 8 Balb/C mice were taken, then the suspension of CHO-K1-BCMA cells expressing the target antigen was injected into the abdominal cavity of the mice with a single-use syringe, and the immunization was repeated for three times; booster immunization and hybridoma preparation were performed.

    [0285] 3) SP2/0-B Cell Fusion

    [0286] Myeloma cells were resuscitated and passaged using 8-azaguanine medium, spleen cells were harvested from immunized mice, and then the SP2/0 cells and the spleen cells were fused by electrofusion. After further selection by HAT (hypoxanthine, aminopterin, and thymine), the hybridoma clones grown in the original 96-well plates were transferred to a new 96-well plate with replaced medium.

    [0287] 4) Hybridoma Flow Cytometry Screening

    [0288] The medium supernatants were taken out from the wells, co-incubated with the CHO-K1-BCMA recombinant cell line expressing the target protein, respectively, and identified by FACS. The FACS-confirmed positive single clones of hybridoma were expanded into 24-well plates according to the cell growth density. In the 24-well plate culture stage, a part of the medium supernatant was collected and re-tested by FACS to determine that the selected hybridomas could be used for secretion of antibodies. Thereby the hybridoma cell line KLB15 was obtained, which was deposited in the China Center for Type Culture Collection (CCTCC) on Nov. 14, 2018, and had the deposit number CCTCC NO. C2018224. Then, the murine monoclonal antibody was isolated and purified from the culture supernatant of the hybridoma cell line.

    [0289] 1.1.2 Affinity Determination of Anti-BCMA Murine Antibody

    [0290] Surface plasmon resonance (SPR) could dynamically reflect the association/dissociation rates and affinity constant of antibody-antigen interaction in real time. If the sample to be tested was an antibody, it could be performed directly using the supernatant of hybridoma cell culture. The operation process was completed on the surface plasmon resonance (SPR) biosensor (BIAcore® T200 (GE HealthCare)). In brief, the antigen (human BCMA) to be recognized by the antibody to be tested (the monoclonal antibody secreted by the hybridoma obtained in Example 1) was coupled to the surface of sensor chip CM5, then the antibody to be tested was injected, and the kinetic parameters of the antibody to be tested could be measured quickly and semi-quantitatively by directly monitoring the antibody-antigen binding process on the surface of the chip. The results showed that the murine monoclonal antibody had an association rate constant ka=4.177×10.sup.5 M.sup.−1s.sup.−1, a dissociation rate constant kdis=0.003709 s.sup.−1, and a dissociation equilibrium constant K.sub.D=8.881×10.sup.−9 M. The above results showed that the murine monoclonal antibody obtained in step 1.1.1 had good binding affinity to human BCMA.

    [0291] 1.1.3 Humanization and Affinity Determination of Anti-BCMA Murine Antibody

    [0292] 1) Determination of Murine Antibody Variable Region Sequence

    [0293] 1×10.sup.6 Hybridoma monoclonal cells obtained in step 1.1.1 were subjected to RNA extraction to prepare cDNA, and the VH and VL were cloned and sequenced to obtain the heavy chain variable region sequence and light chain variable region sequence of the murine BCMA antibody; the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the antibody were as set forth in SEQ ID NOs: 5-7, respectively, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the antibody were as set forth in SEQ ID NOs: 8-10. The above CDR sequences were defined using the Chothia numbering system, and any other CDR sequence determination method known in the art could also be used to identify the amino acid residues of the CDRs in the variable regions.

    [0294] 2) Design and Preparation of Humanized Antibody

    [0295] According to the amino acid sequence of the heavy chain variable region and the light chain variable region of the murine BCMA monoclonal antibody, the humanization design was carried out, and the CDR sequence of the murine monoclonal antibody was retained. The involved CDR sequences were shown in Table 2.

    TABLE-US-00003 TABLE 2 CDR sequences of humanized anti-BCMA antibodies Name Sequence SEQ ID NO: H-BCMA VH CDR1 GYTFTDY  5 H-BCMA VH CDR2 NTETGE  6 H-BCMA VH CDR3 SLYYGYSWFTY  7 H-BCMA VL CDR1 RSSQSLVHSNGNTFLH  8 H-BCMA VL CDR2 KVSNRFS  9 H-BCMA VL CDR3 MQSTHVLT 10

    [0296] The above CDR sequences were defined using the Chothia numbering system, and any other CDR sequence determination method known in the art could also be used to identify the amino acid residues of the CDRs in the variable regions. According to the results of germline alignment and the results of antibody simulation, four different human antibody templates were selected for the heavy chain and light chain respectively, and back mutation could be introduced in the framework region after humanization. Thereby a heavy chain variable region (named as H-BCMA VH, of which amino acid sequence was as set forth in SEQ ID NO: 1, and of which nucleotide sequence was as set forth in SEQ ID NO: 59) and a light chain variable region sequence (named as H-BCMA VL, of which amino acid sequence was as set forth in SEQ ID NO: 2, and of which nucleotide sequence was as set forth in SEQ ID NO: 60) of humanized anti-BCMA antibody were designed and obtained. The humanized anti-BCMA antibody was prepared in the form of scFv and named as H-BCMA scFv, of which the amino acid sequence was as set forth in SEQ ID NO: 27, and the nucleotide sequence encoding H-BCMA scFv was as set forth in SEQ ID NO: 33.

    [0297] 1.2 Production of Murine Anti-CD19 Antibody and Humanized Anti-CD19 Antibody

    [0298] The anti-CD19 antibody in this example was derived from the murine FMC63 antibody, wherein the heavy chain variable region of the murine anti-CD19 antibody (named as murine-CD19 VH, has an amino acid sequence as set forth in SEQ ID NO: 76, and an encoding nucleotide sequence as set forth in SEQ ID NO: 78); and the light chain variable region sequence (named as murine-CD19 VL, has an amino acid sequence as set forth in SEQ ID NO: 77, and an encoding nucleotide sequence as set forth in SEQ ID NO: 79).

    [0299] Further, the humanization design was carried out based on the amino acid sequence of the murine antibody, wherein mutation was introduced in the murine CDR sequence. The affinity of the murine antibody and the humanized antibody was determined, and the results showed that the dissociation equilibrium constant of the murine monoclonal antibody had K.sub.D=2.560×10.sup.−8 M, and the dissociation equilibrium constant of the humanized monoclonal antibody had K.sub.D=2.840×10.sup.−8 M, indicating that the obtained humanized monoclonal antibody had good binding affinity to human CD19. The CDR sequences involved in the anti-CD19 antibody in this example were shown in Table 3.

    TABLE-US-00004 TABLE 3 CDR sequences of murine and humanized anti-CD19 antibodies SEQ SEQ Murine ID Humanized ID Name sequence NO: sequence NO: HCDR1 GVSLPDYG 11 GVSLPDYG 11 HCDR2 IWGSETTYYN 12 IWGSETTYYN 12 HCDR3 AKHYYYGGSYAMDY 13 AKHYYYGGSYAMDY 13 LCDR1 QDISKY 14 QDISKY 14 LCDR2 HTSRLHSGVPS 53 HTSRLHSGIPD 15 LCDR3 QQGNTLPYT 16 QQGNTLPYT 16

    [0300] The above CDR sequences were defined using the Chothia numbering system, and any other CDR sequence determination method known in the art could also be used to identify the amino acid residues of the CDRs in the variable regions. According to the results of germline alignment and the results of antibody simulation, four different human antibody templates were selected for the heavy chain and light chain respectively, and back mutation could be introduced in the framework region after humanization. Thereby a heavy chain variable region (named as H-CD19 VH, of which the amino acid sequence was as set forth in SEQ ID NO: 3, and the encoding nucleotide sequence was as set forth in SEQ ID NO: 61) and a light chain variable region sequence (named as H-CD19 VL, of which the amino acid sequence was as set forth in SEQ ID NO: 4, and the encoding nucleotide sequence was as set forth in SEQ ID NO: 62) of humanized anti-CD19 antibody were designed and obtained.

    [0301] Both the murine and humanized antibodies were prepared in the form of scFv, named as murine-CD19 scFv (of which the amino acid sequence was as set forth in SEQ ID NO: 26, and the nucleotide sequence encoding the murine-CD19 scFv was as set forth in SEQ ID NO: 32) and H-CD19 scFv (of which the amino acid sequence was as set forth in SEQ ID NO: 28, and the nucleotide sequence encoding the CD19 antibody of H-CD19 scFv was as set forth in SEQ ID NO: 34) respectively.

    [0302] 1.3 Construction of BCMA- and CD19-Targeting Antibody or Antigen-Binding Fragment Thereof

    [0303] A first antibody or antigen-binding fragment thereof (specifically binding to BCMA) and a second antigen-binding fragment (specifically binding to CD19), the first antibody has VH and/or VL and the second antibody has VH and/or VL, the VH and VL regions of the first antibody and second antibody could be positioned from N-terminal to C-terminal relative to each other in any suitable arrangement, for example, VH.sub.(first/second)-VL (first/second)-VH.sub.(first/second)-VL (first/second), VH.sub.(first/second)-VL.sub.(first/second)-VL.sub.(first/second)-VH.sub.(first/second), VL.sub.(first/second)-VH.sub.(first/second)-VL.sub.(first/second)-VH.sub.(first/second) or VL (first/second)-VH.sub.(first/second)-VH.sub.(first/second)-VL (first/second), wherein “first/second” in parentheses indicated a choice from “first antigen-binding domain” or “second antigen-binding domain”, and the adjacent variable regions were connected by a linker.

    [0304] The linker sequences of the present application were as follows:

    [0305] Linker 1 has an amino acid sequence (SEQ ID NO: 17): GGGGS; its encoding nucleotide sequence (SEQ ID NO: 54): GGAGGAGGAGGAAGC.

    [0306] Linker 2 has an amino acid sequence (SEQ ID NO: 18): GGGGS GGGGS GGGGS; its encoding nucleotide sequence (SEQ ID NO: 55): GGAGGAGGAGGAAGC GGAGGAGGAGGAAGC GGAGGAGGAGGAAGC.

    [0307] Linker 3 has an amino acid sequence (SEQ ID NO: 19): GGGGS GGGGS GGGGS GGGGS; its encoding nucleotide sequence (SEQ ID NO: 56): GGAGGAGGAGGAAGT GGAGGAGGAGGATCCGGCGGC GGCGGCTCTGGCGGCGGCGGCAGC.

    [0308] Linker 4 has an amino acid sequence (SEQ ID NO: 20): EAAAK EAAAK EAAAK; its encoding nucleotide sequence (SEQ ID NO: 57): GAGGCAGCAGCAAAGGAGGCA GCAGCCAAGGAGGCAGCAGCAAAG.

    [0309] Linker 5 has an amino acid sequence (SEQ ID NO: 68): GSTSGSGKPGSGEGSTKG; its encoding nucleotide sequence (SEQ ID NO: 69): GGGTCTACTTCCGGATCAGGTAAG CCCGGCTCGGGTGAGGGCTCCACGAAGGGT.

    Example 2: Construction of BCMA- and CD19-Targeting Chimeric Antigen Receptor CAR or CAR Construct

    [0310] In this example, anti-BCMA and anti-CD19 chimeric antigen receptor CAR (TanCAR) or CAR construct (BiCAR) was constructed, the CAR or CAR construct comprised a N-signal peptide, an anti-BCMA antigen-binding domain, an anti-CD19 antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain. The anti-CD19 or anti-BCMA antigen-binding domain in this example was a single-chain antibody, specifically scFv, and the antibody sequences were derived from the humanized anti-BCMA antibody and anti-CD19 antibody prepared in Example 1.

    [0311] 2.1 Construction of BCMA- and CD19-Targeting Chimeric Antigen Receptor CAR

    [0312] The chimeric antigen receptor CAR comprised a first antigen-binding domain (specifically binding to BCMA) and a second antigen-binding domain (specifically binding to CD19), the first and the second antigen-binding domains have VH and/or VL, and the VH and VL regions of the first and second antigen-binding domains could be positioned from N-terminal to C-terminal relative to each other in any suitable arrangement, for example, VH.sub.(first/second)-VL.sub.(first/second)-VH.sub.(first/second)-VL.sub.(first/second), VH.sub.(first/second)-VL.sub.(first/second)-VL.sub.(first/second)-VH.sub.(first/second), VL.sub.(first/second)-VH.sub.(first/second)-VL.sub.(first/second)-VH.sub.(first/second) or VL.sub.(first/second)-VH.sub.(first/second)-VH.sub.(first/second)-VL.sub.(first/second), wherein “first/second” in parentheses represented choice from “first antigen-binding domain” or “second antigen-binding domain”, and the adjacent variable regions were connected by a linker.

    [0313] The linker sequences of the present application were as follows:

    [0314] Linker 1 has an amino acid sequence (SEQ ID NO: 17): GGGGS; its encoding nucleotide sequence (SEQ ID NO: 54): GGAGGAGGAGGAAGC.

    [0315] Linker 2 has an amino acid sequence (SEQ ID NO: 18): GGGGS GGGGS GGGGS; its encoding nucleotide sequence (SEQ ID NO: 55): GGAGGAGGAGGAAGC GGAGGAGGAGGAAGC GGAGGAGGAGGAAGC.

    [0316] Linker 3 has an amino acid sequence (SEQ ID NO: 19): GGGGS GGGGS GGGGS GGGGS; its encoding nucleotide sequence (SEQ ID NO: 56): GGAGGAGGAGGAAGTGGA GGAGGAGGATCCGGCGGCGGCGGCTCTGGCGGCGGCGGCAGC.

    [0317] Linker 4 has an amino acid sequence (SEQ ID NO: 20): EAAAK EAAAK EAAAK; its encoding nucleotide sequence (SEQ ID NO: 57): GAGGCAGCAGCAAAGGAGGCA GCAGCCAAGGAGGCAGCAGCAAAG.

    [0318] Linker 5 has an amino acid sequence (SEQ ID NO: 68): GSTSGSGKPGSGEGSTKG; its encoding nucleotide sequence (SEQ ID NO: 69): GGGTCTACTTCCGGATCAGGTAAGCCC GGCTCGGGTGAGGGCTCCACGAAGGGT.

    [0319] N-signal peptide of the present application has an amino acid sequence (SEQ ID NO: 49):

    TABLE-US-00005 MALPVTALLLPLALLLHAARP;

    [0320] Spacer domain CD8α has an amino acid sequence (SEQ ID NO: 21):

    TABLE-US-00006 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

    [0321] IgG4 hinge region (IgG4Hinge) has an amino acid sequence (SEQ ID NO: 70):

    TABLE-US-00007 ESKYGPPCPPCP;

    [0322] CD8 transmembrane domain (CD8TM) has an amino acid sequence (SEQ ID NO: 22):

    TABLE-US-00008 IYIWAPLAGTCGVLLLSLVITLYC;

    [0323] CD28 transmembrane domain (CD28TM) has an amino acid sequence (SEQ ID NO: 72):

    TABLE-US-00009 FWVLVVVGGVLACYSLLVTVAFIIFWV;

    [0324] 4-1BB intracellular signaling domain has an amino acid sequence (SEQ ID NO: 23):

    TABLE-US-00010 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

    [0325] CD3ζ intracellular signaling domain-1 has an amino acid sequence (SEQ ID NO: 24):

    TABLE-US-00011 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR;

    [0326] CD3ζ intracellular signaling domain-2 has an amino acid sequence (SEQ ID NO: 74):

    TABLE-US-00012 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR;

    [0327] The amino acid sequence of the heavy chain variable region (H-CD19 VH) of the humanized anti-CD19 antigen-binding domain was as set forth in SEQ ID NO: 3, and the nucleotide sequence thereof was as set forth in SEQ ID NO: 61; the amino acid sequence of the light chain variable region (H-CD19 VL) was as set forth in SEQ ID NO: 4, and the nucleotide sequence thereof was as set forth in SEQ ID NO: 62;

    [0328] The amino acid sequence of the heavy chain variable region (murine-CD19 VH) of the murine anti-CD19 antigen-binding domain was as set forth in SEQ ID NO: 76, and the nucleotide sequence thereof was as set forth in SEQ ID NO: 78; the amino acid sequence of the light chain variable region (murine-CD19 VL) was as set forth in SEQ ID NO: 77, and the nucleotide sequence thereof was as set forth in SEQ ID NO: 79;

    [0329] The amino acid sequence of the heavy chain variable region (H-BCMA VH) of the anti-BCMA antigen-binding domain was as set forth in SEQ ID NO: 1, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 59; the amino acid sequence of the light chain variable region (H-BCMA VL) was as set forth in SEQ ID NO: 2, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 60;

    [0330] The order of connection of the parts of chimeric antigen receptor was as follows:

    [0331] H-BCMA CAR: N-signal peptide-scFv(H-BCMA)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of H-BCMA CAR was as set forth in SEQ ID NO: 29, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 35;

    [0332] H-CD19 CAR: N-signal peptide-scFv(H-CD19)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of H-CD19 CAR was as set forth in SEQ ID NO: 30, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 36;

    [0333] FMC63 CAR: N-signal peptide-scFv(murine-CD19)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of FMC63 CAR was as set forth in SEQ ID NO: 80, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 81;

    [0334] TanCAR 01: N-signal peptide-H-BCMA scFv(VL-linker2-VH)-linker3-H-CD19 scFv(VH-linker2-VL)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of TanCAR 01 was as set forth in SEQ ID NO: 37, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 43;

    [0335] TanCAR 02: N-signal peptide-H-BCMA scFv(VL-linker2-VH)-linker4-H-CD19 scFv(VH-linker2-VL)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of TanCAR 02 was as set forth in SEQ ID NO: 38, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 44;

    [0336] TanCAR 03: N-signal peptide-H-CD19 scFv(VH-linker2-VL)-linker3-H-BCMA scFv(VL-linker2-VH)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of TanCAR 03 was as set forth in SEQ ID NO: 39, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 45;

    [0337] TanCAR 04: N-signal peptide-H-CD19 scFv(VH-linker2-VL)-linker4-H-BCMA scFv(VL-linker2-VH)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of TanCAR 04 was as set forth in SEQ ID NO: 40, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 46;

    [0338] TanCAR 05: N-signal peptide-H-BCMA scFv(VL-linker1-VH)-linker3-H-CD19 scFv(VH-linker1-VL)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of TanCAR 05 was as set forth in SEQ ID NO: 41, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 47;

    [0339] TanCAR 06: N-signal peptide-H-CD19 VH-linker1-H-BCMA VL-linker3-H-BCMA VH-linker1-H-CD19 VL-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of TanCAR 06 was as set forth in SEQ ID NO:42, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO:48.

    [0340] TanCAR 08: N-signal peptide-H-BCMA scFv(VL-linker2-VH)-linker3-H-CD19 scFv(VH-linker1-VL)-CD8α-CD8TM-4-1BB-CD3zeta-2; the amino acid sequence of TanCAR 08 was as set forth in SEQ ID NO: 64, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 65;

    [0341] TanCAR 10: N-signal peptide-H-BCMA scFv(VL-linker2-VH)-linker3-murine-CD19 scFv(VH-linker5-VL)-IgG4Hinge-CD28TM-4-1BB-CD3zeta-1; the amino acid sequence of TanCAR 10 was as set forth in SEQ ID NO: 66, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 67;

    [0342] The connection order of each part of each BCMA- and CD19-targeting chimeric antigen receptor CAR was shown below:

    TABLE-US-00013 N-terminal .fwdarw. C-terminal, amino acid sequence of each element (SEQ ID NO:) Amino acid sequence of each element from N- terminal to C-terminal of the BCMA and CD19 binding domain Trans- Intracellular Signal Variable Variable Variable Variable Hinge membrane singnaling Nam peptide region Linker region Linker region Linker region region region domain Fu Co TanCAR 01 49 2 18 1 19 3 18 4 21 22 23, 24 37 43 TanCAR 02 49 2 18 1 20 3 18 4 21 22 23, 24 38 44 TanCAR 03 49 3 18 4 19 2 18 1 21 22 23, 24 39 45 TanCAR 04 49 3 18 4 20 2 18 1 21 22 23, 24 40 46 TanCAR 05 49 2 17 1 19 3 17 4 21 22 23, 24 41 47 TanCAR 06 49 3 17 2 19 1 17 4 21 22 23, 24 42 48 TanCAR 08 49 2 18 1 19 3 68 4 21 22 23, 74 64 65 TanCAR 10 49 2 18 1 19 76 68 77 70 72 23, 24 66 67

    [0343] 2.2 Construction of BCMA- and CD19-Targeting Chimeric Antigen Receptor CAR Construct

    [0344] The CAR construct comprised independently a first CAR (H-BCMA CAR) and a second CAR (H-CD19 CAR); the first CAR comprised a signal peptide, an anti-BCMA antibody or antigen-binding fragment thereof, a spacer domain, a transmembrane domain, and an intracellular signaling domain from the N-terminal to the C-terminal; the second CAR comprised a signal peptide, an anti-CD19 antibody or antigen-binding fragment thereof, a spacer domain, a transmembrane domain and an intracellular signaling domain from N-terminal to C-terminal. The nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR were linked by the nucleotide sequence encoding the self-cleaving peptide P2A, so that when the above-mentioned nucleic acid molecule was expressed in a cell, the first CAR and the second CAR could be independently formed. The CAR construct was named as BiCAR.

    [0345] The P2A amino acid sequence of the present application was SEQ ID NO: 50;

    [0346] The nucleotide sequence of the promoter SFFV of the present application was SEQ ID NO: 63;

    [0347] H-BCMA CAR: N-signal peptide-scFv(H-BCMA)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of H-BCMA CAR was as set forth in SEQ ID NO: 29, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 35;

    [0348] H-CD19 CAR: N-signal peptide-scFv(H-CD19)-CD8α-CD8TM-4-1BB-CD3zeta-1; the amino acid sequence of H-CD19 CAR was as set forth in SEQ ID NO: 30, and the coding nucleotide sequence thereof was as set forth in SEQ ID NO: 36;

    [0349] The structure of the nucleic acid molecule encoding BiCAR: N-signal peptide-(H-BCMA CAR)-P2A-(H-CD19 CAR), and the coding nucleotide sequence thereof was as set forth in SEQ ID NO:52.

    Example 3: Construction and Preparation of Lentiviral Expression Vector of Chimeric Antigen Receptor (CAR)/CAR Construct

    [0350] 3.1 Construction of Lentiviral Plasmid

    [0351] Based on the structure of the CAR or CAR construct described in Example 2, a lentiviral expression vector of CAR/CAR construct was further constructed, wherein the nucleic acid sequence encoding the CAR (TanCAR) or CAR construct (BiCAR) was subcloned into Lenti-EF1a-AT-Free vector (produced by Suzhou Aikangde Co., Ltd.); the single clones were picked for culturing and preservation, and the plasmids were finally extracted for sequencing, and the correctly sequenced bacterial liquid was used to prepare lentiviral plasmids. The structure of the chimeric antigen receptor/CAR construct constructed above was shown in FIG. 1, and the order of each element in the lentiviral expression vector encoding the CAR (TanCAR) or CAR construct (BiCAR) was shown in FIG. 2.

    [0352] 3.2 Virus Packaging

    [0353] The mixture of the above-constructed CAR/CAR construct lentiviral plasmid and the transfection reagent was added dropwise to 293T (ATCC) cells, the culture dish was shaken gently to mix well. The culture dish was placed in a 37° C., 5% CO.sub.2 incubator; after being cultured for 6-8 hours, the medium containing the transfection reagent was discarded and replaced with fresh complete medium. After 48 hours of continuous culture, the virus-containing medium supernatant in the culture dish was collected, filtered with a 0.45 μm filter, transferred to a centrifuge tube, and centrifuged after balancing at 20,000×g at 4° C. for 2 hours. After centrifugation, the liquid in the centrifuge tube was carefully removed by sucking in a biological safety cabinet, 500 μL of PBS buffer was added to resuspend the pellet, and the virus was stored at −80° C.

    Example 4: Preparation of CAR-T Cells

    [0354] 1) Isolation of Primary T Cells:

    [0355] Human PBMC cells were isolated by lymphocyte separation medium (GE Healthcare), the PBMC cells were incubated with Dynabeads (Thermo) at room temperature, and subjected to enrichment by magnetic separation. T cells were resuspended in X-vivo 15 medium, and added with 10% FBS, 300 U/mL IL-2, 5 ng/mL IL-15 and 10 ng/mL IL-7 (the IL-2, IL-15, IL-7 were purchased from Novoprotein Technology Co., Ltd.), placed at 37° C., and stored in a 5% CO.sub.2 incubator.

    [0356] 2) Activation of T Cells:

    [0357] The cell density was adjusted to 1×10.sup.6 cells/mL, cytokine and antibody complex (in final concentrations of 300 U/mL IL-2, 10 ng/mL IL-7, 5 ng/mL IL-15, 500 ng/mL anti-CD3 antibody (OKT3), 2 μg/mL anti-CD28 antibody) were added to the 6-well plate, and cultured continuously for 48 hours.

    [0358] 3) Virus Infection:

    [0359] (1) The required amount of virus was calculated according to MOI=20. The calculation formula was as follows: required virus amount (mL)=(MOI×cell number)/virus titer.

    [0360] (2) The virus was rapidly rewarmed to 37° C. The calculated amount of virus was added to the 6-well plate, added with polybrene to reach a final concentration of 6 μg/mL, mixed well, and then centrifuged.

    [0361] (3) After centrifugation, it was continuously cultured in a 37° C., 5% CO.sub.2 incubator for later use.

    [0362] (4) H-BCMA CAR-T, FMC63 CAR-T, H-CD19 CAR-T, TanCAR 01˜06, 08, 10 CAR-T, BiCAR-T cells were obtained.

    Example 5: Detection of Positive Rate of CAR-T Cells

    [0363] The nucleic acid sequence encoding the CAR was expressed under the drive of a promoter, therefore the lentivirus-transfected T cells could be labeled with an antigen or anti-CD19 antibody and measured by flow cytometry, to reflect the expression level of the CAR on the surface of the T cells. The CAR positive rate of the CAR-T cells obtained in Example 4 was detected by the above method, and the FACS test results were shown in Table 4 below. The results showed that the CAR positive rate of all CAR-T cells was greater than 5% 48h after transduction, indicating that the CAR was successfully expressed after effector cells were transfected with lentivirus, and the chimeric antigen receptor T cells expressing BCMA-CAR and CD19-CAR were successfully constructed.

    [0364] The expression of BCMA in CHO-K1-BCMA, RPMI8226 and MM.1S cells and the expression of CD19 in Nalm6 cells were detected by flow cytometry. The results were shown in Table 5, which showed that the CHO-K1-BCMA, RPMI8226 and MM.1S cells had high expression level of BCMA, the Nalm6 cells had high expression level of CD19, and thus they could be used for subsequent detection as target cells.

    TABLE-US-00014 TABLE 4 Results of positive rate test of CAR Chimeric antigen Positive rate of Positive rate of receptor binding to BCMA binding to CD19 H-BCMA CAR 26.94% 0.31% FMC63 CAR 1.01% 15.55% H-CD19 CAR 0.98% 14.67% TanCAR 01 11.34% 10.6% TanCAR 02 14.09% 12.69% TanCAR 03 6.07% 8.99% TanCAR 04 15.87% 16.12% TanCAR 05 14.24% 11.78% TanCAR 06 13.3% 16.44% TanCAR 08 28.54% 24.40% TanCAR 10 39.83% 34.79%

    TABLE-US-00015 TABLE 5 BCMA and CD19 expression rates of target cells Cell line BCMA expression rate CD19 expression rate CHO-K1-BCMA   50% / MM.1S 73.83% / RPMI8226 81.11% / Nalm6 / 88.24%

    Example 6: Evaluation of Killing Activity of CAR-T Cells on Target Cells

    [0365] The killing activity of CAR-T cells was evaluated by measuring the ability of CAR-T cells to lyse target cells and their ability to release cytokines. The specific steps were as follows: 1) Ability of CAR-T to Lyse BCMA Target Cells

    [0366] The cell density of the target cell CHO-K1-BCMA-luc was adjusted to 1×10.sup.5/mL, and the target cell CHO-K1-BCMA-luc was inoculated in a 96-well plate according to the amount of 100 μL/well, and allowed to stand in a 5% CO.sub.2, 37° C. incubator for 30 min. CAR-T was collected, wherein CAR-T cells of H-BCMA CAR, TanCAR 01˜06, TanCAR 08, TanCAR 10, and blank T cells without being transfected with CAR as effector cells were collected by centrifugation and resuspended with F-12K, 10% FBS medium; then they were added to 96-well plates containing CHO-K1-BCMA-luc at E/T (effector cells/target cells) ratios of 2:1, 1:1, 0.5:1, 0.25:1, 100 μL/well, and cultured in a 5% CO.sub.2, 37° C. incubator for 18 to 24 hours. After the culturing, the plates were taken out of the incubator, added with 20 μL of fluorescence detection reagent, and detected by a microplate reader to obtain fluorescence readings.

    [0367] 2) Ability of CAR-T to Lyse CD19 Target Cells

    [0368] The cell density of target cell Nalm6-luc was adjusted to 5×10.sup.4/mL, and the target cell Nalm6-luc was inoculated in a 96-well plate according to the amount of 100 μL/well, and allowed to stand for 30 min in a 5% CO.sub.2, 37° C. incubator. CAR-T was collected, wherein CAR-T cells of H-CD19 CAR, TanCAR 01˜06, TanCAR 08, TanCAR 10, and blank T cells without being transfected with CAR as effector cells were collected by centrifugation and resuspended in RPMI 1640, 10% FBS medium; then they were added to the 96-well plates containing Nalm6-luc at E/T (effector cells/target cells) ratios of 2:1, 1:1, 0.5:1, 0.25:1, 100 μL/well, supplemented to reach a final volume of 200 μL/well, and cultured in a 5% CO.sub.2, 37° C. incubator for 18 to 24 hours. After the culturing, the plates were taken out of the incubator, added with 20 μL of fluorescence detection reagent, and detected by a microplate reader to obtain fluorescence readings.

    [0369] 3) Release of Cytokines

    [0370] According to the steps of 1) and 2), MM.1S-luc (or RPMI8226) and Nalm6-luc were prepared as target cells, H-BCMA CAR or H-CD19 CAR and TanCAR 01˜06, 08, 10 and blank T cells without being transfected with CAR were prepared as effector cells; then the effector cells were added to 96-well plates containing target cells at E/T (effector cells/target cells) ratio of 1:1, 100 μL/well, and supplemented to reach a final volume of 200 μL/well, and cultured in a 5% CO.sub.2, 37° C. incubator overnight. After the culturing, the well plates were taken out of the incubator, centrifuged, and the supernatant was taken, and the release of cytokines (IL-2 and IFN-γ) was detected by ELISA kit.

    [0371] The data obtained after the above processing were plotted using GraphPad 6.0.

    [0372] 4) Result Analysis:

    [0373] The killing activity of CAR-T adopted the following formula:


    Tumor cell lysis rate %=(1−(fluorescence reading with effector cells and target cells/fluorescence reading without effector cells and target cells)/(fluorescence reading with target cells only/fluorescence reading without target cells))×100%.

    [0374] The killing test results were shown in Tables 6 and 7. The detection results of IL-2 secretion levels were shown in FIGS. 3A, 3B, 4A and 4B. The detection results of IFN-γ secretion levels were shown in FIGS. 5A, 5B, 6A and 6B.

    [0375] The results showed that the constructed CAR-T could activate primary T cells and efficiently mediate the killing of targeted tumor cells by T cells (Tables 6 and 7), and caused a significant increase in cytokine secretion (FIGS. 3A, 3B, 4A, 4B, 5A, 5B, 6A, 6B). The above results showed that TanCAR 01˜06, 08, 10 all could specifically lyse tumor cells with targets of BCMA and CD19, and induce a significant increase in cytokine secretion, indicating that they could effectively kill cells expressing CD19 and/or BCMA.

    TABLE-US-00016 TABLE 6 lysis rate of CHO-K1-BCMA-luc cells by CAR-T cells Ratio of CAR-T cells to target cells (CHO-K1-BCMA) CAR-T cells 0.25:1 0.5:1 1:1 2:1 TanCAR 01 31.3% 34.3% 63.2% .sup. 71% TanCAR 02 .sup. 24% 35.9% 60.7% .sup. 59% TanCAR 03 17.8% 27.6% 40.5% 52.1% TanCAR 04 36.4% 52.6% 68.9% 84.1% TanCAR 05 26.6% 39.2% 58.2% 79.8% TanCAR 06 31.9% 48.3% 78.4% 92.9% TanCAR 08 12.3% 38.4% 65.9% / TanCAR 10 17.1% 36.0% 57.9% / H-BCMA CAR   0% .sup. 27% 40.7% 77.9% Blank T   0%   6% 16.4% .sup. 22% “/” indicates that no experiment was performed at the corresponding ratio of CAR-T cells to target cells, and there was no experimental data.

    TABLE-US-00017 TABLE 7 lysis rate of Nalm6-luc cells by CAR-T cells Ratio of CAR-T cells to target cells (Nalm6) CAR-T cells 0.25:1 0.5:1 1:1 2:1 TanCAR 01 46.1% .sup. 40% 56.8% 80.6% TanCAR 02 26.1% 49.8% 70.1% 82.4% TanCAR 03 54.3% 57.7% 72.1% 89.1% TanCAR 04 72.7% 55.8% 68.9% 84.1% TanCAR 05 25.8% 42.4% 67.7% 91.2% TanCAR 06 68.3% 62.9% 88.9% 95.5% TanCAR 08 65.9% 83.8% 92.2% / TanCAR 10 59.6% 77.4% 91.5% / H-CD19 CAR .sup. 48% .sup. 55% 72.8% 86.4% Blank T  2.6%   0%   0%   0% “/” indicates that no experiment was performed at the corresponding ratio of CAR-T cells to target cells, and there was no experimental data.

    Example 7: In Vivo Efficacy Evaluation of CAR-T Cells

    [0376] 7.1 Solid Tumor Model

    [0377] Animal grouping: 30 B-NDG mice were subcutaneously inoculated with the tumor cells of RPMI8226 and Nalm6 (1×10.sup.7/mouse) respectively, which were 6-8 weeks old female mice. The unsuccessfully modeled mice were excluded, and were randomly divided into 7 groups, wherein Group 1 (3 mice) were given H-BCMA CAR-T and H-CD19 CAR-T (BCMA+hCD19), Group 2 (3 mice) were given blank T cells (UTD), Group 3 (4 mice) were given H-BCMA CAR-T (BCMA), Group 4 (4 mice) were given H-CD19 CAR-T (hCD19), Group 5 (4 mice) were given TanCAR 02, Group 6 (4 mice) were given TanCAR 08, and Group 7 (4 mice) were given TanCAR 10. The day of reinfusion of CAR-T was recorded as P0.

    [0378] Treatment:100 mg/kg cyclophosphamide was intraperitoneally injected 24 hours before administration, and CAR-T was re-infused at PO and P3 via tail vein injection at a dose of 3×10.sup.5/mouse. After administration, the tumor volume and body weight of mice were observed and measured regularly.

    [0379] The tumor diameter was measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V=0.5 a×b.sup.2, wherein a and b represented the long diameter and short diameter of the tumor, respectively. The death of animals was observed and recorded every day, until P54.

    [0380] The following formula was used to calculate the tumor growth inhibition rate TGI (%), which was used for evaluating the tumor suppressive efficacy of CAR-T:


    TGI(%)=[1−(V.sub.T-end−V.sub.T-start)/(V.sub.C-end−V.sub.C-start)]×100%

    [0381] wherein

    [0382] V.sub.T-end: mean tumor volume at the end of experiment in the treatment group

    [0383] V.sub.T-start: mean tumor volume at the start of administration in the treatment group

    [0384] V.sub.C-end: mean tumor volume at the end of experiment in the negative control group

    [0385] V.sub.C-start: mean tumor volume at the start of administration in the negative control group

    [0386] Conclusion: As shown in FIGS. 7A to 7B, from P12, TanCAR showed an inhibitory effect on the growth of BCMA and CD19 tumor cells in mice. By P54, TanCAR02, TanCAR08 and TanCAR10 completely eliminated tumor cells in vivo, and these different TanCARs showed no significant difference (the bispecific tumor inhibition rates of TanCAR02 were BCMA: 121%, CD19: 107%; the bispecific tumor inhibition rates of TanCAR08 were BCMA: 116%, CD19: 106%; the bispecific tumor inhibition rates of TanCAR10 were BCMA: 119%, CD19: 105%), and the in vivo efficacy was consistent with the efficacy of mixed refusion of BCMA CAR-T and hCD19 CAR-T (the bispecific tumor inhibition rates were BCMA: 115%, CD19: 106%, respectively).

    [0387] 7.2 Hematological Tumor Model

    [0388] B-NDG mice (body weight 18-22 g) aged 6-8 weeks were taken and randomly divided into 4 groups; inoculation of 2×10.sup.6 Nalm6-BCMA-luc cells/mouse was performed through the tail vein; 7 days after inoculation, CAR-T and UTD were infused intravenously at 3×10.sup.6/mouse, which was recorded as P0; 1 day later (P2), refusion was performed for the second time; the weight of mice was measured by electronic balance twice a week, imaging was performed once a week, and the imaging observation was carried out until P45.

    [0389] Conclusion: As shown in FIG. 8, the mice in the blank control group began to die at P14; TanCAR02, TanCAR08 and TanCAR10 could effectively kill tumor cells in mice and significantly reduce the mortality of mice. Observed until P45, the mice of the TanCAR02, TanCAR08 and TanCAR10 groups survived normally, and no tumor recurrence was found in the TanCAR10 group.

    [0390] Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention. The whole of the present invention is given by the appended claims and any equivalents thereof.