WO2023169555A9 - 靶向bcma和gprc5d的嵌合抗原受体及其应用 - Google Patents

靶向bcma和gprc5d的嵌合抗原受体及其应用 Download PDF

Info

Publication number
WO2023169555A9
WO2023169555A9 PCT/CN2023/080741 CN2023080741W WO2023169555A9 WO 2023169555 A9 WO2023169555 A9 WO 2023169555A9 CN 2023080741 W CN2023080741 W CN 2023080741W WO 2023169555 A9 WO2023169555 A9 WO 2023169555A9
Authority
WO
WIPO (PCT)
Prior art keywords
seq
acid sequence
amino acid
sequence shown
chimeric antigen
Prior art date
Application number
PCT/CN2023/080741
Other languages
English (en)
French (fr)
Other versions
WO2023169555A1 (zh
Inventor
周金财
史中军
何晓文
杨月
陈思晔
Original Assignee
原启生物科技(上海)有限责任公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 原启生物科技(上海)有限责任公司 filed Critical 原启生物科技(上海)有限责任公司
Publication of WO2023169555A1 publication Critical patent/WO2023169555A1/zh
Publication of WO2023169555A9 publication Critical patent/WO2023169555A9/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of biomedicine, specifically a chimeric antigen receptor targeting BCMA and GPRC5D, and its application.
  • MM Multiple myeloma
  • MM is the second most common malignant hematological disease and ranks second in cancer mortality.
  • MM is a plasma cell malignant tumor that is often accompanied by multiple osteolytic lesions, renal damage, bone marrow infiltration, hypercalcemia and anemia.
  • the current main treatment for MM is systemic chemotherapy with severe side effects and cannot be completely cured.
  • BCMA B cell maturation antigen
  • B cell maturation antigen the B cell maturation antigen, mainly regulates the proliferation and survival of B cells, as well as the mature differentiation of B cells into plasma cells (PCs).
  • PCs plasma cells
  • BCMA molecules are gradually induced to express. Since BCMA molecules are only expressed on the surface of PCs and plasmablast membranes and are not expressed on most B cells, hematopoietic stem cells, and other normal tissues, BCMA molecules have become one of the most ideal target molecules for the treatment of MM.
  • G protein-coupled receptor family C group 5 member D (GPRC5D) protein is an atypical surface orphan receptor.
  • GPRCD5 like other C5 family receptors, has a short amino terminus, so it is conformationally very similar to the C4 family. Its expression in normal tissues is limited to hair follicles, but it is also highly expressed specifically in the bone marrow of MM patients and is highly correlated with plasma cell tumor burden and genetic aberrations.
  • CAR-T Chimeric antigen receptor T cell
  • BCMA B cell maturation antigen
  • MM patients with low BCMA expression have target escape problems. Therefore, there is an urgent need to develop more diverse and more effective drugs to treat MM to solve the problems of high relapse and immune escape caused by CAR-T cell therapy in the treatment of multiple myeloma.
  • the present application provides a bispecific chimeric antigen receptor targeting BCMA and GPRC5D, and the present application also provides a cell expressing a chimeric antigen receptor that specifically binds BCMA and specifically binds GPRC5D.
  • This application uses the BCMA antigen and the spatial epitope of GPRC5D as targets, and combines the scFv region of the BCMA antibody, the optimized linker, the V H H region, hinge region, transmembrane region, costimulatory signal region, and intracellular region of the GPRC5D antibody.
  • the constructed chimeric antigen receptor can be used to treat tumors such as multiple myeloma to solve the high relapse and immune escape problems of CAR-T cell therapy in the treatment of multiple myeloma and provide a new treatment strategy for multiple myeloma. , effective treatment of multiple myeloma.
  • the application provides a chimeric antigen receptor comprising a first antigen binding domain and a second antigen binding domain, the first antigen binding domain targeting B cell maturation antigen (BCMA), and The second antigen-binding domain targets the G protein-coupled receptor family group C group 5 member D (GPRC5D) protein.
  • BCMA B cell maturation antigen
  • GPRC5D G protein-coupled receptor family group C group 5 member D
  • the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragments include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH, and/or dAb.
  • the antigen-binding fragment of the first antigen-binding domain comprises a scFv.
  • the first antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VH, which VH comprises the amino acid sequence set forth in SEQ ID NO: 9.
  • the first antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 4.
  • the first antigen binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 3.
  • the first antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the first antigen binding domain includes HCDR1, HCDR2 and HCDR3, the HCDR1 includes the amino acid sequence shown in SEQ ID NO:2, and the HCDR2 includes the amino acid sequence shown in SEQ ID NO:3 sequence, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:4.
  • the first antigen binding domain includes H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 includes SEQ ID NO. :The amino acid sequence shown in 5.
  • the first antigen binding domain comprises H-FR2
  • the H-FR2 is located between the HCDR1 and the HCDR2
  • the H-FR2 comprises SEQ ID NO: 6 amino acid sequence.
  • the first antigen binding domain comprises H-FR3
  • the H-FR3 is located between the HCDR2 and the HCDR3
  • the H-FR3 comprises SEQ ID NO: 7 amino acid sequence.
  • the first antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO:8 The amino acid sequence shown.
  • the first antigen binding domain comprises a VH
  • the VH comprises SEQ ID NO: 9 The amino acid sequence shown.
  • the first antigen binding domain comprises at least one CDR in the antibody light chain variable region VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 17.
  • the first antigen binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the first antigen binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11 (LGS).
  • the first antigen binding domain includes LCDR1, and the LCDR1 includes the amino acid sequence set forth in SEQ ID NO: 10.
  • the first antigen binding domain includes LCDR1, LCDR2 and LCDR3, the LCDR1 includes the amino acid sequence shown in SEQ ID NO:10, and the LCDR2 includes the amino acid sequence shown in SEQ ID NO:11 (LGS) The amino acid sequence shown is, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 12.
  • the first antigen-binding domain includes L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1
  • the L-FR1 includes SEQ ID NO. : The amino acid sequence shown in 13.
  • the first antigen binding domain comprises L-FR2
  • the L-FR2 is located between the LCDR1 and the LCDR2
  • the L-FR2 comprises SEQ ID NO: 14 amino acid sequence.
  • the first antigen binding domain comprises L-FR3
  • the L-FR3 is located between the LCDR2 and the LCDR3
  • the L-FR3 comprises SEQ ID NO: 15 amino acid sequence.
  • the first antigen binding domain comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO. :The amino acid sequence shown in 16.
  • the first antigen binding domain comprises a VL
  • the VL comprises the amino acid sequence set forth in SEQ ID NO: 17.
  • the first antigen binding domain includes VH and VL, the VH and VL are connected by a linker, the linker includes the amino acid sequence shown in SEQ ID NO: 18.
  • the first antigen binding domain comprises a scFv
  • the scFv comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the second antigen-binding domain comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragments include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH, and/or dAb.
  • the antigen-binding fragment of the second antigen-binding domain includes a VHH.
  • the second antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VH of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:39 and SEQ ID NO:46 .
  • the second antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67.
  • the HCDR3 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:37 and SEQ ID NO:43 .
  • the second antigen binding domain comprises HCDR2
  • the HCDR2 comprises SEQ ID NO: 68 (IX 1 is S or W, X 3 is G or S, X 4 is D or G, and X 5 is the amino acid sequence represented by N, S or T).
  • the HCDR2 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:42 .
  • the second antigen binding domain comprises HCDR1
  • the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 69.
  • the HCDR1 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:41 .
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 68 (IX 1 ⁇ 2 X 3 X 4 GX 5 T, X 1 is N or T, X 2 is S or W , X 3 is G or S, X 4 is D or G, sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 67.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:41
  • the amino acid sequence shown in any one of the HCDR2 includes the amino acid sequence shown in any one of SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:42
  • the HCDR3 Contains the amino acid sequence shown in any one of SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:37 and SEQ ID NO:43.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, which comprise an amino acid sequence selected from any of the following groups: (1) HCDR1: SEQ ID NO: 21; HCDR2: SEQ ID NO: 22; and HCDR3: SEQ ID NO: 23; (2) HCDR1: SEQ ID NO: 30; HCDR2: SEQ ID NO: 31; and HCDR3: SEQ ID NO: 32; (3) HCDR1: SEQ ID NO:35; HCDR2: SEQ ID NO:36; and HCDR3: SEQ ID NO:37; and (4) HCDR1: SEQ ID NO:41; HCDR2: SEQ ID NO:42; and HCDR3: SEQ ID NO:43.
  • the second antigen-binding domain includes H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 includes SEQ ID NO. :The amino acid sequence shown in 24.
  • the second antigen binding domain comprises H-FR2
  • the H-FR2 is located between the HCDR1 and the HCDR2
  • the H-FR2 comprises SEQ ID NO:70 amino acid sequence.
  • the H-FR2 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:25, SEQ ID NO:44 and SEQ ID NO:38.
  • the second antigen binding domain comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:71 amino acid sequence.
  • the H-FR3 of the second antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 45.
  • the second antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: 27 The amino acid sequence shown.
  • the second antigen binding domain includes H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 24, so
  • the H-FR2 includes the amino acid sequence shown in SEQ ID NO:70
  • the H-FR3 includes the amino acid sequence shown in SEQ ID NO:71
  • the H-FR4 includes the amino acid sequence shown in SEQ ID NO:27 .
  • the second antigen binding domain includes H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 24, so
  • the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:25, SEQ ID NO:38 and SEQ ID NO:44
  • the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NO:26 or SEQ ID NO:45.
  • the amino acid sequence shown is, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 27.
  • the second antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1, H-FR2, H-FR3 and H-FR4 comprise Selected from any of the following amino acid sequences: (1) H-FR1: SEQ ID NO: 24, H-FR2: SEQ ID NO: 25, H-FR3: SEQ ID NO: 26, and H-FR4: SEQ ID NO: 27; (2) H-FR1: SEQ ID NO: 24, H-FR2: SEQ ID NO: 25, H-FR3: SEQ ID NO: 26, and H-FR4: SEQ ID NO: 27; (3 ) H-FR1: SEQ ID NO: 24, H-FR2: SEQ ID NO: 38, H-FR3: SEQ ID NO: 26, and H-FR4: SEQ ID NO: 27; and (4) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:44, H-FR3: SEQ ID NO:45, and H-FR4: SEQ ID NO:27
  • the second antigen binding domain comprises VH
  • the VH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VH of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:39 and SEQ ID NO:46 .
  • the second antigen binding domain comprises a VHH
  • the VHH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VHH of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:39 and SEQ ID NO:46 .
  • the first antigen binding domain and the second antigen binding domain comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively, the first antigen binding domain and the second antigen binding domain
  • the sequences of HCDR1, HCDR2 and HCDR3 of the two antigen-binding domains are selected from any of the following sets of amino acid sequences: (1) First antigen-binding domain: HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, HCDR3 : SEQ ID NO:4, LCDR1: SEQ ID NO:10, LCDR2: SEQ ID NO:11 (LGS), and LCDR3: SEQ ID NO:12; Second antigen binding domain: HCDR1: SEQ ID NO:21, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:23; (2) First antigen binding domain: HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:3, HCDR3: SEQ ID
  • the C-terminus of the first antigen-binding domain and the N-terminus of the second antigen-binding domain are directly or indirectly connected.
  • the N-terminus of the first antigen-binding domain is directly or indirectly connected to the C-terminus of the second antigen-binding domain.
  • the first antigen binding domain and the second antigen binding domain are connected by a linker.
  • the linker comprises a peptide linker.
  • the linker comprises the amino acid sequence of (GGGGS)n, wherein n is any positive integer from 1 to 10.
  • the linker comprises the amino acid sequence of (EAAAK)n, wherein n is any positive integer from 1 to 10.
  • the chimeric antigen receptor comprises a costimulatory signal domain
  • the costimulatory signal domain comprises an intracellular costimulatory signal domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40 and MyD88.
  • the costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.
  • the costimulatory signal region includes the amino acid sequence shown in SEQ ID NO: 56.
  • the chimeric antigen receptor comprises an intracellular signaling domain comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP-12 and a domain containing at least one ITAM.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpesvirus
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 60.
  • the chimeric antigen receptor includes a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4
  • the transmembrane region is a transmembrane region derived from CD8 ⁇ .
  • the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 52.
  • the chimeric antigen receptor further includes a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region is a hinge region derived from CD8 ⁇ .
  • the hinge region includes the amino acid sequence set forth in SEQ ID NO: 50.
  • the chimeric antigen receptor further comprises a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof is located at the C-terminus of the intracellular signaling region.
  • the low-density lipoprotein receptor-associated protein or fragment thereof comprises one or more selected from the group consisting of: low-density lipoprotein receptor-associated protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 64.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide is a signal peptide derived from a CD8 ⁇ protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 48.
  • the present application also provides isolated one or more nucleic acid molecules encoding the chimeric antigen receptors described herein.
  • the nucleic acid molecule further comprises a sequence encoding a cleavage peptide.
  • the cleaved peptide comprises 2A peptide.
  • the cleavage peptide is selected from the group consisting of P2A, T2A, F2A, and E2A.
  • sequence of the cleavage peptide includes the amino acid sequence shown in SEQ ID NO: 62.
  • sequence encoding the cleavage peptide comprises the nucleic acid sequence set forth in SEQ ID NO: 63.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-associated protein or fragment thereof comprises one or more selected from the group consisting of: low-density lipoprotein receptor-associated protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 64.
  • sequence encoding the low-density lipoprotein receptor-related protein or fragment thereof includes the nucleic acid sequence shown in SEQ ID NO: 65.
  • the present application also provides an expression vector comprising the nucleic acid molecule described in the present application.
  • the present application also provides an expression vector comprising a nucleic acid sequence encoding the chimeric antigen receptor targeting BCMA and a nucleic acid sequence encoding the chimeric antigen receptor targeting GPRC5D.
  • the present application also provides an expression vector, which includes a nucleic acid sequence encoding a chimeric antigen receptor targeting BCMA and a nucleic acid sequence encoding a chimeric antigen receptor targeting GPRC5D.
  • the nucleic acid sequence encoding a chimeric antigen receptor targeting BCMA in the expression vector includes a nucleic acid sequence encoding an antigen-binding domain targeting BCMA.
  • the BCMA-targeting antigen-binding domain comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 9.
  • the BCMA-targeting antigen-binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 4.
  • the BCMA-targeting antigen-binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 3.
  • the BCMA-targeting antigen-binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2.
  • the BCMA-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence shown in SEQ ID NO:2, the HCDR2 comprising the amino acid sequence shown in SEQ ID NO:3
  • the amino acid sequence of HCDR3 includes the amino acid sequence shown in SEQ ID NO:4.
  • the BCMA-targeting antigen-binding domain includes H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 includes SEQ.
  • the BCMA-targeting antigen binding domain comprises H-FR2
  • the H-FR2 is located between the HCDR1 and the HCDR2
  • the H-FR2 comprises SEQ ID NO: 6 The amino acid sequence shown.
  • the BCMA-targeting antigen binding domain comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO: 7 The amino acid sequence shown.
  • the BCMA-targeting antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: The amino acid sequence shown in 8.
  • the BCMA-targeting antigen binding domain comprises VH, and the VH comprises SEQ The amino acid sequence shown in ID NO:9.
  • the BCMA-targeting antigen-binding domain comprises at least one CDR in the heavy chain variable region VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 17.
  • the BCMA-targeting antigen-binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the BCMA-targeting antigen-binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11 (LGS).
  • the BCMA-targeting antigen-binding domain comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10.
  • the BCMA-targeting antigen binding domain comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the LCDR2 comprises SEQ ID NO: 11 (LGS ), and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 12.
  • the BCMA-targeting antigen-binding domain includes L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1
  • the L-FR1 includes SEQ.
  • the BCMA-targeting antigen binding domain comprises L-FR2
  • the L-FR2 is located between the LCDR1 and the LCDR2
  • the L-FR2 comprises SEQ ID NO: 14 The amino acid sequence shown.
  • the BCMA-targeting antigen binding domain comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO: 15 The amino acid sequence shown.
  • the BCMA-targeting antigen binding domain comprises L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO: The amino acid sequence shown in 16.
  • the BCMA-targeting antigen-binding domain in the expression vector includes VL, and the VL includes the amino acid sequence shown in SEQ ID NO: 17.
  • the BCMA-targeting antigen-binding domain in the expression vector includes an scFv, and the scFv includes the amino acid sequence shown in SEQ ID NO: 19.
  • the sequence encoding the scFv in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 20.
  • the BCMA-targeting chimeric antigen receptor comprises a costimulatory signal region, wherein the costimulatory signal region comprises an intracellular co-stimulatory signal region derived from one or more proteins selected from the group consisting of: Stimulation signal area: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88.
  • Stimulation signal area CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-
  • the costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.
  • the costimulatory signal region is an intracellular costimulatory signal region derived from CD28.
  • the sequence encoding the costimulatory signal region in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 59.
  • the BCMA-targeting chimeric antigen receptor includes an intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of: Signal region: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma herpesvirus (HSKV) ), DAP10, DAP-12 and domains containing at least one ITAM.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpesvirus
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the sequence encoding the intracellular signaling domain in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 61.
  • the chimeric antigen receptor targeting BCMA includes a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ ,
  • the transmembrane region is a transmembrane region derived from CD8 ⁇ .
  • the transmembrane region is a transmembrane region derived from CD28.
  • sequence encoding the transmembrane region in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 55.
  • the chimeric antigen receptor targeting BCMA includes a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4 , IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4 , IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region is a hinge region derived from CD8 ⁇ .
  • sequence encoding the hinge region in the expression vector includes the sequence shown in SEQ ID NO: 51 Nucleic acid sequence.
  • the BCMA-targeting chimeric antigen receptor includes a signal peptide.
  • the signal peptide comprises a signal peptide derived from a CD8 ⁇ protein.
  • sequence encoding the signal peptide in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 49.
  • the GPRC5D-targeting antigen-binding domain comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragments include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH, and/or dAb.
  • the antigen-binding fragment targeting the antigen-binding domain of GPRC5D comprises a VHH.
  • the GPRC5D-targeting antigen binding domain comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VH targeting the antigen-binding domain of GPRC5D comprises any one of SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: 46 The amino acid sequence shown.
  • the sequence encoding the VH targeting the antigen-binding domain of GPRC5D in the expression vector includes SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 40, and SEQ ID NO : The nucleic acid sequence shown in any one of 47.
  • the GPRC5D-targeting antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67.
  • the HCDR3 targeting the antigen-binding domain of GPRC5D comprises any one of SEQ ID NO: 23, SEQ ID NO: 32, SEQ ID NO: 37, and SEQ ID NO: 43 The amino acid sequence shown.
  • the GPRC5D-targeting antigen binding domain comprises HCDR2, and the HCDR2 comprises SEQ ID NO: 68 ( IX 1 X 2 is S or W, X 3 is G or S, X 4 is D or G, and X 5 is the amino acid sequence represented by N, S or T).
  • the HCDR2 targeting the antigen-binding domain of GPRC5D comprises any one of SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 36, and SEQ ID NO: 42. The amino acid sequence shown.
  • the GPRC5D-targeting antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 69.
  • the HCDR1 targeting the antigen-binding domain of GPRC5D comprises any one of SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:35, and SEQ ID NO:41 The amino acid sequence shown.
  • the GPRC5D-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 includes the amino acid sequence shown in SEQ ID NO:69, the HCDR2 includes SEQ ID NO:68 (IX 1 X 2 X 3 X 4 GX 5 T, X 1 is N or T, X 2 is S Or W, X3 is G or S, X4 is D or G, X5 is N, S or T), and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 67.
  • the GPRC5D-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:
  • the The HCDR3 includes the amino acid sequence shown in any one of SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:37 and SEQ ID NO:43.
  • the GPRC5D-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3, which comprise an amino acid sequence selected from any of the following groups: (1) HCDR1: SEQ ID NO:21; HCDR2: SEQ ID NO:22; and HCDR3: SEQ ID NO:23; (2) HCDR1: SEQ ID NO:30; HCDR2: SEQ ID NO:31; and HCDR3: SEQ ID NO:32; ( 3) HCDR1: SEQ ID NO:35; HCDR2: SEQ ID NO:36; and HCDR3: SEQ ID NO:37; and (4) HCDR1: SEQ ID NO:41; HCDR2: SEQ ID NO:42; and HCDR3: SEQ ID NO:43.
  • the GPRC5D-targeting antigen-binding domain comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 comprises SEQ.
  • the GPRC5D-targeting antigen binding domain comprises H-FR2
  • the H-FR2 is located between the HCDR1 and the HCDR2
  • the H-FR2 comprises SEQ ID NO: 70 The amino acid sequence shown.
  • the H-FR2 targeting the antigen-binding domain of GPRC5D comprises the amino acid sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 38 .
  • the GPRC5D-targeting antigen binding domain comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO: 71 The amino acid sequence shown.
  • the H-FR3 targeting the antigen-binding domain of GPRC5D comprises the amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 45.
  • the GPRC5D-targeting antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: The amino acid sequence shown in 27.
  • the GPRC5D-targeting antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 24 , the H-FR2 includes the amino acid sequence shown in SEQ ID NO:70, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:71, and the H-FR4 includes Contains the amino acid sequence shown in SEQ ID NO:27.
  • the GPRC5D-targeting antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 24 , the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:25, SEQ ID NO:38 and SEQ ID NO:44, and the H-FR3 includes SEQ ID NO:26 or SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 27, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 27.
  • the GPRC5D-targeting antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1, H-FR2, H-FR3 and H- FR4 includes any amino acid sequence selected from the following group: (1) H-FR1: SEQ ID NO: 24, H-FR2: SEQ ID NO: 25, H-FR3: SEQ ID NO: 26, and H-FR4: SEQ ID NO:27; (2) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:25, H-FR3: SEQ ID NO:26, and H-FR4: SEQ ID NO:27; (3) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:38, H-FR3: SEQ ID NO:26, and H-FR4: SEQ ID NO:27; and (4) H- FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:44, H-FR3: SEQ ID NO:45, and H
  • the GPRC5D-targeting antigen binding domain comprises a VH
  • the VH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VH targeting the antigen-binding domain of GPRC5D comprises any one of SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: 46 The amino acid sequence shown.
  • sequence encoding the VH in the expression vector includes any one of SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 40 and SEQ ID NO: 47. Nucleic acid sequence.
  • the GPRC5D-targeting antigen binding domain comprises a VHH
  • the VHH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VHH targeting the antigen-binding domain of GPRC5D comprises any one of SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: 46. The amino acid sequence shown.
  • sequence encoding the VHH in the expression vector includes any one of SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 40 and SEQ ID NO: 47. Nucleic acid sequence.
  • the chimeric antigen receptor targeting GPRC5D comprises a costimulatory signal region, wherein the costimulatory signal region comprises an intracellular co-stimulatory signal region derived from one or more proteins selected from the group consisting of: Stimulation signal area: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40 and MyD88.
  • Stimulation signal area CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B
  • the costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.
  • the costimulatory signal region is an intracellular costimulatory domain derived from CD28.
  • the costimulatory signal region includes the amino acid sequence shown in SEQ ID NO: 56 or SEQ ID NO: 58.
  • the sequence encoding the costimulatory signal region in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 59.
  • the chimeric antigen receptor targeting GPRC5D includes an intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of: Signal region: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma herpesvirus (HSKV) ), DAP10, DAP-12 and domains containing at least one ITAM.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpesvirus
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 60.
  • the sequence encoding the intracellular signaling domain in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 61.
  • the chimeric antigen receptor targeting GPRC5D includes a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD
  • the transmembrane region is a transmembrane region derived from CD8 ⁇ .
  • the transmembrane region is a transmembrane region derived from CD28.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:52 or SEQ ID NO:54.
  • sequence encoding the transmembrane region in the expression vector includes the nucleic acid sequence shown in any one of SEQ ID NO: 53 or SEQ ID NO: 55.
  • the chimeric antigen receptor targeting GPRC5D includes a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4 , IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4 , IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and L
  • the hinge region is a hinge region derived from CD8 ⁇ .
  • the hinge region includes the amino acid sequence set forth in SEQ ID NO: 50.
  • sequence encoding the hinge region in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 51.
  • the chimeric antigen receptor targeting GPRC5D includes a signal peptide.
  • the signal peptide is a signal peptide derived from a CD8 ⁇ protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 48.
  • sequence encoding the signal peptide in the expression vector includes the nucleic acid sequence shown in SEQ ID NO: 49.
  • the nucleic acid sequence encoding a BCMA-targeting chimeric antigen receptor and the nucleic acid sequence encoding a GPRC5D-targeting chimeric antigen receptor are expressed by encoding a cleavage peptide. sequence connection.
  • the cleaved peptide is a 2A peptide.
  • the cleavage peptide is selected from the group consisting of P2A, T2A, F2A, and E2A.
  • the cleavage peptide comprises the amino acid sequence set forth in SEQ ID NO: 62.
  • the sequence encoding the cleavage peptide in the expression vector includes the amino acid sequence shown in SEQ ID NO: 63.
  • the 3' end of the nucleic acid sequence encoding a chimeric antigen receptor targeting BCMA and the 3' end of the nucleic acid sequence encoding a chimeric antigen receptor targeting GPRC5D The 5' ends are connected via a cleaved peptide.
  • the 3' end of the nucleic acid sequence encoding a chimeric antigen receptor targeting GPRC5D and the 3' end of the nucleic acid sequence encoding a chimeric antigen receptor targeting BCMA The 5' ends are connected via a cleaved peptide.
  • the expression vector further comprises a nucleic acid sequence encoding a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-associated protein or fragment thereof comprises one or more selected from the group consisting of: low-density lipoprotein receptor-associated protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 64.
  • the low-density lipoprotein receptor-related protein or fragment thereof is encoded
  • the sequence of contains the nucleic acid sequence shown in SEQ ID NO:65.
  • the 5' end of the nucleic acid sequence encoding low-density lipoprotein receptor-related protein or a fragment thereof is identical to the nucleic acid encoding a chimeric antigen receptor targeting GPRC5D.
  • the 3' ends of the sequences are connected by a cleaved peptide.
  • the 5' end of the nucleic acid sequence encoding low-density lipoprotein receptor-related protein or a fragment thereof is identical to the nucleic acid encoding a chimeric antigen receptor targeting GPRC5D.
  • the 3' ends of the sequences are connected by a cleaved peptide.
  • the expression vector contains, from the 5' end to the 3' end, a sequence encoding a signal peptide, a sequence encoding a chimeric antigen receptor targeting BCMA, a sequence encoding a spliced peptide, and a sequence encoding a target. Sequences of chimeric antigen receptors to GPRC5D, sequences encoding cleavage peptides, and sequences encoding low-density lipoprotein receptor-related proteins or fragments thereof.
  • the expression vector contains, from the 5' end to the 3' end, a sequence encoding a signal peptide, a sequence encoding a chimeric antigen receptor targeting GPRC5D, a sequence encoding a spliced peptide, and a sequence encoding a target. Sequences of chimeric antigen receptors to BCMA, sequences encoding cleavage peptides, and sequences encoding low-density lipoprotein receptor-related proteins or fragments thereof.
  • the present application also provides a cell comprising the chimeric antigen receptor, the nucleic acid molecule, or the expression vector.
  • the present application also provides a cell comprising and/or expressing a chimeric antigen receptor targeting BCMA, and a chimeric antibody receptor targeting GPRC5D.
  • the BCMA-targeting chimeric antibody receptor comprises an antigen-binding domain that targets BCMA.
  • the BCMA-targeting antigen-binding domain comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment includes Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antigen-binding fragment targeting the antigen-binding domain of BCMA includes a scFv.
  • the BCMA-targeting antigen binding domain comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid set forth in SEQ ID NO: 9 sequence.
  • the BCMA-targeting antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 4.
  • the BCMA-targeting antigen binding domain comprises HCDR2, And the HCDR2 includes the amino acid sequence shown in SEQ ID NO:3.
  • the BCMA-targeting antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2.
  • the antigen-binding domain of BCMA comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence shown in SEQ ID NO:2, the HCDR2 comprising SEQ ID NO : The amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 4.
  • the antigen-binding domain of BCMA comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 -FR1 contains the amino acid sequence shown in SEQ ID NO:5.
  • the antigen binding domain of BCMA comprises H-FR2
  • the H-FR2 is located between the HCDR1 and the HCDR2
  • the H-FR2 comprises SEQ The amino acid sequence shown in ID NO:6.
  • the antigen-binding domain of BCMA comprises H-FR3
  • the H-FR3 is located between the HCDR2 and the HCDR3
  • the H-FR3 comprises SEQ.
  • the antigen-binding domain of BCMA comprises H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises The amino acid sequence shown in SEQ ID NO:8.
  • the BCMA-targeting antigen-binding domain comprises a VH
  • the VH comprises the amino acid sequence set forth in SEQ ID NO: 9.
  • the BCMA-targeting antigen binding domain comprises at least one CDR in the heavy chain variable region VL, and the VL comprises the amino acid set forth in SEQ ID NO: 17 sequence.
  • the BCMA-targeting antigen-binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the BCMA-targeting antigen-binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11 (LGS).
  • the BCMA-targeting antigen-binding domain comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10.
  • the BCMA-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, the LCDR2 comprising The amino acid sequence shown in SEQ ID NO: 11 (LGS), and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 12.
  • the BCMA-targeting antigen-binding domain comprises L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1
  • the The L-FR1 includes the amino acid sequence shown in SEQ ID NO:13.
  • the BCMA-targeting antigen binding domain comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 Contains the amino acid sequence shown in SEQ ID NO:14.
  • the BCMA-targeting antigen binding domain comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 Contains the amino acid sequence shown in SEQ ID NO:15.
  • the BCMA-targeting antigen binding domain comprises L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L- FR4 contains the amino acid sequence shown in SEQ ID NO:16.
  • the BCMA-targeting antigen-binding domain comprises a VL
  • the VL comprises the amino acid sequence set forth in SEQ ID NO: 17.
  • the BCMA-targeting antigen-binding domain scFv, and the scFv comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the BCMA-targeting chimeric antigen receptor comprises a costimulatory signal region, wherein the costimulatory signal region comprises one or more genes selected from the group consisting of: Intracellular costimulatory signal region of species proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40 and MyD88.
  • the costimulatory signal region comprises one or more genes selected from the group consisting of: Intracellular costimulatory signal region of species proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3,
  • the costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.
  • the costimulatory signal region is an intracellular costimulatory domain derived from CD28.
  • the costimulatory signal region includes the amino acid sequence shown in SEQ ID NO: 56 or SEQ ID NO: 58.
  • the chimeric antigen receptor targeting BCMA includes an intracellular signaling domain comprising one derived from the group consisting of or Intracellular signaling regions of multiple proteins: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP -12 and a domain containing at least one ITAM.
  • intracellular signaling domain comprising one derived from the group consisting of or Intracellular signaling regions of multiple proteins: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV)
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 60.
  • the chimeric antigen receptor targeting BCMA includes a transmembrane region comprising a protein derived from one or more proteins selected from the group consisting of: Transmembrane domain: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • Transmembrane domain CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CT
  • the transmembrane region is a transmembrane region derived from CD8 ⁇ .
  • the transmembrane region is a transmembrane region derived from CD28.
  • the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 54.
  • the chimeric antigen receptor targeting BCMA includes a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: : CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM , CD30 and LIGHT.
  • a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: : CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM , CD30 and LIGHT
  • the hinge region is a hinge region derived from CD8 ⁇ .
  • the hinge region includes the amino acid sequence set forth in SEQ ID NO: 50.
  • the chimeric antigen receptor targeting BCMA includes a signal peptide.
  • the signal peptide comprises a signal peptide derived from a CD8 ⁇ protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 48.
  • the antigen-binding domain targeting GPRC5D comprises an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment includes Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antigen-binding fragment targeting the antigen-binding domain of GPRC5D includes VHH.
  • the antigen-binding domain targeting GPRC5D comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid set forth in SEQ ID NO:72 sequence.
  • the VH targeting the antigen binding domain of GPRC5D comprises SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: The amino acid sequence shown in any one of 46.
  • the antigen-binding domain targeting GPRC5D comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67.
  • the HCDR3 targeting the antigen-binding domain of GPRC5D comprises SEQ ID NO: 23, SEQ ID NO: 32, SEQ ID NO: 37, and SEQ ID NO: The amino acid sequence shown in any one of 43.
  • the GPRC5D-targeting antigen binding domain comprises HCDR2, and the HCDR2 comprises SEQ ID NO: 68 ( IX 1 1 is N or T, X 2 is S or W, X 3 is G or S, X 4 is D or G, X 5 is the amino acid sequence represented by N, S or T).
  • the HCDR2 targeting the antigen binding domain of GPRC5D comprises SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 36, and SEQ ID NO: The amino acid sequence shown in any one of 42.
  • the antigen-binding domain targeting GPRC5D comprises HCDR1
  • the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 69.
  • the HCDR1 targeting the antigen-binding domain of GPRC5D comprises SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:35, and SEQ ID NO: The amino acid sequence shown in any one of 41.
  • the antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69, the HCDR2 comprising SEQ ID NO:68(IX 1 X 2 X 3 X 4 GX 5 T, X 1 is N or T, X 2 is S or W, X 3 is G or S, S or T), and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 67.
  • the antigen binding domain targeting GPRC5D comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 35 and the amino acid sequence shown in any one of SEQ ID NO:41, the HCDR2 includes the amino acid sequence shown in any one of SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:42 The amino acid sequence of , and said HCDR3 includes SEQ The amino acid sequence shown in any one of ID NO:23, SEQ ID NO:32, SEQ ID NO:37 and SEQ ID NO:43.
  • the antigen binding domain targeting GPRC5D comprises HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from any of the following: ( 1) HCDR1: SEQ ID NO: 21; HCDR2: SEQ ID NO: 22; and HCDR3: SEQ ID NO: 23; (2) HCDR1: SEQ ID NO: 30; HCDR2: SEQ ID NO: 31; and HCDR3: SEQ ID NO:32; (3) HCDR1: SEQ ID NO:35; HCDR2: SEQ ID NO:36; and HCDR3: SEQ ID NO:37; and (4) HCDR1: SEQ ID NO:41; HCDR2: SEQ ID NO: :42; and HCDR3: SEQ ID NO:43.
  • the antigen-binding domain targeting GPRC5D comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the The H-FR1 includes the amino acid sequence shown in SEQ ID NO:24.
  • the GPRC5D-targeting antigen binding domain comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 Contains the amino acid sequence shown in SEQ ID NO:70.
  • the H-FR2 targeting the antigen-binding domain of GPRC5D comprises any of SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 38 The amino acid sequence shown in the item.
  • the GPRC5D-targeting antigen binding domain comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 Contains the amino acid sequence shown in SEQ ID NO:71.
  • the H-FR3 targeting the antigen-binding domain of GPRC5D comprises the amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 45.
  • the antigen-binding domain targeting GPRC5D comprises H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H- FR4 contains the amino acid sequence shown in SEQ ID NO:27.
  • the GPRC5D-targeting antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprising SEQ ID NO: The amino acid sequence shown in 24, the H-FR2 includes the amino acid sequence shown in SEQ ID NO:70, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:71, and the H-FR4 includes SEQ ID The amino acid sequence shown in NO:27.
  • the GPRC5D-targeting antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprising SEQ ID NO:
  • the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 38 and SEQ ID NO: 44
  • the H- FR3 includes the amino acid sequence shown in SEQ ID NO:26 or SEQ ID NO:45
  • the H-FR4 includes the amino acid sequence shown in SEQ ID NO:27.
  • the antigen binding domain targeting GPRC5D comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1, H-FR2, H-FR3 and H-FR4 comprise any one of the following amino acid sequences: (1) H-FR1: SEQ ID NO: 24, H-FR2: SEQ ID NO: 25, H-FR3: SEQ ID NO: 26 , and H-FR4: SEQ ID NO:27; (2) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:25, H-FR3: SEQ ID NO:26, and H-FR4: SEQ ID NO:27; (3) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:38, H-FR3: SEQ ID NO:26, and H-FR4: SEQ ID NO:27; and (4) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:44, H-FR3: SEQ ID NO:45,
  • the GPRC5D-targeting antigen binding domain comprises a VH
  • the VH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VH targeting the antigen binding domain of GPRC5D comprises SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: The amino acid sequence shown in any one of 46.
  • the antigen-binding domain targeting GPRC5D comprises a VHH
  • the VHH comprises the amino acid sequence set forth in SEQ ID NO: 72.
  • the VHH targeting the antigen binding domain of GPRC5D comprises SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: The amino acid sequence shown in any one of 46.
  • the chimeric antigen receptor targeting GPRC5D comprises a costimulatory signal region, wherein the costimulatory signal region comprises one or more genes selected from the group consisting of: Intracellular costimulatory signal region of species proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40 and MyD88.
  • the costimulatory signal region comprises one or more genes selected from the group consisting of: Intracellular costimulatory signal region of species proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3,
  • the costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.
  • the costimulatory signal region is an intracellular costimulatory domain derived from CD28.
  • the costimulatory signal region comprises the amino acid sequence set forth in SEQ ID NO: 56 or SEQ ID NO: 58.
  • the chimeric antigen receptor targeting GPRC5D includes an intracellular signaling conduction domain, the intracellular signaling domain comprising an intracellular signaling region derived from one or more proteins selected from the following group: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine Leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12 and domains containing at least one ITAM.
  • the intracellular signaling domain comprising an intracellular signaling region derived from one or more proteins selected from the following group: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine Leukemia virus gp30
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 60.
  • the chimeric antigen receptor targeting GPRC5D includes a transmembrane region comprising a protein derived from one or more proteins selected from the group consisting of: Transmembrane domain: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • Transmembrane domain CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3
  • the transmembrane region is a transmembrane region derived from CD8 ⁇ .
  • the transmembrane region is a transmembrane region derived from CD28.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:52 or SEQ ID NO:54.
  • the chimeric antigen receptor targeting GPRC5D includes a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: : CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM , CD30 and LIGHT.
  • a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: : CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM , CD30
  • the hinge region is a hinge region derived from CD8 ⁇ .
  • the hinge region includes the amino acid sequence set forth in SEQ ID NO: 50.
  • the chimeric antigen receptor targeting GPRC5D includes a signal peptide.
  • the signal peptide comprises a signal peptide derived from a CD8 ⁇ protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 48.
  • the cells further comprise and/or express low-density lipoprotein receptor-related protein or fragments thereof.
  • the low-density lipoprotein receptor-associated protein or fragment thereof comprises one or more selected from the group consisting of: low-density lipoprotein receptor-associated protein 1-12 and its functional fragments.
  • the low-density lipoprotein receptor-associated protein or fragment thereof is low-density lipoprotein receptor-associated protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 64.
  • the cells comprise immune effector cells.
  • the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, Peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • NK cells natural killer cells
  • NKT cells monocytes, dendritic cells
  • monocytes granulocytes
  • lymphocytes lymphocytes
  • leukocytes leukocytes
  • Peripheral blood mononuclear cells embryonic stem cells
  • lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells are T cells.
  • the present application also provides a method for preparing the chimeric antigen receptor, which method includes culturing the cells under conditions such that the chimeric antigen receptor is expressed.
  • the present application also provides a method for preparing modified immune effector cells, which includes introducing the expression vector into the immune effector cells.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor, the nucleic acid molecule, the expression vector, and/or the cell, and optionally a pharmaceutically acceptable Carrier.
  • this application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the cell, and/or the pharmaceutical composition in the preparation of medicines. These medicines are used to prevent, treat and/or alleviate diseases and/or conditions.
  • the present application also provides a method for preventing, treating and/or alleviating diseases and/or disorders, the method comprising administering the cells, and/or the medicine to a subject in need combination.
  • this application also provides the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the cell, and/or the pharmaceutical composition, which is used for prevention, treatment and /or alleviation of disease and/or condition.
  • the diseases and/or disorders include diseases and/or disorders associated with aberrant expression of GPRC5D.
  • the diseases and/or conditions include those associated with abnormal expression of BCMA.
  • the disease and/or disorder includes tumors.
  • the tumor includes a solid tumor.
  • the tumors include non-solid tumors.
  • the tumors include hematologic tumors and/or lymphomas.
  • the tumor includes myeloma.
  • the myeloma includes refractory/relapsed multiple myeloma.
  • Figure 1 shows a schematic flow chart based on cell panning and solid phase panning technology.
  • Figure 2 shows a schematic diagram of the structural connection of the bispecific chimeric antigen receptor.
  • Figure 3 shows the molecular schematic diagram of the tandem and parallel structures of the bispecific chimeric antigen receptor.
  • Figure 4 shows flow cytometric detection of BCMA and GPRC5D antigen expression on the target cell surface.
  • Figure 5 shows the expression of CAR genes on the surface of T cells 72 hours after lentiviral transduction of dual-specific chimeric antigen receptors.
  • Figure 6 shows the positive rate of T cells after exemplary lentivirus infection with dual-specific chimeric antigen receptor OriC321 CAR.
  • Figure 7 shows the in vitro proliferation curve of bispecific CAR-T cells targeting BCMA and GPRC5D.
  • Figure 8 shows the in vitro expansion activity rate of bispecific CAR-T cells targeting BCMA and GPRC5D.
  • Figure 9 shows an exemplary in vitro proliferation curve of dual-specific chimeric antigen receptor OriC321 CAR-T cells.
  • Figure 10 shows the killing efficiency of bispecific CAR-T cells targeting BCMA and GPRC5D at an effect-to-target ratio of 3:1.
  • Figure 11 shows the killing efficiency of bispecific CAR-T cells targeting BCMA and GPRC5D under different effective-to-target ratios.
  • Figure 12 shows the killing efficiency of exemplary dual-specific chimeric antigen receptor OriC321 CAR-T cells under different efficacy-to-target ratios.
  • Figure 13 shows the degranulated CD107a expression levels of bispecific CAR-T cells co-cultured with different target cells.
  • Figure 14 shows an exemplary dual-specific chimeric antigen receptor OriC321 CAR-T cell immune synapse confocal imaging image.
  • Figure 15 shows quantitative analysis of microtubule organizing center (MTOC) and synaptic distance.
  • MTOC microtubule organizing center
  • Figure 16 shows the level of cytokine IL-2 secretion after bispecific CAR-T cells were co-incubated with target cells.
  • Figure 17 shows the level of cytokine IFN- ⁇ secretion after bispecific CAR-T cells were co-incubated with target cells.
  • Figure 18 shows the targeted proliferation curve of bispecific CAR-T cells targeting BCMA and GPRC5D.
  • Figure 19 shows the flow chart of the in vivo tumor inhibition experiment in NSG mice targeting BCMA and GPRC5D bispecific CAR-T cells.
  • Figure 20 shows the in vivo anti-tumor activity of NSG mice targeting BCMA and GPRC5D bispecific CAR-T cells.
  • Figure 21 shows the gene knockout efficiency of BCMA and GPRC5D in MM.1S target cells.
  • Figure 22 shows the flow chart of in vivo tumor inhibition experiments in NSG mice bearing tumor-bearing MM.1S target cells based on gene knockout.
  • Figure 23 shows the anti-tumor activity of bispecific CAR-T cells targeting BCMA and GPRC5D on target gene knockout in vivo tumor models.
  • Figure 24 shows the cross-binding activity of GPRC5D VHH -hFc antibodies to target antigens of different species.
  • Figure 25 shows a summary of the cross-binding activities of GPRC5D VHH -hFc antibodies with target antigens of different species.
  • antibody is used to include intact antibodies and binding fragments thereof. Typically, a fragment competes with the intact antibody from which it is derived for specific binding to the antigen.
  • the antibody or binding fragment thereof can be chemically conjugated to other proteins, or expressed as a fusion protein with other proteins.
  • the antibody can be a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
  • the binding protein of the antibody or binding fragment thereof may include GPRC5D.
  • the antibody or binding fragment thereof may be specific for GPRC5D.
  • antigen-binding fragment refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody.
  • the antigen-binding fragments may include Fab, Fab', F(ab')2, Fv fragments and single chain Fv fragments, tandem Fv fragments, VHH, bispecific antibodies.
  • the antigen-binding fragment may be VHH.
  • the antigen-binding fragment can bind GPRC5D.
  • the antigen-binding fragment may be specific for GPRC5D.
  • VHH generally refers to an antibody comprising the variable antigen binding domain of a heavy chain antibody.
  • VHH can also be called Nanobody (Nb) and/or single domain antibody.
  • Nb Nanobody
  • the VHH can bind GPRC5D.
  • the VHH may be specific for GPRC5D.
  • scFv generally refers to a single-chain antibody, which is an antibody composed of a heavy chain variable region and a light chain variable region connected directly or through a linking molecule (eg, a linking peptide).
  • the structure of the scFv, from the N end to the C end, can be heavy chain variable region - light chain variable region, light chain variable region - heavy chain variable region, heavy chain variable region - connecting peptide - light chain.
  • Variable region, or light chain variable region-linker peptide-heavy chain variable region can be heavy chain variable region - light chain variable region, light chain variable region - heavy chain variable region, heavy chain variable region - connecting peptide - light chain.
  • the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region.
  • the term "heavy chain constant region” consists of three domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region.
  • the term "light chain constant region” consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs).
  • CDRs complementarity-determining regions
  • Each VH and VL consists of three CDRs arranged from the amino terminus to the carboxyl terminus in the following order It consists of four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors.
  • G protein-coupled receptor family C group 5 member D (GPRC5D) protein is an atypical surface orphan receptor.
  • GPRCD5 like other C5 family receptors, has a short amino terminus, so it is conformationally very similar to the C4 family. Its expression in normal tissues is restricted to hair follicles, but it is also significantly expressed in the bone marrow of multiple myeloma patients and is highly correlated with plasma cell tumor burden and genetic aberrations.
  • GPRC5D in this application may specifically refer to GPRC5D expressed in MM patients.
  • BCMA is used interchangeably with “CD269", “BCM” and “TNFRSF17” and generally refers to B cell maturation antigen.
  • BCMA protein is a member of the tumor necrosis factor receptor family.
  • the term “BCMA” may include proteins containing mutations, for example, may include proteins containing point mutations, fragments, insertions, deletions and splice variants of full-length wild-type BCMA.
  • the BCMA may include those from any vertebrate, for example, primates (eg, humans or monkeys), rodents (mouse or rat), avian and/or livestock, and the like.
  • BCMA may also include a portion of the intact BCMA protein, as long as the relevant biological activity is retained.
  • the BCMA can be human BCMA, and its GenBank accession number is BAB60895.1.
  • human BCMA is typically a 184 amino acid long protein encoded by a 994 nucleotide long primary mRNA transcript (NM_001192.2).
  • chimeric antigen receptor generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds an antigen or target.
  • a hinge region is included between the extracellular domain and the transmembrane region.
  • the chimeric antigen receptor may also include low-density lipoprotein receptor-related protein or a fragment thereof.
  • the chimeric antigen receptor may include a signal peptide. Binding of the extracellular domain of CAR to target antigens on the surface of target cells causes CAR clustering and delivers activating stimuli to CAR-containing cells.
  • CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis and/or production of molecules capable of mediating death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner .
  • MHC major histocompatibility
  • the extracellular structure can specifically bind GPRC5D.
  • intracellular domain is meant to include any truncated portion of an intracellular domain that is sufficient to transduce an activation signal.
  • the intracellular domain may include an intracellular signaling domain and/or a costimulatory signaling region.
  • intracellular signaling domain refers to an intracellular region that can generate signals that promote immune effector function of a CAR-containing cell (eg, a CART cell or a CAR-expressing NK cell).
  • the intracellular signaling region may comprise an intracellular signaling region of one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30 , Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP-12 and domains containing at least one ITAM.
  • the intracellular signaling region may be a signaling domain derived from CD3 ⁇ .
  • costimulatory signaling region refers to the intracellular signaling region The part of the CAR domain that is capable of transducing effector signals.
  • the costimulatory signal region may include an intracellular costimulatory signal region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40 and MyD88.
  • the costimulatory signal region may be an intracellular costimulatory signal region derived from 4-1BB.
  • the term "transmembrane region” refers to a domain of a peptide, polypeptide or protein capable of spanning the plasma membrane of a cell. These domains can be used to anchor the extracellular domain to the cell membrane.
  • the transmembrane region may comprise a transmembrane domain of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • the transmembrane region
  • the term "hinge region” refers to the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain, e.g., from about position 216 to about position 230 according to the EU numbering system of Kabat.
  • the hinge region is usually a dimer molecule composed of two polypeptides with the same amino acid sequence.
  • the hinge region generally includes about 25 amino acid residues and is flexible, allowing independent movement of the antigen-binding region.
  • the hinge region can be subdivided into 3 domains: upper, middle and lower hinge domains.
  • the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region may be derived from the hinge region of CD8.
  • Low-density lipoprotein receptor-related protein refers to a cell surface protein that is an endocytic receptor and is widely distributed in the body and has a large inter-tissue structure. Difference, the main function is to absorb cholesterol into cells for cell proliferation and synthesis of sterol hormones and bile salts.
  • the low-density lipoprotein receptor-related protein can come from any vertebrate.
  • the The low-density lipoprotein receptor-related protein or its fragment may be located at the C-terminus of the intracellular signaling region.
  • the low-density lipoprotein receptor-related protein or its fragment may comprise one or more selected from the group consisting of: : low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof may be low-density lipoprotein receptor-related protein 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof may be low-density lipoprotein receptor-related protein 5 or a fragment thereof.
  • signal peptide refers to the leader sequence at the amino terminus (N-terminus) of the nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequent surface expression during or after translation.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • nucleic acid molecule includes DNA molecules and RNA molecules.
  • a nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • promoter generally refers to a DNA sequence that regulates the expression of a selected DNA sequence operably linked to the promoter, thereby affecting the expression of the selected DNA sequence in a cell.
  • the nucleic acid molecule may encode the corresponding antigen-binding protein and/or the chimeric antigen receptor.
  • the nucleic acid molecule may include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the term "vector” generally refers to a molecule to which one or more nucleic acid molecules of the invention can be attached.
  • the vector may be a viral vector.
  • the vector may be a lentiviral vector.
  • the term “cell” refers to a cell into which a nucleic acid can be transfected, and the term “cell” includes prokaryotic cells for propagation of plasmids and eukaryotic cells for expression of nucleic acids and production of encoded polypeptides.
  • cells may include the chimeric antigen receptor, the nucleic acid molecule and/or the vector.
  • the cells may be immune effector cells.
  • immune effector cells generally refers to immune cells that participate in immune responses and perform effector functions. For example, the exercise of effector functions may include clearing foreign antigens or promoting immune effector responses.
  • immune effector cells can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells , embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • immune effector cells can be T cells.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a mammalian subject.
  • the pharmaceutical composition may include the antigen-binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable Carrier.
  • the pharmaceutical composition can be used to prevent, treat and/or alleviate diseases or conditions associated with abnormal expression of GPRC5D.
  • the disease or disorder associated with abnormal expression of GPRC5D may include tumors.
  • the tumor includes solid tumors and/or non-solid tumors.
  • the tumors may include hematomas and/or lymphomas.
  • the tumor may include myeloma.
  • pharmaceutically acceptable carrier generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • homologs When referring to the amino acid sequence of a protein or the nucleotide sequence of a nucleic acid molecule, the present application also includes homologs of these sequences.
  • the term “homologue” generally refers to an amino acid sequence or a nucleotide sequence that has certain homology to a wild-type amino acid sequence and a wild-type nucleotide sequence.
  • the term “homology” may be equated with sequence "identity.”
  • Homology sequence Columns may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence.
  • homologs will contain active sites, etc. that are identical to the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or homology can be expressed in terms of sequence identity.
  • the term "about” generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • Chimeric antigen receptors comprising a first antigen binding domain and a second antigen binding domain
  • the present application provides a chimeric antigen receptor comprising a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain targeting B cell maturation antigen (BCMA), and the The second antigen-binding domain targets the G protein-coupled receptor family group C group 5 member D (GPRC5D) protein.
  • BCMA B cell maturation antigen
  • GPRC5D G protein-coupled receptor family group C group 5 member D
  • the CAR targeting BCMA and the CAR targeting GPRC5D can exert a synergistic effect and improve the tumor killing effect to a greater extent.
  • targeting BCMA and GPRC5D simultaneously can effectively solve the immune escape problem in tumor treatment.
  • targeting BCMA and GPRC5D simultaneously can effectively reduce the recurrence rate of tumor treatment.
  • the first antigen-binding domain may be an antigen-binding domain targeting BCMA.
  • the second antigen binding agent may comprise an antigen binding domain targeting GPRC5D.
  • the C-terminus of the first antigen-binding domain may be directly or indirectly connected to the N-terminus of the second antigen-binding domain.
  • the N-terminus of the first antigen-binding domain may be directly or indirectly connected to the C-terminus of the second antigen-binding domain.
  • the linker may comprise a peptide linker.
  • the linker may comprise the amino acid sequence of (GGGGS)n, where n may be any positive integer from 1 to 10.
  • the linker may comprise the amino acid sequence of (EAAAK)n, wherein n may be any positive integer from 1 to 10.
  • the chimeric antigen receptor may further comprise a costimulatory signal region.
  • the chimeric antigen receptor may further comprise an intracellular signaling domain.
  • the chimeric antigen receptor may further comprise a transmembrane region.
  • the chimeric antigen receptor may further comprise a hinge region.
  • the chimeric antigen receptor may further comprise a signal peptide.
  • the chimeric antigen receptor may also comprise low-density lipoprotein receptor-related protein or a fragment thereof.
  • the application also provides isolated nucleic acid molecule(s) encoding the chimeric antigen receptor.
  • the nucleic acid molecule may also comprise a sequence encoding a cleavage peptide.
  • the nucleic acid molecule may further comprise a nucleic acid sequence encoding a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the present application also provides expression vectors, which may comprise the nucleic acid molecules.
  • the chimeric antigen receptor may sequentially include a signal peptide, an antigen-binding domain targeting BCMA, a peptide linker, an antigen-binding domain targeting GPRC5D, a hinge region, and a trans-antigen binding domain from the N-terminus to the C-terminus. membrane region, costimulatory signaling region, and intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 3B5 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 3B5 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 3B5 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 3E7 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 3E7 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 3E7 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 4A2 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 4A2 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 4A2 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (GGGGS)n, 4B3 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (EAAAK)n, 3B5 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (EAAAK)n, 3E7 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (EAAAK)n, 4A2 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from N-terminus to C-terminus. signal region and CD3 ⁇ Intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, hBCMA22 scFv, (EAAAK)n, 4B3 VHH, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, 3B5 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, 3B5 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, 3B5 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • the chimeric antigen receptor can sequentially include signal peptide, 3E7 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, 3E7 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, 3E7 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • the chimeric antigen receptor can sequentially include signal peptide, 4A2 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • Signal region and amino acid sequence of CD3 ⁇ intracellular signaling domain can sequentially include signal peptide, 4A2 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus.
  • the chimeric antigen receptor can sequentially include signal peptide, 4B3 VHH, (GGGGS)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, 3B5 VHH, (EAAAK)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, 3E7 VHH, (EAAAK)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, 4A2 VHH, (EAAAK)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor can sequentially include signal peptide, 4B3 VHH, (EAAAK)n, hBCMA22 scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, and 4-1BB costimulation from the N-terminus to the C-terminus. signaling region as well as the CD3 ⁇ intracellular signaling domain.
  • the present application also provides an expression vector, which may include a nucleic acid encoding a chimeric antigen receptor targeting BCMA Sequences and nucleic acid sequences encoding chimeric antigen receptors targeting GPRC5D.
  • the present application also provides cells, which may comprise the expression vector.
  • the present application also provides cells that may comprise and/or express chimeric antigen receptors targeting BCMA, and chimeric antibody receptors targeting GPRC5D.
  • the cells may further comprise and/or express low-density lipoprotein receptor-related protein or fragments thereof.
  • the chimeric antigen receptor targeting BCMA may comprise an antigen-binding domain targeting BCMA.
  • the chimeric antigen receptor targeting BCMA may comprise a costimulatory signal region.
  • the chimeric antigen receptor targeting BCMA may comprise an intracellular signaling domain.
  • the chimeric antigen receptor targeting BCMA may comprise a transmembrane region.
  • the chimeric antigen receptor targeting BCMA may comprise a hinge region.
  • the chimeric antigen receptor targeting BCMA may comprise a signal peptide.
  • the chimeric antigen receptor targeting GPRC5D may comprise an antigen-binding domain targeting GPRC5D.
  • the chimeric antigen receptor targeting GPRC5D may comprise a costimulatory signal region.
  • the chimeric antigen receptor targeting GPRC5D may comprise an intracellular signaling domain.
  • the chimeric antigen receptor targeting GPRC5D may comprise a transmembrane region.
  • the chimeric antigen receptor targeting GPRC5D may comprise a hinge region.
  • the chimeric antigen receptor targeting GPRC5D may comprise a signal peptide.
  • the nucleic acid sequence encoding the BCMA-targeting chimeric antigen receptor in the expression vector can be connected to the nucleic acid sequence encoding the GPRC5D-targeting chimeric antigen receptor through a sequence encoding a cleavage peptide. .
  • the 3' end of the nucleic acid sequence encoding the chimeric antigen receptor targeting BCMA in the expression vector can be the same as the 5' end of the nucleic acid sequence encoding the chimeric antigen receptor targeting GPRC5D. Linked by cleaved peptides.
  • the 3' end of the nucleic acid sequence encoding the chimeric antigen receptor targeting GPRC5D and the 5' end of the nucleic acid sequence encoding the chimeric antigen receptor targeting BCMA in the expression vector are passed through Cleaved peptides are linked.
  • the expression vector may further comprise a nucleic acid sequence encoding a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the 5' end of the nucleic acid sequence encoding low-density lipoprotein receptor-related protein or its fragment in the expression vector can be the same as the 5' end of the nucleic acid sequence encoding the chimeric antigen receptor targeting GPRC5D.
  • the 3' ends are connected via a cleaved peptide.
  • the 5' end of the nucleic acid sequence encoding low-density lipoprotein receptor-related protein or its fragment in the expression vector is identical to the 3' end of the nucleic acid sequence encoding the chimeric antigen receptor targeting GPRC5D. ' ends are connected by cleaving the peptide.
  • the expression vector can sequentially contain a sequence encoding a signal peptide, a sequence encoding a chimeric antigen receptor targeting BCMA, a sequence encoding a spliced peptide, and a sequence encoding a targeting BCMA, respectively, from the 5' end to the 3' end.
  • the expression vector can sequentially contain a sequence encoding a signal peptide, a sequence encoding a chimeric antigen receptor targeting GPRC5D, a sequence encoding a spliced peptide, and a sequence encoding a targeting GPRC5D, respectively, from the 5' end to the 3' end.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the 3B5 VHH, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the 3E7 VHH, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the 4A2 VHH, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the 4B3 VHH, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD28 hinge region, a sequence encoding a CD28 transmembrane region, and a sequence encoding a CD28 transmembrane region from the 5' end to the 3' end.
  • Sequence of 4-1BB costimulatory signal region, sequence encoding CD3 ⁇ intracellular signaling domain, sequence encoding spliced peptide, sequence encoding CD8 The sequence of ⁇ signal peptide, the sequence encoding 3B5 VHH, the sequence encoding CD8 ⁇ hinge region, the sequence encoding CD8 ⁇ transmembrane region, the sequence encoding 4-1BB costimulatory signal region, the sequence encoding CD3 ⁇ intracellular signaling domain, the sequence encoding splicing Sequences of cleaved peptides, sequences encoding low-density lipoprotein receptor-related proteins or fragments thereof.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD28 hinge region, a sequence encoding a CD28 transmembrane region, and a sequence encoding a CD28 transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the 3E7 VHH, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD828 hinge region, a sequence encoding a CD28 transmembrane region, and an encoding sequence from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the 4A2 VHH, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding an hBCMA22 scFv, a sequence encoding a CD28 hinge region, a sequence encoding a CD28 transmembrane region, and a sequence encoding a CD28 transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the 4B3 VHH, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 3B5 VHH, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the hBCMA22 scFv, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 3E7 VHH, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • Sequences of the 4-1BB costimulatory signal region sequences encoding the intracellular signaling domain of CD3 ⁇ , sequences encoding spliced peptides, and sequences encoding CD8 ⁇ Sequence of signal peptide, sequence encoding hBCMA22 scFv, sequence encoding CD8 ⁇ hinge region, sequence encoding CD8 ⁇ transmembrane region, sequence encoding 4-1BB costimulatory signal region, sequence encoding CD3 ⁇ intracellular signaling domain, encoding splicing A sequence of a peptide, a sequence encoding a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 4A2 VHH, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the hBCMA22 scFv, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 4B3 VHH, a sequence encoding a CD8 ⁇ hinge region, a sequence encoding a CD8 ⁇ transmembrane region, and a sequence encoding a CD8 ⁇ transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the hBCMA22 scFv, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 3B5 VHH, a sequence encoding a CD28 hinge region, a sequence encoding a CD28 transmembrane region, and a sequence encoding a CD28 transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the hBCMA22 scFv, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 3E7 VHH, a sequence encoding a CD28 hinge region, a sequence encoding a CD28 transmembrane region, and a sequence encoding a CD28 transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the hBCMA22 scFv, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 4A2 VHH, a sequence encoding a CD28 hinge region, a sequence encoding a CD28 transmembrane region, and a sequence encoding a CD28 transmembrane region from the 5' end to the 3' end.
  • sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal The sequence of peptide No., the sequence encoding hBCMA22 scFv, the sequence encoding CD8 ⁇ hinge region, the sequence encoding CD8 ⁇ transmembrane region, the sequence encoding 4-1BB costimulatory signal region, the sequence encoding CD3 ⁇ intracellular signaling domain, the sequence encoding splicing
  • the expression vector can sequentially contain a sequence encoding a CD8 ⁇ signal peptide, a sequence encoding a 4B3 VHH, a sequence encoding a CD28 hinge region, a sequence encoding a CD28 transmembrane region, and a sequence encoding a CD28 transmembrane region from the 5' end to the 3' end.
  • the sequence of the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the CD8 ⁇ signal peptide, the sequence encoding the hBCMA22 scFv, the sequence encoding the CD8 ⁇ hinge region, the sequence encoding the CD8 ⁇ trans The sequence of the membrane region, the sequence encoding the 4-1BB costimulatory signal region, the sequence encoding the CD3 ⁇ intracellular signaling domain, the sequence encoding the spliced peptide, the sequence encoding the low-density lipoprotein receptor-related protein or its fragment.
  • the expression vector may include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the EF1 ⁇ promoter may comprise the sequence shown in SEQ ID NO: 66.
  • the expression vector may include the nucleic acid molecule.
  • the expression vector can transform, transduce or transfect host cells so that the genetic material elements they carry can be expressed in the host cells.
  • vectors can include promoters, transcripts, enhancers, replicons, selection elements, and reporter genes.
  • the vector may include components that facilitate entry into cells.
  • the 5' end and 3' end of the nucleic acid molecule may also contain long terminal repeat sequences.
  • the BCMA-targeting antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the BCMA-targeting antigen-binding domain may comprise a BCMA-targeting scFv.
  • the BCMA-targeting antigen-binding domain may comprise at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 9.
  • the BCMA-targeting antigen-binding domain may comprise HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4.
  • the BCMA-targeting antigen-binding domain may comprise HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3.
  • the BCMA-targeting antigen-binding domain may comprise HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2.
  • the antigen-binding domain of BCMA may include HCDR1, HCDR2 and HCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO:2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO:3.
  • Amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the antigen-binding domain of BCMA may include H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may include SEQ ID NO. :The amino acid sequence shown in 5.
  • the antigen-binding domain of BCMA may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise what is shown in SEQ ID NO: 6 amino acid sequence.
  • the antigen-binding domain of BCMA may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise what is shown in SEQ ID NO: 7 amino acid sequence.
  • the antigen-binding domain of BCMA may comprise H-FR4, the N-terminus of H-FR4 is connected to the C-terminus of HCDR3, and the H-FR4 may comprise SEQ ID NO:8 The amino acid sequence shown.
  • the BCMA-targeting antigen-binding domain may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 9.
  • the BCMA-targeting antigen-binding domain may comprise at least one CDR in the heavy chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 17.
  • the BCMA-targeting antigen-binding domain may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the BCMA-targeting antigen-binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11 (LGS).
  • the BCMA-targeting antigen-binding domain may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • the BCMA-targeting antigen-binding domain may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:10, and the LCDR2 may comprise SEQ ID NO:11 ( LGS), and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 12.
  • the BCMA-targeting antigen-binding domain may comprise L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise SEQ.
  • the amino acid sequence shown in ID NO:13 the BCMA-targeting antigen-binding domain may comprise L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise SEQ ID NO: 14 The amino acid sequence shown.
  • the BCMA-targeting antigen-binding domain may comprise L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise SEQ ID NO: 15 The amino acid sequence shown.
  • the BCMA-targeting antigen-binding domain may comprise L-FR4, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 may comprise SEQ ID NO: The amino acid sequence shown in 16.
  • the BCMA-targeting antigen-binding domain may comprise a VL, and the VL may comprise SEQ ID The amino acid sequence shown in NO:17.
  • the VH and VL of the BCMA-targeting antigen-binding domain can be connected through a linker.
  • the linker may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the BCMA-targeting antigen-binding domain may comprise scFv, and the scFv may comprise the amino acid sequence shown in SEQ ID NO: 19.
  • nucleic acid sequence encoding the scFv may comprise the nucleic acid sequence shown in SEQ ID NO: 20.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may contact the antigen or antigenic epitope.
  • Antibody CDRs can be determined through a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, taking Kabat/Chothia into consideration, etc. These encoding systems are known in the art and can be found, for example, at http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different encoding systems, there may be differences in the CDR area.
  • the CDR covers CDR sequences classified according to any CDR classification method; it also covers its variants, which include substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR. .
  • the homologues may be at least about 85% identical to the amino acid sequence of the CDR (for example, having at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences that have about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater) sequence homology.
  • the isolated antigen-binding proteins described herein are defined by the IMGT encoding system.
  • the scFv targeting the antigen-binding domain of BCMA may comprise hBCMA22.
  • the HCDR1 of hBCMA22 can include the amino acid sequence shown in SEQ ID NO:2
  • HCDR2 can include the amino acid sequence shown in SEQ ID NO:3
  • HCDR3 can include the amino acid sequence shown in SEQ ID NO:4
  • LCDR1 can Contains the amino acid sequence shown in SEQ ID NO:10
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:11 (LGS)
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:12
  • VH can include SEQ ID NO:
  • the amino acid sequence shown in SEQ ID NO: 17, VL may include the amino acid sequence shown in SEQ ID NO: 17.
  • the scFv of hBCMA22 may comprise the amino acid sequence shown in SEQ ID NO: 19.
  • the antigen-binding domain targeting GPRC5D may comprise an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the GPRC5D-targeting antigen-binding domain may comprise a GPRC5D-targeting scFv.
  • the CDR covers CDR sequences classified according to any CDR classification method; it also covers its variants, which include substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR. .
  • the homologues may be at least about 85% identical to the amino acid sequence of the CDR (for example, having at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences that have about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater) sequence homology.
  • the isolated antigen-binding proteins described herein are defined by the IMGT encoding system.
  • the antigen-binding domain targeting GPRC5D may comprise at least one CDR in the heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 72.
  • the VH targeting the antigen-binding domain of GPRC5D may comprise any one of SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39 and SEQ ID NO: 46 amino acid sequence.
  • the antigen-binding domain targeting GPRC5D may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 67.
  • the HCDR3 targeting the antigen-binding domain of GPRC5D may comprise any one of SEQ ID NO: 23, SEQ ID NO: 32, SEQ ID NO: 37 and SEQ ID NO: 43. amino acid sequence.
  • the antigen-binding domain targeting GPRC5D may comprise HCDR2, and the HCDR2 may comprise SEQ ID NO: 68 (IX 1 X 2 is S or W, X 3 is G or S, X 4 is D or G, and X 5 is the amino acid sequence represented by N, S or T).
  • the HCDR2 targeting the antigen-binding domain of GPRC5D may comprise any one of SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 36 and SEQ ID NO: 42 amino acid sequence.
  • the antigen-binding domain targeting GPRC5D may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 69.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:41.
  • the antigen-binding domain targeting GPRC5D may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 69, and the HCDR2 may comprise SEQ ID NO: 68 ( IX 1 X 2 X 3 X 4 GX 5 T, X 1 is N or T , X 2 is S or W, X 3 is G or S, The amino acid sequence shown in SEQ ID NO: 67, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 67.
  • the antigen-binding domain targeting GPRC5D may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:
  • the amino acid sequence shown in any one of 41, the HCDR2 may include SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 36 and the amino acid sequence shown in any one of SEQ ID NO:42, and the HCDR3 may comprise any one of SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:37 and SEQ ID NO:43
  • the amino acid sequence shown may comprise HCDR1, HCDR2 and HCDR3
  • the amino acid sequence shown in any one of 41, the HCDR2 may include SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 36 and the amino acid sequence shown in any one of SEQ ID NO:42, and the HCDR3 may comprise any one of SEQ ID NO:23, SEQ ID NO
  • the antigen-binding domain targeting GPRC5D may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise an amino acid sequence selected from any of the following groups: (1) HCDR1: SEQ ID NO:21; HCDR2: SEQ ID NO:22; and HCDR3: SEQ ID NO:23; (2) HCDR1: SEQ ID NO:30; HCDR2: SEQ ID NO:31; and HCDR3: SEQ ID NO:32; ( 3) HCDR1: SEQ ID NO:35; HCDR2: SEQ ID NO:36; and HCDR3: SEQ ID NO:37; and (4) HCDR1: SEQ ID NO:41; HCDR2: SEQ ID NO:42; and HCDR3: SEQ ID NO:43.
  • the antigen-binding domain targeting GPRC5D may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 contains SEQ ID The amino acid sequence shown in NO:24.
  • the antigen-binding domain targeting GPRC5D may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise SEQ ID NO: 70 The amino acid sequence shown.
  • the H-FR2 targeting the antigen-binding domain of GPRC5D may comprise the amino acid sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 44 and SEQ ID NO: 38.
  • the antigen-binding domain targeting GPRC5D may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO: 71 The amino acid sequence shown.
  • the H-FR3 targeting the antigen-binding domain of GPRC5D may comprise the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 45.
  • the antigen-binding domain targeting GPRC5D may comprise H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may comprise SEQ ID NO: The amino acid sequence shown in 27.
  • the antigen-binding domain targeting GPRC5D may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 24 , the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:70, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:71, and the H-FR4 may comprise SEQ ID NO:27 The amino acid sequence shown.
  • the antigen-binding domain targeting GPRC5D may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 24 , the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:25, SEQ ID NO:38 and SEQ ID NO:44, and the H-FR3 may comprise SEQ ID NO:26 or SEQ ID The amino acid sequence shown in NO:45, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO:27.
  • the antigen-binding domain targeting GPRC5D may comprise H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1, H-FR2, H-FR3 and H-FR4 It may include any amino acid sequence selected from the following groups: (1) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:25, H-FR3: SEQ ID NO:26, and H-FR4: SEQ ID NO:27; (2) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:25, H-FR3: SEQ ID NO:26, and H-FR4: SEQ ID NO:27; (3) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:38, H-FR3: SEQ ID NO:26, and H-FR4: SEQ ID NO:27; and (4) H-FR1: SEQ ID NO:24, H-FR2: SEQ ID NO:44, H -FR3: SEQ ID NO: SEQ ID
  • the antigen-binding domain targeting GPRC5D may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 72.
  • the VH targeting the antigen-binding domain of GPRC5D may comprise any one of SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39 and SEQ ID NO: 46 amino acid sequence.
  • sequence encoding the VH in the expression vector may comprise the nucleic acid shown in any one of SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 40 and SEQ ID NO: 47 sequence.
  • the antigen-binding domain targeting GPRC5D may comprise a VHH, and the VHH may comprise the amino acid sequence shown in SEQ ID NO: 72.
  • the VHH targeting the antigen-binding domain of GPRC5D may comprise any one of SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:39 and SEQ ID NO:46 amino acid sequence.
  • sequence encoding the VHH in the expression vector may comprise the nucleic acid shown in any one of SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 40 and SEQ ID NO: 47 sequence.
  • the CDR1 of 3B5 includes the amino acid sequence shown in SEQ ID NO:21
  • the CDR2 includes the amino acid sequence shown in SEQ ID NO:22
  • the CDR3 includes the amino acid sequence shown in SEQ ID NO:23
  • the VHH includes the amino acid sequence shown in SEQ ID NO:23.
  • the CDR1 of 3E7 includes the amino acid sequence shown in SEQ ID NO:30
  • the CDR2 includes the amino acid sequence shown in SEQ ID NO:31
  • the CDR3 includes the amino acid sequence shown in SEQ ID NO:32
  • the VHH includes the amino acid sequence shown in SEQ ID NO:32.
  • the CDR1 of 4A2 includes the amino acid sequence shown in SEQ ID NO:35
  • the CDR2 includes the amino acid sequence shown in SEQ ID NO:36
  • the CDR3 includes the amino acid sequence shown in SEQ ID NO:37
  • the VHH includes the amino acid sequence shown in SEQ ID NO:37.
  • the CDR1 of 4B3 includes the amino acid sequence shown in SEQ ID NO:41
  • the CDR2 includes the amino acid sequence shown in SEQ ID NO:42
  • the CDR3 includes the amino acid sequence shown in SEQ ID NO:43
  • the VHH includes the amino acid sequence shown in SEQ ID NO:42.
  • the chimeric antigen receptor, BCMA-targeting chimeric antigen receptor and/or GPRC5D-targeting chimeric antigen receptor may comprise a costimulatory signal region.
  • the costimulatory signal region may comprise an intracellular costimulatory signal region derived from one or more proteins selected from the following group: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40 , CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1 , LIGHT, JAML, CD244, CD100, ICOS, ligands of CD83, CD40 and MyD88.
  • proteins selected from the following group: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40 , CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP
  • the costimulatory signal region may be an intracellular costimulatory signal region derived from 4-1BB.
  • the costimulatory signal region may be an intracellular costimulatory domain derived from CD28.
  • the 4-1BB costimulatory signal region may include the amino acid sequence shown in SEQ ID NO: 56.
  • the CD28 costimulatory signal region may include the amino acid sequence shown in SEQ ID NO: 58.
  • sequence encoding the 4-1BB costimulatory signal region may comprise the nucleic acid sequence shown in SEQ ID NO: 57.
  • sequence encoding the CD28 costimulatory signal region may comprise the nucleic acid sequence shown in SEQ ID NO: 59.
  • the chimeric antigen receptor, BCMA-targeting chimeric antigen receptor and/or GPRC5D-targeting chimeric antigen receptor may comprise an intracellular signaling domain.
  • the intracellular signaling domain may comprise an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and domains containing at least one ITAM.
  • EBV Epstein-Barr virus
  • HSKV Kaposi's sarcoma herpesvirus
  • the intracellular signaling domain may be a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 60.
  • sequence encoding the intracellular signaling domain may comprise the nucleic acid sequence shown in SEQ ID NO: 61.
  • the chimeric antigen receptor, BCMA-targeting chimeric antigen receptor and/or GPRC5D-targeting chimeric antigen receptor may comprise a transmembrane region.
  • the transmembrane region may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1 , TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L , TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • proteins selected from the group consisting of: CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1 , TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD
  • the transmembrane region may be a transmembrane region derived from CD8 ⁇ .
  • the CD8 ⁇ transmembrane region may comprise the amino acid sequence shown in SEQ ID NO: 52.
  • the sequence encoding the CD8 ⁇ transmembrane region may comprise the nucleic acid sequence shown in SEQ ID NO: 53.
  • the transmembrane region may be a transmembrane region derived from CD28.
  • the CD28 transmembrane region may comprise the amino acid sequence shown in SEQ ID NO: 54.
  • the sequence encoding the CD28 transmembrane region may comprise the nucleic acid sequence shown in SEQ ID NO: 55.
  • the chimeric antigen receptor, BCMA-targeting chimeric antigen receptor and/or GPRC5D-targeting chimeric antigen receptor may comprise a hinge region.
  • the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region may be a hinge region derived from CD8 ⁇ .
  • the hinge region of CD8 ⁇ may comprise the amino acid sequence shown in SEQ ID NO: 50.
  • the sequence encoding the hinge region of CD8 ⁇ may comprise the nucleic acid sequence shown in SEQ ID NO: 53.
  • the chimeric antigen receptor, BCMA-targeting chimeric antigen receptor and/or GPRC5D-targeting chimeric antigen receptor may comprise a signal peptide.
  • the signal peptide is a signal peptide derived from CD8 ⁇ protein.
  • the signal peptide includes the amino acid sequence shown in SEQ ID NO: 48.
  • the sequence encoding the signal peptide includes the nucleic acid sequence shown in SEQ ID NO: 49.
  • the cleaved peptide may be a 2A peptide.
  • the cleavage peptide may be selected from the group consisting of: P2A, T2A, F2A and E2A.
  • the cleavage peptide may comprise the amino acid sequence shown in SEQ ID NO: 62.
  • the sequence encoding the cleavage peptide may comprise the nucleic acid sequence shown in SEQ ID NO: 63.
  • the low-density lipoprotein receptor-related protein or fragment thereof may comprise one or more selected from the group consisting of: low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof includes the amino acid sequence shown in SEQ ID NO: 64.
  • the sequence encoding the low-density lipoprotein receptor-related protein or fragment thereof includes the nucleic acid sequence shown in SEQ ID NO: 65.
  • the present application also provides cells, which may include the chimeric antigen receptor, the nucleic acid molecule and/or the expression vector.
  • the cells may include progeny of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical (either in morphology of the total DNA complement or in genome) to the original parent cell.
  • the cells may be immune effector cells.
  • the cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes , peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells may be T cells.
  • the present application also provides a pharmaceutical composition, which may include the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable adjuvant. agent.
  • the pharmaceutical composition may further comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable preparation of preservatives.
  • Acceptable ingredients of the composition are preferably non-toxic to the recipient at the doses and concentrations used.
  • Pharmaceutical compositions of the invention may include liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration and are generally safe, non-toxic , and is neither biologically nor otherwise undesirable.
  • the pharmaceutical compositions may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • administration of the pharmaceutical composition can be by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application also provides a method for preparing the chimeric antigen receptor.
  • the method may include culturing the cells under conditions such that the chimeric antigen receptor is expressed.
  • the present application also provides methods for preparing modified immune effector cells, which may include introducing the vector into immune cells.
  • this application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the cell, and/or the pharmaceutical composition in the preparation of medicines. These medicines are used to prevent, treat and/or alleviate diseases and/or conditions.
  • the present application also provides a method for preventing, treating and/or alleviating diseases and/or disorders, the method comprising administering the cells, and/or the medicine to a subject in need combination.
  • this application also provides the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the cell, and/or the pharmaceutical composition, which is used for prevention, treatment and /or alleviation of disease and/or condition.
  • the diseases and/or disorders may include diseases and/or disorders associated with abnormal expression of GPRC5D.
  • the diseases and/or disorders may include diseases and/or disorders associated with abnormal expression of BCMA.
  • the disease and/or condition may include tumors.
  • the tumor includes solid tumors.
  • the tumor includes non-solid tumors.
  • the tumor includes hematoma and/or lymphoma.
  • the tumor includes myeloma.
  • the myeloma includes refractory/relapsed multiple myeloma.
  • the bispecific chimeric antigen receptor sequence in this example is briefly described as follows: BCMA antigen and the spatial epitope of GPRC5D are used as target antigens, and the anti-BCMA scFv sequence is obtained by screening the natural phage antibody library based on solid-phase panning technology.
  • VH sequence is SEQ ID NO: 9
  • VL sequence is SEQ ID NO: 17
  • the anti-GPRC5D V H H sequence was obtained through screening of the synthetic nanobody library NanoOri_1.0, named 3B5 VHH (SEQ ID NO:28), 3E7 VHH (SEQ ID NO:33), 4A2 VHH (SEQ ID NO:39), 4B3 VHH (SEQ ID NO:46), specific phage antibodies
  • the library panning process is shown in Figure 1.
  • This embodiment constructs a bispecific chimeric antigen receptor CAR structure targeting BCMA and GPRC5D, an anti-BCMA chimeric antigen receptor BCMA CAR, and an anti-GPRC5D chimeric antigen receptor GPRC5D CAR.
  • the bispecific chimeric antigen receptor Antigen receptors include the signal peptide sequence (SP)* (SEQ ID NO:48) of CD8 ⁇ , the single chain antibody scFv against BCMA and the single domain antibodies V H H, (G 4 S)n and (EAAAK)n against GPRC5D.
  • the connecting peptide Linker, n is an integer from 1 to 10, the hinge region (Hinge) of CD8 ⁇ or CD28, the transmembrane region sequence (TM) of CD8 ⁇ (SEQ ID NO:52) or CD28 (SEQ ID NO:54), 4 -1BB (SEQ ID NO:56) or CD28 (SEQ ID NO:58) costimulatory domain sequence and CD3 ⁇ (SEQ ID NO:60) signaling domain sequence, the low-density lipoprotein receptor-related protein fragment is the Ori element (SEQ ID NO:64),
  • the structure of the bispecific chimeric antigen receptor includes two forms: series and parallel.
  • the structural connection diagram of the bispecific chimeric antigen receptor is shown in Figure 2.
  • the molecular diagram of the series and parallel structures of the chimeric antigen receptor As shown in Figure 3.
  • This example uses PE anti-human BCMA (Biolegend) and APC anti-human GPRC5D (R&D) antibodies to detect BCMA and GPRC5D antigen expression on the surface of the wild-type human myeloma cell line MM.1S and the constructed overexpression stable cell line CHO cells. Staining, BD flow cytometry was performed to detect the expression of BCMA and GPRC5D in target cells.
  • CAR lentiviral empty vector The existing pCore-CAR-Ori plasmid (original biotechnology, containing Ori elements, hereinafter referred to as CAR lentiviral empty vector) is used as the enzyme cutting template, and the restriction endonucleases SphI-HF and NotI-HF (purchased from NEB ), digested at 37°C, and 8900 bp linearized fragment containing sticky ends was recovered from 1% agarose gel;
  • the plasmid was transformed into competent E.coli (DH5 ⁇ ). After sequencing was correct, the plasmid was extracted and purified using a plasmid purification kit (Qiagen Company), and the plasmid concentration was measured.
  • the lentiviral vector system of the dual-specific chimeric antigen receptor belongs to the third generation.
  • the system consists of four plasmids, namely the packaging plasmid pRH1 encoding the Gag-Pol protein, the packaging plasmid pVH3 encoding the Rev protein and The pMH2 plasmid encoding the envelope protein VSV-G, and the core plasmid (BCMA and GPRC5D dual-targeting CAR encoding gene lentiviral plasmid, BCMA single-targeting CAR encoding gene lentiviral plasmid, GPRC5D single-targeting CAR encoding gene lentiviral plasmid Viral plasmid), the CAR gene in each CAR lentiviral plasmid is expressed under the control of the elongation factor-1 ⁇ (EF-1 ⁇ ) promoter.
  • EF-1 ⁇ elongation factor-1 ⁇
  • a total of 1 ⁇ 10 6 293T cells were suspended in 2 mL of DMEM medium containing 10% FBS, and cultured in a 6-well plate overnight; after about 20-24 hours, the cell confluence was observed to be about 80-90%, and the helper plasmid gag/ Add pol, Rev, VSV-G and recombinant lentiviral plasmid in corresponding proportions to serum-free medium Opti-MEM with a volume of 50x the total plasmid mass, and mix thoroughly.
  • the total plasmid volume: transfection reagent is 1:3.
  • transfection reagent FUGENE HD (Promega, E2311), mix evenly, and incubate at room temperature for 10 minutes; aspirate the appropriate amount of culture medium in the 6-well cell culture plate, and add the plasmid/FUGENE HD transfection complex dropwise to the culture plate. Shake to mix, and culture overnight at 37°C and 5% CO2 ; remove the plasmid-containing culture medium about 20 hours after transfection, replace it with 2 mL of DMEM culture medium containing 5% FBS, and culture for 48 hours; collect 2 mL of virus supernatant, 3000 rpm, 10 min Centrifuge, aliquot and freeze at -80°C.
  • FUGENE HD Promega, E2311
  • the virus liquid prepared in 4.1 suspend a total of 5 ⁇ 10 5 293T cells in 2.7 mL of DMEM medium containing 10% FBS, and inoculate it into a 6-well cell culture plate; add Polybrene reagent with a final concentration of 10 ⁇ g/mL. ; Add 2 ⁇ L, 5 ⁇ L and 10 ⁇ L of virus-containing stock solution, set 1-2 blank wells without adding virus solution as negative controls for flow cytometry testing, mix thoroughly, and incubate statically at 37°C, 5% CO 2 ; aspirate after 48 hours Remove the original culture medium, wash with 1xPBS, digest with trypsin, and take an appropriate amount.
  • the cell suspension into a flow tube, wash with 1 ⁇ PBS (containing 2% FBS), and centrifuge to remove the supernatant; add detection antibodies and incubate at 4°C for 60 minutes.
  • BD flow cytometer detects the cell positivity rate and uses FlowJo-V10 software for data processing. , the results are available only when the positive rate of each test tube - the positive rate of the negative control is 5%-20%; the virus titer calculation formula is as follows:
  • the above-mentioned CAR lentivirus titer range is 1-10 ⁇ 10 6 TU/mL.
  • the CAR-T cell preparation methods for each combination of bispecific chimeric antigen receptor CAR structures and monospecific chimeric antigen receptors BCMA CAR and GPRC5D CAR are as follows:
  • the CD3 MicroBeads kit (Miltenyi Biotec) was used to sort CD3 + T cells in PBMC. Specifically, PBMC (Ficoll density gradient centrifugation) was centrifuged at 500g for 5 minutes to remove the cryopreservation solution; the total number of cells was 1 ⁇ 10 7 Add 80 ⁇ L of pre-cooled sorting solution to resuspend the cells; add 20 ⁇ L of CD3MicroBeads to the total number of 1 ⁇ 10 7 cells, mix thoroughly, and incubate at 4°C for 15 minutes; after the incubation, add 1.5 mL of pre-cooled sorting solution to the total number of 1 ⁇ 10 7 cells.
  • PBMC Ficoll density gradient centrifugation
  • CD3/CD28 DynaBeads activate T cells, culture at 37°C, 5% CO 2 , and PBMC are sorted by CD3 positive magnetic beads to obtain CD3 + T cells with a purity of >95%.
  • Example 6 T cell CAR gene expression efficiency and in vitro amplification detection
  • CD3/CD28 DynaBeads activated T cells, after lentivirus infection, cultured for 72 hours to perform CAR-T cell CAR Gene expression efficiency detection, details are as follows:
  • CD3/CD28 DynaBeads-activated CAR-T cells lentivirus infection, cell passage according to culture time D5, D7, D9, and D13; mix the CAR-T cells thoroughly, take 20 ⁇ L cell suspension into a 1.5 mL EP tube, and add Mix equal volumes of AO/PI reagents, add 20 ⁇ L of the mixed solution to the counting plate, count under the fluorescence background of a cell counter (Auto2000), record the cell growth and draw a growth curve according to the obtained cell density, and adjust the cell passage density to 7 ⁇ 10 5 cells/mL, inoculated in a 6-well plate, and the culture period was 10-13 days; the obtained data were processed and analyzed using GraphPad 6.0.
  • OriCAR001 is OriCAR-dual06 in Figure 2
  • the structure of OriCAR002 is OriCAR-dual02 in Figure 2
  • the structure of OriCAR003 is OriCAR-dual02 in Figure 2.
  • the linker is replaced with GGGGSEAAAK
  • OriCAR004 The structure is OriCAR-dual14 in Figure 2.
  • the structure of OriCAR005 is OriCAR-dual10 in Figure 2.
  • the structure of OriCAR006 is OriCAR-dual10 in Figure 2.
  • the linker is replaced with GGGGSEAAAK.
  • the structure of OriCAR007 is OriCAR-Dual10 in Figure 2.
  • the structure of OriCAR008 is OriCAR-dual21 in Figure 2.
  • the structure of OriC321 is OriCAR-dual02 in Figure 2.
  • the results of this example are shown in Figure 7.
  • the total amplification factor of bispecific chimeric antigen receptor CAR-T cells after activation and culture for 13 days is between 100-350 times; as shown in Figure 8, after activation and culture for 13 days
  • the viability of bispecific chimeric antigen receptor CAR-T cells gradually increased with the extension of culture time, and the cell viability was maintained at about 90%; as shown in Figure 9, an exemplary bispecific chimeric antigen receptor CAR-T cell body
  • the total expansion fold of OriC321 CAR-T cells is about 400 times; compared with Mock T control cells, there is no significant difference in the expansion fold in vitro and can be used for cytological functional experiments.
  • CD3/CD28 DynaBeads activated CAR-T cells, lentivirus infection, CAR-T cytotoxicity experiment was performed according to culture time D9, the target cells were CHO-BCMA cells overexpressing BCMA and CHO-GPRC5D cells overexpressing GPRC5D; negative control The cells are blank CHO cells. CytoTox 96 non-radioactive cytotoxicity detection kit (Promega Company) was used for detection. The target cell system was mixed evenly at a ratio of CHO-BCMA:CHO-GPRC5D of 1:1; the killing efficiency of CAR-T cells was tested as follows:
  • the total number of target cells is 2 ⁇ 10 4 cells, spread in a 96-well V-bottom plate, and set different effective target ratios;
  • the obtained data were processed and analyzed using GraphPad 6.0, and unpaired t test was used to determine whether there were statistical differences. P ⁇ 0.05 was considered a significant difference.
  • the results of this example are shown in Figure 10-11. Compared with blank effector T cells, the results show that bispecific CAR-T cells specifically kill target cells with two different targets, BCMA CAR-T and GPRC5D CAR-T cells only specifically kill the corresponding target cells and have no killing activity against CHO-negative cells, revealing the targeting specificity of different bispecific CAR-Ts; as shown in Figure 12, an exemplary OriC321 CAR-T cell The killing efficiency of target cells is significantly better than that of BCMA CAR and GPRC5D CAR, with an effective-to-target ratio of 5:1 and a killing efficiency of 70%.
  • Example 8 Determination of the expression level of bispecific CAR-T cell degranulation molecule CD107a
  • This example is to detect the expression level of CD107a under the stimulation of positive target cells CHO-BCMA and CHO-GPRC5D by bispecific CAR-T targeting BCMA and GPRC5D.
  • Bispecific CAR-T cells activated by CD3/CD28 DynaBeads and control Mock T cells were cultured on D9 to detect cell degranulation level CD107a.
  • the corresponding positive rate of CAR-T cells and target cells were at an effect-to-target ratio of 1:1.
  • target cells and positive CAR-T cells were both 5 ⁇ 10 6 cells/mL, 0.6mL/well, inoculated into a 12-well cell culture plate, mixed, 37°C, 5% Incubate in CO2 , co-culture for 4-6 hours, collect cells, wash with 1 ⁇ PBS (2% FBS), centrifuge to remove the supernatant; add PE-labeled mouse anti-human CD107a antibody, APC/Cy7-labeled mouse anti-human CD3 antibody , incubate at 4°C in the dark for 30 minutes, and detect the proportion of CD107a in CD3 + T cells using BD flow cytometry.
  • the results of this example are shown in Figure 13.
  • the CD107a expression of bispecific CAR-T cells co-cultured with target cells CHO-BCMA and CHO-GPRC5D is about 15-40%.
  • the positive rate of CD107a expression of dCAR02 and dCAR09 is close to 40%.
  • Mock T control cells have almost no CD107a expression.
  • the immune synapse is the contact area where T cells and antigen-presenting cells combine, including T cell antigen receptors, corresponding antigens, and some auxiliary molecules.
  • the formation of immune synapse is also a key step for CAR-T cells to recognize antigens, proliferate and activate.
  • the formation of the immune synapse is related to the effector function of CAR-T cells.
  • the polarization of the microtubule organizing center (MTOC) of the immune synapse structure is measured to further evaluate the functional advantage of CAR-T cells.
  • MTOC microtubule organizing center
  • CAR-T cells and target cells were co-cultured at a ratio of 1:1 for 5 minutes, then transferred to Nunc chamber slides, incubated for 20 minutes, fixed with 4% PFA at room temperature, washed three times with PBS, and immunostained with permeabilization solution (Triton X-100) ), incubate at room temperature; 10% horse serum/immunostaining permeabilization solution (Triton X-100), incubate at room temperature; add primary antibody diluted in 2% horse serum/immunostaining permeabilization solution (Triton Incubate at room temperature with secondary antibodies diluted in 2% horse serum/immunostaining permeabilization solution (Triton 1024x1024, Z-axis scanning 0.4 ⁇ m, superimposed to obtain 3D images and videos.
  • the MTOC polarization level of OriC321 CAR T cells to IS is significantly increased, which is manifested as a reduction in the distance between MTOC and IS, thus obtaining superior and more stable IS structure, and therefore has superior functional advantages, indicating that OriC321 CAR T cell-mediated cytotoxicity is superior to single-targeted BCMA CAR and GPRC5D CAR-T cells.
  • Bispecific CAR-T cells were cultured on D9 to detect the factors IL-2 and IFN- ⁇ released by CAR-T cells, using R&D Company's IL-2 and IFN- ⁇ cytokine ELISA detection kits.
  • the positive rate and cell density of CAR-T cells were pre-detected, and blank T cells were used to adjust the CAR-T cells after lentivirus infection to make the total number of cells consistent between groups;
  • CAR-T cells and MM.1S cells were compared with an effect-to-target ratio of 1 Co-incubate at a ratio of :1, that is, the total number of CAR-positive cells is 2 ⁇ 10 4 and the total number of MM1S cells is 2 ⁇ 10 4.
  • the kit detects the secretion of cytokines as follows: Coat diluted capture antibodies on a 96-well ELISA plate at a concentration of 1 ⁇ g/ml and incubate at 4°C overnight. Then use 10% skim milk powder to block the non-specific binding site.
  • untreated MM.1S cells were selected as activation target cells for the detection of the targeted proliferation ability of CAR-T cells.
  • CAR-T cells activated by CD3/CD28 DynaBeads and control Mock T cells were infected with lentivirus, and the targeted proliferation ability of CAR-T cells was tested around the culture time of D9 or D13.
  • CAR-T cells and target cells were subjected to the first round of targeted stimulation at an effect-to-target ratio of 1:1, and cultured at 37°C and 5% CO2 ; after 5 days of co-incubation, all cells were collected and counted with AO/PI reagent.
  • Bispecific CAR-T cells have varying degrees of targeted proliferation capabilities after being stimulated by target cells. There is no significant difference compared with BCMA CAR-T and GPRC5D CAR-T. The proliferation times There is a significant difference between 200-800 times compared with control Mock T cells, indicating that bispecific CAR-T cells can effectively and rapidly expand against specific tumors in vivo.
  • CAR-T cells activated by CD3/CD28 DynaBeads, infected with lentivirus, and cultured on D9 for cryopreservation of CAR-T cells.
  • the positive rate and cell density (AO/PI) of CAR-T cells are pre-detected, and blank T cells are used to adjust the total number of cells between groups; the corresponding CAR-T cells are cryopreserved according to the required density of CAR-T cells and collected as needed.
  • cryopreservation solution A (Yuanqi Bio) at a volume ratio of 1:2 to resuspend the cells
  • cryopreservation solution B (Yuanqi Bio)
  • mix and aliquot Place it in a 2 mL cryopreservation tube in a programmed cooling box, freeze it at -80°C, and transfer the frozen cells to liquid nitrogen for storage after 24 hours.
  • mice (Biocytogen) were subcutaneously inoculated with MM.1S cells at a dose of 1 ⁇ 10 7 cells/200 ⁇ L/mouse. After 14 days of tumor bearing, the tumor volume of the mice was measured to be approximately 50-150 mm 3 .
  • mice were randomly divided Divided into 5 groups, with 5 animals in each group, they were control Mock T group, bispecific CAR-T group, BCMA CAR-T group; GPRC5D CAR-T group; tail vein injection method, T cells were injected into the Mock T group ( The cryopreserved dose is 6 ⁇ 10 6 cells/200 ⁇ L/bird), the corresponding CAR + T cells are injected into the bispecific CAR-T group (the cryopreserved dose is 6 ⁇ 10 6 cells/200 ⁇ L/bird), and the BCMA CAR-T The corresponding CAR + T cells were injected into the GPRC5D CAR-T group (the cryopreserved dose was 6 ⁇ 10 6 cells/200 ⁇ L/animal), and the corresponding CAR + T cells were injected into the GPRC5D CAR-T group (the cryopreserved dose was 6 ⁇ 10 6 cells/200 ⁇ L/animal).
  • CAR-T cell reinfusion After CAR-T cell reinfusion, the tumor volume was measured every Monday, Wednesday, and Friday, and vital signs such as mouse coat color, excrement, food and water intake, body movement, and mouse death were observed; after CAR-T cell reinfusion Blood was collected from the tail vein on D7, D14, and D21, and the CBA Human Th1/Th2/Th17 Cytokine Kit (BD Biosciences) was used to detect the expression of cytokines such as IL2, IL4, IL6, IL10, TNF- ⁇ , IFN- ⁇ , and IL17A in the serum. , BD flow cytometry to detect the proportion of CD3 + CD8 + T cells.
  • cytokines such as IL2, IL4, IL6, IL10, TNF- ⁇ , IFN- ⁇ , and IL17A
  • mice in the OriC321 CAR-T cell group had bright coat color and normal vital signs such as mouse behavior after injection, indicating that OriC321 CAR- Good safety of T cells in mice ( Figure 20B); Compared with the Mock T cell group, OriC321 CAR-T cells have a significant tumor elimination effect, and the tumor inhibition rate can be close to 100% (Figure 20C); Exemplary bispecificity Chimeric antigen receptor OriC321 CAR-T cells can significantly prolong the survival rate of mice ( Figure 20C); it illustrates that OriC321 CAR-T cells provide greater efficacy and survival benefits when treating MM in mouse models, and can be used in clinical applications May exhibit superior antitumor activity.
  • Example 13 Gene-knockout MM.1S target cells verify the anti-tumor activity of OriC321 CAR-T cells in vivo
  • CRISPR-Cas9 technology was used to knock out the BCMA and GPRC5D genes in the target cells respectively, and the positive cells of BCMA KO and GPRC5D KO were sorted by flow cytometry.
  • MM.1S -BCMA KO , MM.1S-GPRC5D KO , and MM.1S-WT cells are mixed evenly in an equal ratio of 1:1:1. They are mouse tumor-bearing cells for animal experiments and an exemplary bispecific chimeric antigen for in vivo verification in NSG mice.
  • tumor-eliminating and tumor-inhibiting activities of the recipient OriC321 CAR-T cells please refer to the anti-tumor activity of the 12MM.1S tumor transplanted mouse model in this example for specific steps.
  • the in vivo tumor inhibition experimental process of NSG mice based on gene knockout MM.1S target cell modeling is shown in Figure 22; as shown in Figure 23, Mock T has no tumor inhibitory effect, BCMA CAR-T and GPRC5D After CAR-T exerts mild anti-tumor activity, it eventually fails to inhibit tumor growth over time.
  • the exemplary bispecific OriC321 CAR-T cells completely inhibit tumor growth and have significant tumor elimination effects, with the tumor inhibition rate approaching 100%.
  • FIG. 23B Exemplary bispecific chimeric antigen receptor OriC321 CAR-T cells can significantly prolong the survival rate of mice ( Figure 23C), illustrating the role of exemplary bispecific chimeric antigen receptor OriC321 CAR-T cells in Gene-knockout MM.1S target cell mouse model has significant therapeutic efficacy and survival benefit in the treatment of MM.
  • the flow cytometry binding activity detection results of this example are shown in Figures 24 and 25.
  • the binding characteristics are summarized in Table 3. It can be seen that the GPRC5D V H H-hFc antibody shows crossover with human GPRC5D, mouse GPRC5D, and cynomolgus monkey GPRC5D.
  • Binding activity among which, GPRC5D V H H-hFc antibody has strong binding activity to human GPRC5D and cynomolgus monkey GPRC5D, but has relatively weak binding activity to mouse GPRC5D; no GPRC5D V H H-hFc antibody was detected with blank CHO Cell binding, no binding of the GPRC5D V H H-hFc antibody to CHO cells expressing different isoforms of GPRC5A from the same family of human GPRC5D was detected, indicating the high specificity of the GPRC5D V H H-hFc antibody applied to this product. It can reduce the risk of various off-target side effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本申请涉及一种嵌合抗原受体，其包含第一抗原结合结构域和第二抗原结合结构域，所述第一抗原结合结构域靶向B细胞成熟抗原(BCMA)，且所述第二抗原结合结构域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。本申请还提供了一种细胞，其包含和/或表达靶向BCMA的嵌合抗原受体以及靶向GPRC5D的嵌合抗原受体。

Description

靶向BCMA和GPRC5D的嵌合抗原受体及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向BCMA和GPRC5D嵌合抗原受体，及其应用。
背景技术
多发性骨髓瘤(Multiple Myeloma，MM)是第二常见的恶性血液病，并且在癌症致死率中居于第2位。MM属于浆细胞恶性肿瘤，常伴有多发性溶骨性损害、肾脏功能损害、骨髓浸润、高钙血症和贫血。MM在目前的主要治疗方法是副作用严重的全身化疗，并且无法彻底治愈。
BCMA(B cell maturation antigen)，即B细胞成熟抗原，主要调控B细胞的增殖、存活，以及B细胞向浆细胞(PCs)的成熟分化，在PCs分化过程中，BCMA分子逐渐被诱导表达。由于BCMA分子仅表达于PCs和浆母细胞膜表面，不表达于大多数B细胞、造血干细胞，以及其它正常组织上，使得BCMA分子成为治疗MM的最理想的靶标分子之一。
G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白，是一种非典型的表面孤儿受体。GPRCD5与其他C5家族受体一样拥有过短的氨基末端，因此在构象上与C4家族十分类似。其在正常组织中表达仅限于毛囊，但在MM患者的骨髓中也有特异性高表达，并与浆细胞肿瘤负荷与遗传畸变高度相关。
随着蛋白酶抑制剂(PI)、免疫调节药物(IMiD)和单克隆抗体(mAb)等新型药物的介入，MM的治疗方式得到扩充，但依然有着极高的复发率并产生耐药性。而嵌合抗原受体T细胞(CAR-T)治疗对MM产生了显著疗效和可控的毒性，其中主流的以B细胞成熟抗原(BCMA)为靶点的CAR-T细胞疗法对于BCMA阴性或BCMA低表达的MM病患存在靶向逃逸问题。因此，亟需开发更多样、更有效的治疗MM的药物，以解决CAR-T细胞疗法在治疗多发性骨髓瘤中的高复发及免疫逃逸问题。
发明内容
本申请提供了一种靶向BCMA和GPRC5D的双特异性嵌合抗原受体，本申请还提供了一种表达特异性结合BCMA和特异性结合GPRC5D的嵌合抗原受体的细胞。本申请以BCMA抗原和GPRC5D的空间表位为靶点，将BCMA抗体的scFv区，优化后的连接子，GPRC5D抗体的VHH区，铰链区，跨膜区，共刺激信号区域，胞内信号传导域，进行串联或者并联， 构建的嵌合抗原受体可以用于治疗例如多发性骨髓瘤的肿瘤，以解决CAR-T细胞疗法在治疗多发性骨髓瘤中的高复发及免疫逃逸问题，提供多发性骨髓瘤新的治疗策略，有效治疗多发性骨髓瘤。
一方面，本申请提供了一种嵌合抗原受体，其包含第一抗原结合结构域和第二抗原结合结构域，所述第一抗原结合结构域靶向B细胞成熟抗原(BCMA)，且所述第二抗原结合结构域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。
在某些实施方式中，所述第一抗原结合结构域包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述第一抗原结合结构域的抗原结合片段包含scFv。
在某些实施方式中，所述第一抗原结合结构域包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含SEQ ID NO:3所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含VH，且所述VH包含SEQ ID NO:9 所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含抗体轻链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:11(LGS)所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含SEQ ID NO:11(LGS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含L-FR1，所述L-FR1的C端与所述LCDR1的N端直接或间接相连，且所述L-FR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含L-FR2，所述L-FR2位于所述LCDR1和所述LCDR2之间，且所述L-FR2包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含L-FR3，所述L-FR3位于所述LCDR2和所述LCDR3之间，且所述L-FR3包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含L-FR4，所述L-FR4的N端与所述LCDR3的C端直接或间接相连，且所述L-FR4包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含VL，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含VH和VL，所述VH和VL通过连接子相连，所述连接子包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述第二抗原结合结构域的抗原结合片段包括VHH。
在某些实施方式中，所述第二抗原结合结构域包含抗体重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域的VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域的HCDR3包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域的HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:69所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域的HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下述任一组的氨基酸序列：(1)HCDR1：SEQ ID NO:21；HCDR2：SEQ ID NO:22；和HCDR3：SEQ ID NO:23；(2)HCDR1：SEQ ID NO:30；HCDR2：SEQ ID NO:31；和HCDR3：SEQ ID NO:32；(3)HCDR1：SEQ ID NO:35；HCDR2： SEQ ID NO:36；和HCDR3：SEQ ID NO:37；以及(4)HCDR1：SEQ ID NO:41；HCDR2：SEQ ID NO:42；和HCDR3：SEQ ID NO:43。
在某些实施方式中，所述第二抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，
在某些实施方式中，所述第二抗原结合结构域的H-FR2包含SEQ ID NO:25、SEQ ID NO:44和SEQ ID NO:38中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域的H-FR3包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2包含SEQ ID NO:70所示的氨基酸序列，所述H-FR3包含SEQ ID NO:71所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2包含SEQ ID NO:25、SEQ ID NO:38和SEQ ID NO:44中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4包含选自下述任一组氨基酸序列：(1)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(2)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(3)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:38，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；以及(4)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:44，H-FR3：SEQ ID NO:45，和H-FR4：SEQ ID NO:27。
在某些实施方式中，所述第二抗原结合结构域包含VH，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域的VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域包含VHH，且所述VHH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合结构域的VHH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合结构域和所述第二抗原结合结构域分别包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3，所述第一抗原结合结构域和所述第二抗原结合结构域的HCDR1，HCDR2和HCDR3的序列选自下述任一组氨基酸序列：(1)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:22，和HCDR3：SEQ ID NO:23；(2)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:31，和HCDR3：SEQ ID NO:32；(3)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:35，HCDR2：SEQ ID NO:36，和HCDR3：SEQ ID NO:37；以及(4)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:41，HCDR2：SEQ ID NO:42，和HCDR3：SEQ ID NO:43。
在某些实施方式中，所述第一抗原结合结构域的C端和所述第二抗原结合结构域的N端直接或间接相连。
在某些实施方式中，所述第一抗原结合结构域的N端与所述第二抗原结合结构域的C端直接或间接相连。
在某些实施方式中，所述第一抗原结合结构域和所述第二抗原结合结构域通过连接子相连。
在某些实施方式中，所述连接子包含肽接头。
在某些实施方式中，所述连接子包含(GGGGS)n的氨基酸序列，其中所述n为1-10中的任意正整数。
在某些实施方式中，所述连接子包含(EAAAK)n的氨基酸序列，其中所述n为1-10中的任意正整数。
在某些实施方式中，所述嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述共刺激信号区域包含SEQ ID NO:56所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述胞内信号传导域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述跨膜区为源自CD8α的跨膜区。
在某些实施方式中，所述跨膜区包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、 TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述铰链区为源自CD8α的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段位于所述胞内信号区域的C端。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含信号肽。
在某些实施方式中，所述信号肽为源自CD8α蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:48所示的氨基酸序列。
另一方面，本申请还提供了分离的一种或多种核酸分子，其编码本申请所述的嵌合抗原受体。
在某些实施方式中，所述核酸分子还包含编码剪切肽的序列。
在某些实施方式中，所述剪切肽包含2A肽。
在某些实施方式中，所述剪切肽选自下组：P2A、T2A、F2A和E2A。
在某些实施方式中，所述剪切肽的序列包含SEQ ID NO:62所示的氨基酸序列。
在某些实施方式中，编码所述剪切肽的序列包含SEQ ID NO:63所示的核酸序列。
在某些实施方式中，所述核酸分子还包含编码低密度脂蛋白受体相关蛋白或其片段的核酸序列。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，编码所述低密度脂蛋白受体相关蛋白或其片段的序列包含SEQ ID NO:65所示的核酸序列。
另一方面，本申请还提供了表达载体，其包含本申请所述的核酸分子。
另一方面，本申请还提供了表达载体，其包含编码所述靶向BCMA的嵌合抗原受体的核酸序列和编码所述靶向GPRC5D的嵌合抗原受体的核酸序列。
另一方面，本申请还提供了表达载体，其包括编码靶向BCMA的嵌合抗原受体的核酸序列和编码靶向GPRC5D的嵌合抗原受体的核酸序列。
在某些实施方式中，所述表达载体中所述编码靶向BCMA的嵌合抗原受体的核酸序列包含编码靶向BCMA的抗原结合结构域的核酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含SEQ ID NO:3所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含VH，且所述VH包含SEQ  ID NO:9所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含重链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:11(LGS)所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含SEQ ID NO:11(LGS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述靶向BCMA的抗原结合结构域包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述表达载体中所述靶向BCMA的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述表达载体中所述靶向BCMA的抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述表达载体中编码所述scFv的序列包含SEQ ID NO:20所示的核酸序列。
在某些实施方式中，所述靶向BCMA的嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、 B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述共刺激信号区域为源自CD28的胞内共刺激信号区域。
在某些实施方式中，所述表达载体中编码所述共刺激信号区域的序列包含SEQ ID NO:57或SEQ ID NO:59所示的核酸序列。
在某些实施方式中，所述靶向BCMA的嵌合抗原受体包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述胞内信号传导域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述表达载体中编码所述胞内信号传导域的序列包含SEQ ID NO:61所示的核酸序列。
在某些实施方式中，所述靶向BCMA的嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述跨膜区为源自CD8α的跨膜区。
在某些实施方式中，所述跨膜区为源自CD28的跨膜区。
在某些实施方式中，所述表达载体中编码所述跨膜区的序列包含SEQ ID NO:53或SEQ ID NO:55所示的核酸序列。
在某些实施方式中，所述靶向BCMA的嵌合抗原受体包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述铰链区为源自CD8α的铰链区。
在某些实施方式中，所述表达载体中编码所述铰链区的序列包含SEQ ID NO:51所示的 核酸序列。
在某些实施方式中，所述靶向BCMA的嵌合抗原受体包括信号肽。
在某些实施方式中，所述信号肽包含为源自CD8α蛋白的信号肽。
在某些实施方式中，所述表达载体中编码所述信号肽的序列包含SEQ ID NO:49所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的抗原结合片段包含VHH。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述靶向GPRC5D的抗原结合结构域的VH的序列包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
在某些实施方式中，，所述靶向GPRC5D的抗原结合结构域的所述HCDR3包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的所述HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:69所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的所述HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和 HCDR3，所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下述任一组的氨基酸序列：(1)HCDR1：SEQ ID NO:21；HCDR2：SEQ ID NO:22；和HCDR3：SEQ ID NO:23；(2)HCDR1：SEQ ID NO:30；HCDR2：SEQ ID NO:31；和HCDR3：SEQ ID NO:32；(3)HCDR1：SEQ ID NO:35；HCDR2：SEQ ID NO:36；和HCDR3：SEQ ID NO:37；以及(4)HCDR1：SEQ ID NO:41；HCDR2：SEQ ID NO:42；和HCDR3：SEQ ID NO:43。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的所述H-FR2包含SEQ ID NO:25、SEQ ID NO:44和SEQ ID NO:38中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的所述H-FR3包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2包含SEQ ID NO:70所示的氨基酸序列，所述H-FR3包含SEQ ID NO:71所示的氨基酸序列，且所述H-FR4包 含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2包含SEQ ID NO:25、SEQ ID NO:38和SEQ ID NO:44中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4包含选自下述任一组氨基酸序列：(1)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(2)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(3)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:38，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；以及(4)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:44，H-FR3：SEQ ID NO:45，和H-FR4：SEQ ID NO:27。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述VH的序列包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域包含VHH，且所述VHH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述靶向GPRC5D的抗原结合结构域的所述VHH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述VHH的序列包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述共刺激信号区域为源自CD28的胞内共刺激域。
在某些实施方式中，所述共刺激信号区域包含SEQ ID NO:56或SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述共刺激信号区域的序列包含SEQ ID NO:57或SEQ ID NO:59所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的嵌合抗原受体包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述胞内信号传导域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述胞内信号传导域的序列包含SEQ ID NO:61所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述跨膜区为源自CD8α的跨膜区。
在某些实施方式中，所述跨膜区为源自CD28的跨膜区。
在某些实施方式中，所述跨膜区包含SEQ ID NO:52或SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述跨膜区的序列包含SEQ ID NO:53或SEQ ID NO:55中任一项所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的嵌合抗原受体包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、 CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述铰链区为源自CD8α的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述铰链区的序列包含SEQ ID NO:51所示的核酸序列。
在某些实施方式中，所述靶向GPRC5D的嵌合抗原受体包括信号肽。
在某些实施方式中，所述信号肽为源自CD8α蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述信号肽的序列包含SEQ ID NO:49所示的核酸序列。
在某些实施方式中，在所述表达载体中，所述编码靶向BCMA的嵌合抗原受体的核酸序列和所述编码靶向GPRC5D的嵌合抗原受体的核酸序列通过编码剪切肽的序列连接。
在某些实施方式中，所述剪切肽为2A肽。
在某些实施方式中，所述剪切肽选自下组：P2A、T2A、F2A和E2A。
在某些实施方式中，所述剪切肽包含SEQ ID NO:62所示的氨基酸序列。
在某些实施方式中，在所述表达载体中编码所述剪切肽的序列包含SEQ ID NO:63所示的氨基酸序列。
在某些实施方式中，在所述表达载体中，所述编码靶向BCMA的嵌合抗原受体的核酸序列的3’端和所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的5’端通过剪切肽相连。
在某些实施方式中，在所述表达载体中，所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端和所述编码靶向BCMA的嵌合抗原受体的核酸序列的5’端通过剪切肽相连。
在某些实施方式中，所述表达载体还包含编码低密度脂蛋白受体相关蛋白或其片段的核酸序列。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，在所述表达载体中，编码所述低密度脂蛋白受体相关蛋白或其片段 的序列包含SEQ ID NO:65所示的核酸序列。
在某些实施方式中，在所述表达载体中，所述编码低密度脂蛋白受体相关蛋白或其片段的核酸序列的5’端与所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端通过剪切肽相连。
在某些实施方式中，在所述表达载体中，所述编码低密度脂蛋白受体相关蛋白或其片段的核酸序列的5’端与所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端通过剪切肽相连。
在某些实施方式中，所述表达载体从5’端至3’端分别依次包含编码信号肽的序列、编码靶向BCMA的嵌合抗原受体的序列、编码剪切肽的序列、编码靶向GPRC5D的嵌合抗原受体的序列、编码剪切肽的序列，以及编码低密度脂蛋白受体相关蛋白或其片段的序列。
在某些实施方式中，所述表达载体从5’端至3’端分别依次包含编码信号肽的序列、编码靶向GPRC5D的嵌合抗原受体的序列、编码剪切肽的序列、编码靶向BCMA的嵌合抗原受体的序列、编码剪切肽的序列，以及编码低密度脂蛋白受体相关蛋白或其片段的序列。
另一方面，本申请还提供了一种细胞，其包含所述嵌合抗原受体，所述核酸分子，或所述表达载体。
另一方面，本申请还提供了一种细胞，其包含和/或表达靶向BCMA的嵌合抗原受体，以及靶向GPRC5D的嵌合抗体受体。
在某些实施方式中，在所述细胞中，所述靶向BCMA的嵌合抗体受体包含靶向BCMA的抗原结合结构域。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含抗体或其抗原结合片段。
在某些实施方式中，在所述细胞中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域的抗原结合片段包括scFv。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含HCDR2， 且所述HCDR2包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述BCMA的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含SEQ ID NO:3所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述BCMA的抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述BCMA的抗原结合结构域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述BCMA的抗原结合结构域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述BCMA的抗原结合结构域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含重链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:11(LGS)所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含 SEQ ID NO:11(LGS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的抗原结合结构域scFv，且所述scFv包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述共刺激信号区域为源自CD28的胞内共刺激域。
在某些实施方式中，所述共刺激信号区域包含SEQ ID NO:56或SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的嵌合抗原受体包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、 CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述胞内信号传导域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述跨膜区为源自CD8α的跨膜区。
在某些实施方式中，所述跨膜区为源自CD28的跨膜区。
在某些实施方式中，在所述细胞中，所述跨膜区包含SEQ ID NO:52或SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的嵌合抗原受体包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述铰链区为源自CD8α的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向BCMA的嵌合抗原受体包括信号肽。
在某些实施方式中，所述信号肽包含为源自CD8α蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含抗体或其抗原结合片段。
在某些实施方式中，在所述细胞中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述抗原结合片段包括VHH。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述HCDR3包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:69所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列，且所述HCDR3包含SEQ  ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下述任一组的氨基酸序列：(1)HCDR1：SEQ ID NO:21；HCDR2：SEQ ID NO:22；和HCDR3：SEQ ID NO:23；(2)HCDR1：SEQ ID NO:30；HCDR2：SEQ ID NO:31；和HCDR3：SEQ ID NO:32；(3)HCDR1：SEQ ID NO:35；HCDR2：SEQ ID NO:36；和HCDR3：SEQ ID NO:37；以及(4)HCDR1：SEQ ID NO:41；HCDR2：SEQ ID NO:42；和HCDR3：SEQ ID NO:43。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述H-FR2包含SEQ ID NO:25、SEQ ID NO:44和SEQ ID NO:38中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述H-FR3包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2包含SEQ ID NO:70所示的氨基酸序列，所述H-FR3包含SEQ ID NO:71所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2包含SEQ ID NO:25、SEQ ID NO:38和SEQ ID NO:44中任一项所示的氨基酸序列，所述H- FR3包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4包含选自下述任一组氨基酸序列：(1)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(2)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(3)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:38，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；以及(4)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:44，H-FR3：SEQ ID NO:45，和H-FR4：SEQ ID NO:27。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域包含VHH，且所述VHH包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的抗原结合结构域的所述VHH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，在所述共刺激信号区域为源自CD28的胞内共刺激域。
在某些实施方式中，在所述细胞中，所述共刺激信号区域包含SEQ ID NO:56或SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的嵌合抗原受体包括胞内信号传 导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述胞内信号传导域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述跨膜区为源自CD8α的跨膜区。
在某些实施方式中，所述跨膜区为源自CD28的跨膜区。
在某些实施方式中，所述跨膜区包含SEQ ID NO:52或SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的嵌合抗原受体包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述铰链区为源自CD8α的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，在所述细胞中，所述靶向GPRC5D的嵌合抗原受体包括信号肽。
在某些实施方式中，所述信号肽包含为源自CD8α蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，所述细胞还包含和/或表达低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，在所述细胞中，所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，在所述细胞中，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
在某些实施方式中，在所述细胞中，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，所述细胞包含免疫效应细胞。
在某些实施方式中，所述细胞包含包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
在某些实施方式中，所述细胞为T细胞。
另一方面，本申请还提供了制备所述嵌合抗原受体的方法，所述方法包括在使得所述嵌合抗原受体表达的条件下，培养所述的细胞。
另一方面，本申请还提供了制备经修饰的免疫效应细胞的方法，其包括向免疫效应细胞中引入所述的表达载体。
另一方面，本申请还提供了药物组合物，其包含所述嵌合抗原受体，所述核酸分子，所述的表达载体，和/或所述的细胞，以及任选地药学上可接受的载体。
另一方面，本申请还提供了所述嵌合抗原受体，所述核酸分子，所述的表达载体，所述的细胞，和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、治疗和/或缓解疾病和/或病症。
另一方面，本申请还提供了一种预防、治疗和/或缓解疾病和/或病症的方法，所述方法包括向有需要的受试者施用所述的细胞，和/或所述的药物组合物。
另一方面，本申请还提供了所述嵌合抗原受体，所述核酸分子，所述的表达载体，所述的细胞，和/或所述的药物组合物，其用于预防、治疗和/或缓解疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括与BCMA异常表达相关的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤和/或淋巴瘤。
在某些实施方式中，所述肿瘤包括骨髓瘤。
在某些实施方式中，所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本 申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示基于细胞淘选和固相淘选技术的流程示意图。
图2显示所述双特异性嵌合抗原受体的结构连接示意图。
图3显示所述双特异性嵌合抗原受体串联和并联结构的分子示意图。
图4显示流式检测靶细胞表面BCMA和GPRC5D抗原表达。
图5显示慢病毒转导双特异性嵌合抗原受体72h后T细胞表面CAR基因表达。
图6显示示例性慢病毒感染双特异性嵌合抗原受体OriC321 CAR后T细胞的阳性率。
图7显示靶向BCMA和GPRC5D双特异性CAR-T细胞体外增殖曲线。
图8显示靶向BCMA和GPRC5D双特异性CAR-T细胞体外扩增活率。
图9显示示例性双特异性嵌合抗原受体OriC321 CAR-T细胞体外增殖曲线。
图10显示靶向BCMA和GPRC5D双特异性CAR-T细胞在效靶比3:1的杀伤效率。
图11显示靶向BCMA和GPRC5D双特异性CAR-T细胞不同效靶比下的杀伤效率。
图12显示示例性双特异性嵌合抗原受体OriC321 CAR-T细胞不同效靶比下的杀伤效率。
图13显示双特异性CAR-T细胞与不同靶细胞共培养脱颗粒CD107a表达水平。
图14显示示例性双特异性嵌合抗原受体OriC321 CAR-T细胞免疫突触共聚焦成像图。
图15显示微管组织中心(MTOC)与突触距离的定量分析。
图16显示双特异性CAR-T细胞与靶细胞共孵育后细胞因子IL-2分泌水平。
图17显示双特异性CAR-T细胞与靶细胞共孵育后细胞因子IFN-γ分泌水平。
图18显示靶向BCMA和GPRC5D双特异性CAR-T细胞靶向增殖曲线。
图19显示靶向BCMA和GPRC5D双特异性CAR-T细胞NSG小鼠体内抑瘤实验流程图。
图20显示靶向BCMA和GPRC5D双特异性CAR-T细胞NSG小鼠体内抗肿瘤活性。
图21显示MM.1S靶细胞BCMA和GPRC5D的基因敲除效率。
图22显示基于基因敲除的MM.1S靶细胞荷瘤NSG小鼠体内抑瘤实验流程图。
图23显示靶向BCMA和GPRC5D双特异性CAR-T细胞在靶基因敲除的体内肿瘤模型上的抗肿瘤活性。
图24显示GPRC5D VHH-hFc抗体与不同种属靶抗原的交叉结合活性。
图25显示GPRC5D VHH-hFc抗体与不同种属靶抗原的交叉结合活性汇总。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
使用的术语“抗体”包括完整抗体和其结合片段。通常，片段与其来源的完整抗体竞争性地与抗原特异性结合。任选，抗体或其结合片段可以与其它蛋白质化学结合，或者与其它蛋白质以融合蛋白质的形式表达。例如，所述抗体可以是单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。例如，所述抗体或其结合片段的结合蛋白质可以包括GPRC5D。例如，所述抗体或其结合片段对GPRC5D可以有特异性。
术语“抗原结合片段”是指完整抗体的一部分并且是指完整抗体的抗原决定可变区。例如，所述抗原结合片段的可以包括Fab、Fab'、F(ab')2、Fv片段和单链Fv片段、串联Fv片段、VHH、双特异性抗体。例如，所述抗原结合片段可以是VHH。例如，所述抗原结合片段可以结合GPRC5D。例如，所述抗原结合片段对GPRC5D可以有特异性。
在本申请中，术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体。VHH也可称为纳米抗体(Nanobody)(Nb)和/或单域抗体。例如，所述VHH可以结合GPRC5D。例如，所述VHH对GPRC5D可以有特异性。
在本申请中，术语“scFv“通常是指单链抗体，是由重链可变区和轻链可变区直接连接或通过连接分子(例如，连接肽)连接而成的抗体。所述scFv的结构自N端至C端，可以是重链可变区-轻链可变区，轻链可变区-重链可变区，重链可变区-连接肽-轻链可变区，或轻链可变区-连接肽-重链可变区。
在本申请中，所述抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链。每条重链由重链可变区(VH)和重链恒定区组成。术语“重链恒定区”由三个结构域CH1，CH2和CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区组成。术语“轻链恒定区”由一个结构域CL组成。VH和VL区可以进一步细分为高变区，称为互补决定区(CDR)，散布有更保守的区域，称为构架区(FR)。每个VH和VL由以下列顺序从氨基末端到羧基末端排列的三个CDR 和四个FR组成:FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合。
在本申请中，术语“G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白”是一种非典型的表面孤儿受体。GPRCD5与其他C5家族受体一样拥有过短的氨基末端，因此在构象上与C4家族十分类似。其在正常组织中表达仅限于毛囊，但在多发性骨髓瘤患者的骨髓中也有显著表达，并与浆细胞肿瘤负荷与遗传畸变高度相关。例如，在本申请中的GPRC5D可以特指在MM患者体内表达的GPRC5D。
在本申请中，术语“BCMA”可与“CD269”、“BCM”“TNFRSF17”互换使用，通常是指B细胞成熟抗原。BCMA蛋白是肿瘤坏死因子受体家族成员。在本申请中，术语“BCMA”可包括包含突变的蛋白质，例如可包括包含全长野生型BCMA的点突变、片段、插入、缺失和剪接变体的蛋白质。所述BCMA可以包括来自任何脊椎动物，例如，灵长类发动物(例如，人或猴)、啮齿类动物(小鼠或大鼠)、禽和/或畜等。在本申请中，术语“BCMA”还可包括完整BCMA蛋白的一部分，只要保留相关的生物活性即可。在本申请中，所述BCMA可以为人BCMA，其GenBank登录号为BAB60895.1。例如，人BCMA通常是由994个核苷酸长的初级mRNA转录物(NM_001192.2)编码的184个氨基酸长的蛋白质。
在本申请中，术语“嵌合抗原受体”(CAR)通常是指至少包含特异性地结合抗原或靶标的胞外结构域、跨膜区以及胞内结构域的重组多肽。例如，所述胞外结构域与所述跨膜区之间包括铰链区。例如，所述嵌合抗原受体还可以包括低密度脂蛋白受体相关蛋白或其片段。例如，所述嵌合抗原受体可以包括信号肽。CAR的胞外结构域与靶细胞表面上的靶抗原结合导致CAR聚簇并将激活刺激传送给含CAR细胞。CAR重定向免疫效应细胞的特异性，并且触发了增殖、细胞因子产生、能够以不依赖主要组织相容性(MHC)的方式介导表达靶抗原的细胞死亡的分子的吞噬作用和/或产生。例如，所述胞外结构可以特异性结合GPRC5D。
在本申请中，术语“胞内结构域”指意思是包括足以转导活化信号的任何截短部分的细胞内结构域。所述胞内结构域可以包括胞内信号传导域和/或共刺激信号区域。术语“胞内信号传导域”指可以产生促进含CAR细胞(例如CART细胞或表达CAR的NK细胞)的免疫效应子功能的信号的胞内区域。例如，所述胞内信号区域可以包括选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。例如，所述胞内信号区域可以是源自CD3ζ的信号传导结构域。术语“共刺激信号区域”是指所述胞内信号区 域中能够转导效应信号的CAR的一部分。例如，所述共刺激信号区域可以包括源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，所述共刺激信号区域可以是源自4-1BB的胞内共刺激信号区域。
在本申请中，术语“跨膜区”指能够跨越细胞质膜的肽、多肽或蛋白质的结构域。可采用这些结构域将胞外结构域锚定在细胞膜上。例如，所述跨膜区可以包含选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。例如，所述跨膜区可以源自CD8的跨膜区。
在本申请中，术语“铰链区”表示抗体重链多肽中连接CH1结构域和CH2结构域的一部分，例如，从根据Kabat的EU编号系统的约第216位至约第230位。铰链区通常是由两条具有相同氨基酸序列的多肽组成的二聚体分子。铰链区一般包括约25个氨基酸残基，是柔性的，允许抗原结合区独立的移动。铰链区可以细分为3个结构域：上、中、下铰链结构域。例如，所述铰链区可以包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。例如，所述铰链区可以源自CD8的铰链区。
在本申请中，术语“低密度脂蛋白受体相关蛋白(Low-density lipoproteinreceptor-related protein)指一种属于内吞性受体的细胞表面蛋白，在生物体内广泛分布并有很大的组织间差异性，主要功能是摄取胆固醇进入细胞内用以细胞增殖和固醇类激素及胆汁酸盐的合成。例如，所述低密度脂蛋白受体相关蛋白可以来自于任何脊椎动物。例如，所述低密度脂蛋白受体相关蛋白或其片段可以位于所述胞内信号区域的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白6或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白5或其片段。
在本申请中，术语“信号肽”是指处于新生CAR蛋白的氨基末端(N-末端)的前导序列，其在翻译时或在翻译后将新生蛋白引导到内质网并后续表面表达。例如，所述信号肽源自CD8蛋白的信号肽。
在本申请中，术语“核酸分子”包括DNA分子和RNA分子。一个核酸分子可以是单链或双链的，但优选为双链DNA。术语“启动子”通常是指一个DNA序列，可以调节与启动子操作性连接的所选DNA序列的表达，从而影响细胞中所选DNA序列的表达。例如，所述核酸分子可以编码包括所属抗原结合蛋白和/或所述嵌合抗原受体。例如，所述核酸分子可以包括启动子。例如，所述启动子可以是组成型启动子。例如，所述启动子可以是EF1α启动子。
在本申请中，术语“载体”通常是指可以附着本申请的一种或多种核酸分子的分子。例如，所述载体可以是病毒载体。例如，所述载体可以是慢病毒载体。
在本申请中，术语“细胞”指可以向其转染核酸的细胞，术语“细胞”包括用于质粒繁殖的原核细胞，和用于核酸表达和编码多肽产生的真核细胞。例如，细胞可以包括所述的嵌合抗原受体，所述的核酸分子和/或所述的载体。例如，所述细胞可以是免疫效应细胞。术语“免疫效应细胞”通常是指参与免疫应答，行使效应功能的免疫细胞。例如，所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等。例如，免疫效应细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如，免疫效应细胞可以是T细胞。
在本申请中，术语“药物组合物”通常指适合施用于哺乳动物个体的化学或生物组合物。例如，所述药物组合物可以包括所述抗原结合蛋白，所述嵌合抗原受体，所述多肽，所述核酸分子，所述载体和/或所述细胞，以及可选的药学上可接受的载体。所述药物组合物可以用于预防、治疗和/或缓解与GPRC5D异常表达相关的疾病或病症。例如，所述与GPRC5D异常表达相关的疾病或病症可以包括肿瘤。例如，所述肿瘤包括实体瘤和/或非实体瘤。例如，所述肿瘤包可以括血液瘤和/或淋巴瘤。例如，所述肿瘤可以包括骨髓瘤。
在本申请中，术语“药学上可接受的载剂”通常是指不干扰活性成分的生物活性的有效性的一种或多种非毒性材料。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选地其他治疗剂。
在提到蛋白质的氨基酸序列或核酸分子的核苷酸序列时，本申请还包括这些序列的同源物。在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序 列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
包含第一抗原结合结构域和第二抗原结合结构域的嵌合抗原受体
一方面，本申请提供一种嵌合抗原受体，其包含第一抗原结合结构域和第二抗原结合结构域，所述第一抗原结合结构域靶向B细胞成熟抗原(BCMA)，且所述第二抗原结合结构域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。
在本申请中，靶向BCMA的CAR和靶向GPRC5D的CAR能够发挥协同作用，更大程度上提高了肿瘤杀伤作用。另一方面，同时靶向BCMA和GPRC5D能够有效解决肿瘤治疗中的免疫逃逸问题。同时，同时靶向BCMA和GPRC5D能够有效减少肿瘤治疗的复发率。
在本申请中，所述第一抗原结合结构域可以为靶向BCMA的抗原结合结构域。在本申请中，所述第二抗原结合与可以包含靶向GPRC5D的抗原结合结构域。
在本申请中，所述第一抗原结合结构域的C端可以和所述第二抗原结合结构域的N端直接或间接相连。
在本申请中，所述第一抗原结合结构域的N端可以和所述第二抗原结合结构域的C端直接或间接相连。
在本申请中，所述第一抗原结合结构域和所述第二抗原结合结构域通过连接子相连。例如，所述连接子可包含肽接头。例如，所述连接子可包含(GGGGS)n的氨基酸序列，其中所述n可以为1-10中的任意正整数。例如，所述连接子可包含(EAAAK)n的氨基酸序列，其中所述n可以为1-10中的任意正整数。
在本申请中，所述嵌合抗原受体还可包含共刺激信号区域。
在本申请中，所述嵌合抗原受体还可包含胞内信号传导域。
在本申请中，所述嵌合抗原受体还可包含跨膜区。
在本申请中，所述嵌合抗原受体还可包含铰链区。
在本申请中，所述嵌合抗原受体还可包含信号肽。
在本申请中，所述嵌合抗原受体还可包含低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供了分离的一种或多种核酸分子，其可编码所述嵌合抗原受体。
在本申请中，所述核酸分子还可包含编码剪切肽的序列。
在本申请中，所述核酸分子还可包含编码低密度脂蛋白受体相关蛋白或其片段的核酸序列。
另一方面，本申请还提供了表达载体，其可包含所述核酸分子。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、靶向BCMA的抗原结合结构域、肽接头、靶向GPRC5D的抗原结合结构域、铰链区、跨膜区、共刺激信号区域以及胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(GGGGS)n、3B5 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域的氨基酸序列。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(GGGGS)n、3E7 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域的氨基酸序列。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(GGGGS)n、4A2 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域的氨基酸序列。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(GGGGS)n、4B3 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(EAAAK)n、3B5 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(EAAAK)n、3E7 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(EAAAK)n、4A2 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ 胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、hBCMA22 scFv、(EAAAK)n、4B3 VHH、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、3B5 VHH、(GGGGS)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域的氨基酸序列。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、3E7 VHH、(GGGGS)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域的氨基酸序列。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、4A2 VHH、(GGGGS)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域的氨基酸序列。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、4B3 VHH、(GGGGS)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、3B5 VHH、(EAAAK)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、3E7 VHH、(EAAAK)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、4A2 VHH、(EAAAK)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
在本申请中，所述嵌合抗原受体从N端至C端可分别依次包含信号肽、4B3 VHH、(EAAAK)n、hBCMA22 scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激信号区域以及CD3ζ胞内信号传导域。
表达载体、细胞
另一方面，本申请还提供了表达载体，其可包括编码靶向BCMA的嵌合抗原受体的核酸 序列和编码靶向GPRC5D的嵌合抗原受体的核酸序列。
另一方面，本申请还提供了细胞，其可包含所述表达载体。
另一方面，本申请还提供了细胞，其可包含和/或表达靶向BCMA的嵌合抗原受体，以及靶向GPRC5D的嵌合抗体受体。在本申请中，所述细胞还可包含和/或表达低密度脂蛋白受体相关蛋白或其片段。
在本申请中，所述靶向BCMA的嵌合抗原受体可包含靶向BCMA的抗原结合结构域。在本申请中，所述靶向BCMA的嵌合抗原受体可包含共刺激信号区域。在本申请中，所述靶向BCMA的嵌合抗原受体可包含胞内信号传导域。在本申请中，所述靶向BCMA的嵌合抗原受体可包含跨膜区。在本申请中，所述靶向BCMA的嵌合抗原受体可包含铰链区。在本申请中，所述靶向BCMA的嵌合抗原受体可包含信号肽。
在本申请中，所述靶向GPRC5D的嵌合抗原受体可包含靶向GPRC5D的抗原结合结构域。在本申请中，所述靶向GPRC5D的嵌合抗原受体可包含共刺激信号区域。在本申请中，所述靶向GPRC5D的嵌合抗原受体可包含胞内信号传导域。在本申请中，所述靶向GPRC5D的嵌合抗原受体可包含跨膜区。在本申请中，所述靶向GPRC5D的嵌合抗原受体可包含铰链区。在本申请中，所述靶向GPRC5D的嵌合抗原受体可包含信号肽。
在本申请中，所述表达载体中所述编码靶向BCMA的嵌合抗原受体的核酸序列可以和所述编码靶向GPRC5D的嵌合抗原受体的核酸序列通过编码剪切肽的序列连接。
在本申请中，所述表达载体中所述编码靶向BCMA的嵌合抗原受体的核酸序列的3’端可以和所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的5’端通过剪切肽相连。
在本申请中，所述表达载体中所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端和所述编码靶向BCMA的嵌合抗原受体的核酸序列的5’端通过剪切肽相连。
在本申请中，所述表达载体还可包含编码低密度脂蛋白受体相关蛋白或其片段的核酸序列。
在本申请中，所述表达载体中的所述编码低密度脂蛋白受体相关蛋白或其片段的核酸序列的5’端可以与所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端通过剪切肽相连。
在本申请中，所述表达载体中的所述编码低密度脂蛋白受体相关蛋白或其片段的核酸序列的5’端与所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端通过剪切肽相连。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码信号肽的序列、编码靶向BCMA的嵌合抗原受体的序列、编码剪切肽的序列、编码靶向GPRC5D的嵌合抗原受体的序列、编码剪切肽的序列，以及编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码信号肽的序列、编码靶向GPRC5D的嵌合抗原受体的序列、编码剪切肽的序列、编码靶向BCMA的嵌合抗原受体的序列、编码剪切肽的序列，以及编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码3B5 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码3E7 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码4A2 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码4B3 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD28铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8 α信号肽的序列、编码3B5 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD28铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码3E7 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD828铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码4A2 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD28铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码4B3 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码3B5 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码3E7 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α 信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码4A2 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码4B3 VHH的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码3B5 VHH的序列、编码CD28铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码3E7 VHH的序列、编码CD28铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码4A2 VHH的序列、编码CD28铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信 号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体从5’端至3’端可分别依次包含编码CD8α信号肽的序列、编码4B3 VHH的序列、编码CD28铰链区的序列、编码CD28跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码CD8α信号肽的序列、编码hBCMA22 scFv的序列、编码CD8α铰链区的序列、编码CD8α跨膜区的序列、编码4-1BB共刺激信号区域的序列、编码CD3ζ胞内信号传导域的序列、编码剪切肽的序列、编码低密度脂蛋白受体相关蛋白或其片段的序列。
在本申请中，所述表达载体可以包括启动子。例如，所述启动子可以是组成型启动子。例如，所述启动子可以是EF1α启动子。例如，所述EF1α启动子可包含SEQ ID NO:66所示的序列。
在本申请中，所述表达载体可以包括所述的核酸分子。所述表达载体可以转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达。例如，载体可以包括启动子、转录子、增强子、复制子、选择元件和报告基因。例如，载体可以包括协助进入细胞的成分。为了使所述核酸分子在表达载体中复制，所述核酸分子的5’端和3’端还可以包含长末端重复序列。
靶向BCMA的抗原结合结构域
在本申请中，所述靶向BCMA的抗原结合结构域可包含抗体或其抗原结合片段。
在本申请中，所述抗原结合片段可包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在本申请中，所述靶向BCMA的抗原结合结构域可包含靶向BCMA的scFv。
在本申请中，所述靶向BCMA的抗原结合结构域可包含重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含HCDR3，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。在本申请中，所述靶向BCMA的抗原结合结构域可包含HCDR2，且所述HCDR2包含SEQ ID NO:3所示的氨基酸序列。在本申请中，所述靶向BCMA的抗原结合结构域可包含HCDR1，且所述HCDR1包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述BCMA的抗原结合结构域可包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:3所示的 氨基酸序列，且所述HCDR3可包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述BCMA的抗原结合结构域可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可包含SEQ ID NO:5所示的氨基酸序列。在本申请中，所述BCMA的抗原结合结构域可包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可包含SEQ ID NO:6所示的氨基酸序列。在本申请中，所述BCMA的抗原结合结构域可包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可包含SEQ ID NO:7所示的氨基酸序列。在本申请中，所述BCMA的抗原结合结构域可包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4可包含SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含VH，且所述VH可包含SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含重链可变区VL中的至少一个CDR，且所述VL可包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含LCDR3，且所述LCDR3可包含SEQ ID NO:12所示的氨基酸序列。在本申请中，所述靶向BCMA的抗原结合结构域可包含LCDR2，且所述LCDR2可包含SEQ ID NO:11(LGS)所示的氨基酸序列。在本申请中，所述靶向BCMA的抗原结合结构域可包含LCDR1，且所述LCDR1可包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含LCDR1，LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:11(LGS)所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:12所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1可包含SEQ ID NO:13所示的氨基酸序列。在本申请中，所述靶向BCMA的抗原结合结构域可包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2可包含SEQ ID NO:14所示的氨基酸序列。在本申请中，所述靶向BCMA的抗原结合结构域可包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3可包含SEQ ID NO:15所示的氨基酸序列。在本申请中，所述靶向BCMA的抗原结合结构域可包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4可包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含VL，且所述VL可包含SEQ ID  NO:17所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域的VH和VL可通过连接子相连。例如，所述连接子可包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述靶向BCMA的抗原结合结构域可包含scFv，且所述scFv可包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，编码所述scFv的核酸序列可包含SEQ ID NO:20所示的核酸序列。
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。
在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，本申请所述分离的抗原结合蛋白通过IMGT编码系统定义。
在本申请中，所述靶向BCMA的抗原结合结构域的scFv可包含hBCMA22。例如，所述hBCMA22的HCDR1可包含SEQ ID NO:2所示的氨基酸序列，HCDR2可包含SEQ ID NO:3所示的氨基酸序列，HCDR3可包含SEQ ID NO:4所示的氨基酸序列，LCDR1可包含SEQ ID NO:10所示的氨基酸序列，LCDR2可包含SEQ ID NO:11(LGS)所示的氨基酸序列，LCDR3可包含SEQ ID NO:12所示的氨基酸序列，VH可包含SEQ ID NO:9所示的氨基酸序列，VL可包含SEQ ID NO:17所示的氨基酸序列。例如，所述hBCMA22的scFv可包含SEQ ID NO:19所示的氨基酸序列。
靶向GPRC5D的抗原结合结构域
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含抗体或其抗原结合片段。
在本申请中，所述抗原结合片段可包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含靶向GPRC5D的scFv。
在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，本申请所述分离的抗原结合蛋白通过IMGT编码系统定义。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含重链可变区VH中的至少一个CDR，且所述VH可包含SEQ ID NO:72所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域的所述VH可包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含HCDR3，且所述HCDR3可包含SEQ ID NO:67所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域的所述HCDR3可包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含HCDR2，且所述HCDR2可包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域的所述HCDR2可包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含HCDR1，且所述HCDR1可包含SEQ ID NO:69所示的氨基酸序列。在本申请中，所述HCDR1可包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:69所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:67所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO: 36和SEQ ID NO:42中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3可包含选自下述任一组的氨基酸序列：(1)HCDR1：SEQ ID NO:21；HCDR2：SEQ ID NO:22；和HCDR3：SEQ ID NO:23；(2)HCDR1：SEQ ID NO:30；HCDR2：SEQ ID NO:31；和HCDR3：SEQ ID NO:32；(3)HCDR1：SEQ ID NO:35；HCDR2：SEQ ID NO:36；和HCDR3：SEQ ID NO:37；以及(4)HCDR1：SEQ ID NO:41；HCDR2：SEQ ID NO:42；和HCDR3：SEQ ID NO:43。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:24所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域可包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可包含SEQ ID NO:70所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域的所述H-FR2可包含SEQ ID NO:25、SEQ ID NO:44和SEQ ID NO:38中任一项所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域可包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可包含SEQ ID NO:71所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域的所述H-FR3可包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列。在本申请中，所述靶向GPRC5D的抗原结合结构域可包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4可包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:70所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:71所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可包含SEQ ID NO:24所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:25、SEQ ID NO:38和SEQ ID NO:44中任一项所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:26或SEQ ID NO:45所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4可包含选自下述任一组氨基酸序列：(1)H-FR1： SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(2)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:25，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；(3)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:38，H-FR3：SEQ ID NO:26，和H-FR4：SEQ ID NO:27；以及(4)H-FR1：SEQ ID NO:24，H-FR2：SEQ ID NO:44，H-FR3：SEQ ID NO:45，和H-FR4：SEQ ID NO:27。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含VH，且所述VH可包含SEQ ID NO:72所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域的所述VH可包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在本申请中，在所述表达载体中编码所述VH的序列可包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域可包含VHH，且所述VHH可包含SEQ ID NO:72所示的氨基酸序列。
在本申请中，所述靶向GPRC5D的抗原结合结构域的所述VHH可包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
在本申请中，在所述表达载体中编码所述VHH的序列可包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
例如，所述3B5的CDR1包含SEQ ID NO:21所示的氨基酸序列，CDR2包含SEQ ID NO:22所示的氨基酸序列，CDR3包含SEQ ID NO:23所示的氨基酸序列，VHH包含SEQ ID NO:28所示的氨基酸序列。
例如，所述3E7的CDR1包含SEQ ID NO:30所示的氨基酸序列，CDR2包含SEQ ID NO:31所示的氨基酸序列，CDR3包含SEQ ID NO:32所示的氨基酸序列，VHH包含SEQ ID NO:33所示的氨基酸序列。
例如，所述4A2的CDR1包含SEQ ID NO:35所示的氨基酸序列，CDR2包含SEQ ID NO:36所示的氨基酸序列，CDR3包含SEQ ID NO:37所示的氨基酸序列，VHH包含SEQ ID NO:39所示的氨基酸序列。
例如，所述4B3的CDR1包含SEQ ID NO:41所示的氨基酸序列，CDR2包含SEQ ID NO:42所示的氨基酸序列，CDR3包含SEQ ID NO:43所示的氨基酸序列，VHH包含SEQ ID NO:46所示的氨基酸序列。
靶向BCMA的嵌合抗原受体、靶向GPRC5D的嵌合抗原受体
在本申请中，所述嵌合抗原受体、靶向BCMA的嵌合抗原受体和/或靶向GPRC5D的嵌合抗原受体可包含共刺激信号区域。
在本申请中，所述共刺激信号区域可包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在本申请中，所述共刺激信号区域可以为源自4-1BB的胞内共刺激信号区域。
在本申请中，所述共刺激信号区域可以为源自CD28的胞内共刺激域。
在本申请中，所述4-1BB共刺激信号区域可包含SEQ ID NO:56所示的氨基酸序列。
在本申请中，所述CD28共刺激信号区域可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，编码所述4-1BB共刺激信号区域的序列可包含SEQ ID NO:57所示的核酸序列。
在本申请中，编码所述CD28共刺激信号区域的序列可包含SEQ ID NO:59所示的核酸序列。
在本申请中，所述嵌合抗原受体、靶向BCMA的嵌合抗原受体和/或靶向GPRC5D的嵌合抗原受体可包含胞内信号传导结构域。
在本申请中，所述胞内信号传导域可包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在本申请中，所述胞内信号传导域可以为源自CD3ζ的信号传导结构域。
在本申请中，所述胞内信号传导域可包含SEQ ID NO:60所示的氨基酸序列。
在本申请中，编码所述胞内信号传导域的序列可包含SEQ ID NO:61所示的核酸序列。
在本申请中，所述嵌合抗原受体、靶向BCMA的嵌合抗原受体和/或靶向GPRC5D的嵌合抗原受体可包含跨膜区。
在本申请中，所述跨膜区可包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、 CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在本申请中，所述跨膜区可以为源自CD8α的跨膜区。例如，所述CD8α跨膜区可包含SEQ ID NO:52所示的氨基酸序列。例如，编码所述CD8α跨膜区的序列可包含SEQ ID NO:53所示的核酸序列。
在本申请中，所述跨膜区可以为源自CD28的跨膜区。例如，所述CD28跨膜区可包含SEQ ID NO:54所示的氨基酸序列。例如，编码所述CD28跨膜区的序列可包含SEQ ID NO:55所示的核酸序列。
在本申请中，所述嵌合抗原受体、靶向BCMA的嵌合抗原受体和/或靶向GPRC5D的嵌合抗原受体可包含铰链区。
在本申请中，所述铰链区可包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在本申请中，所述铰链区可以为源自CD8α的铰链区。例如，所述CD8α的铰链区可包含SEQ ID NO:50所示的氨基酸序列。例如，编码所述CD8α的铰链区的序列可包含SEQ ID NO:53所示的核酸序列。
在本申请中，所述嵌合抗原受体、靶向BCMA的嵌合抗原受体和/或靶向GPRC5D的嵌合抗原受体可包含信号肽。例如，所述信号肽为源自CD8α蛋白的信号肽。例如，所述信号肽包含SEQ ID NO:48所示的氨基酸序列。例如，编码所述信号肽的序列包含SEQ ID NO:49所示的核酸序列。
剪切肽、低密度脂蛋白受体相关蛋白或其片段
在本申请中，所述剪切肽可以为2A肽。
在本申请中，所述剪切肽可以选自下组：P2A、T2A、F2A和E2A。
在本申请中，所述剪切肽可包含SEQ ID NO:62所示的氨基酸序列。在本申请中，编码所述剪切肽的序列可包含SEQ ID NO:63所示的核酸序列。
在本申请中，所述低密度脂蛋白受体相关蛋白或其片段可包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。例如，编码所述低密度脂蛋白受体相关蛋白或其片段的序列包含SEQ ID NO:65所示的核酸序列。
细胞
另一方面，本申请还提供了细胞，所述细胞可以包括所述的嵌合抗原受体、所述的核酸分子和/或所述的表达载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。
在某些实施方案中，所述的细胞可以是免疫效应细胞。在某些实施方案中，所述的细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如，所述的细胞可以是T细胞。
药物组合物
另一方面，本申请还提供了一种药物组合物，可以包括所述嵌合抗原受体、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的佐剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物可以包括液体、冷冻和冻干组合物。
在某些实施方案中，所述药学上可接受的佐剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
制备方法
另一方面，本申请还提供了制备所述嵌合抗原受体的方法。所述方法可以包括在使得所述嵌合抗原受体表达的条件下，培养所述细胞。
本申请还提供了制备经修饰的免疫效应细胞的方法，所述方法可以包括向免疫细胞中引入所述载体。
用途
另一方面，本申请还提供了所述嵌合抗原受体，所述核酸分子，所述的表达载体，所述的细胞，和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、治疗和/或缓解疾病和/或病症。
另一方面，本申请还提供了一种预防、治疗和/或缓解疾病和/或病症的方法，所述方法包括向有需要的受试者施用所述的细胞，和/或所述的药物组合物。
另一方面，本申请还提供了所述嵌合抗原受体，所述核酸分子，所述的表达载体，所述的细胞，和/或所述的药物组合物，其用于预防、治疗和/或缓解疾病和/或病症。
在本申请中，所述疾病和/或病症可包括与GPRC5D异常表达相关的疾病和/或病症。
在本申请中，所述疾病和/或病症可包括与BCMA异常表达相关的疾病和/或病症。
在本申请中，所述疾病和/或病症可包括肿瘤。
在本申请中，所述肿瘤包括实体瘤。
在本申请中，所述肿瘤包括非实体瘤。
在本申请中，所述肿瘤包括血液瘤和/或淋巴瘤。
在本申请中，所述肿瘤包括骨髓瘤。
在本申请中，所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的嵌合抗原受体、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1.双特异性嵌合抗原受体的设计
本实施例双特异性嵌合抗原受体序列的获得简述如下：以BCMA抗原和GPRC5D的空间表位为靶抗原，基于固相淘选技术，通过天然噬菌体抗体文库筛选获得抗BCMA scFv序列，命名为hBCMA22(VH序列为SEQ ID NO:9，VL序列为SEQ ID NO:17)；基于细胞淘选技术，通过人工合成纳米抗体库NanoOri_1.0筛选获得抗GPRC5D VHH序列，命名为3B5 VHH(SEQ ID NO:28)，3E7 VHH(SEQ ID NO:33)，4A2 VHH(SEQ ID NO:39)，4B3 VHH(SEQ ID NO:46)，具体噬菌体抗体文库淘选流程如图1所示。
本实施例构建靶向BCMA和GPRC5D的双特异性嵌合抗原受体CAR结构、抗BCMA的嵌合抗原受体BCMA CAR和抗GPRC5D的嵌合抗原受体GPRC5D CAR，所述双特异性嵌合抗原受体包括CD8α的信号肽序列(SP)*(SEQ ID NO:48)，抗BCMA的单链抗体scFv和抗GPRC5D的单域抗体VHH，(G4S)n和(EAAAK)n的连接肽Linker，n为1-10的整数，CD8α或CD28的铰链区(Hinge)，CD8α(SEQ ID NO:52)或CD28(SEQ ID NO:54)的跨膜区序列(TM)，4-1BB(SEQ ID NO:56)或CD28(SEQ ID NO:58)共刺激域序列和CD3ζ(SEQ ID NO:60)信号传导域序列，低密度脂蛋白受体相关蛋白片段即Ori元件(SEQ ID NO:64)， 此外，所述双特异性嵌合抗原受体结构包含串联和并联两种形式，双特异性嵌合抗原受体结构连接示意图如图2所示，嵌合抗原受体串联和并联结构的分子示意如图3所示。
实施例2.靶细胞表面BCMA及GPRC5D抗原表达
本实施例以检测野生型人骨髓瘤细胞系MM.1S和构建过表达稳定细胞系CHO细胞表面BCMA和GPRC5D抗原表达，通过PE anti-human BCMA(Biolegend)和APC anti-human GPRC5D(R&D)抗体染色，BD流式细胞仪检测靶细胞BCMA和GPRC5D表达。
本实施例结果如图4所示，可知阴性对照细胞均不表达BCMA和GPRC5D；CHO-GPRC5D细胞特异性表达GPRC5D；CHO-BCMA细胞特异性表达BCMA；野生型MM.1S细胞同时特异性表达BCMA和GPRC5D，且表达效率达到99％以上。
实施例3.CAR慢病毒载体构建
(1)现有的BCMA CAR、GPRC5D CAR质粒(原启生物)为模板，PCR克隆获得含BCMA scFv序列、含不同Linker序列、GPRC5D VHH序列的核酸片段；
(2)现有pCore-CAR-Ori质粒(原启生物，包含Ori元件，以下简称CAR慢病毒空载体)为酶切模板，限制性核酸内切酶SphI-HF、NotI-HF(购自NEB)，37℃酶切，1％琼脂糖凝胶回收8900bp含粘性末端线性化片段；
(3)以含有与骨架载体质粒同源序列的正反向引物F1，R1，重叠延申PCR克隆获得同时含BCMA scFv、连接肽Linker、GPRC5D VHH序列的重组核酸片段，如表1所示；
表1 示例性Overlap PCR体系
(4)Overlap PCR片段同源重组到线性化载体上，如表2所示，构建得到同时含BCMA和GPRC5D双特异性的重组CAR分子慢病毒质粒载体，定义为OriCAR-dual01……OriCAR-dual35，其编号分别对应dCAR01……dCAR35，例如，OriCAR-dual15的编号为dCAR15，具体如图1所示；
表2示例性同源重组体系
质粒转化感受态E.coli(DH5α)，测序正确后，质粒纯化试剂盒(Qiagen公司)抽提纯化质粒，测定质粒浓度。
实施例4.慢病毒包装及滴度测定
作为示例，所述双特异性嵌合抗原受体的慢病毒载体系统属于第三代，该系统由四个质粒组成，即编码Gag-Pol蛋白的包装质粒pRH1、编码Rev蛋白的包装质粒pVH3和编码包膜蛋白VSV-G的pMH2质粒，以及核心质粒(BCMA和GPRC5D双靶向的CAR编码基因慢病毒质粒、BCMA单靶向的CAR编码基因慢病毒质粒、GPRC5D单靶向的CAR编码基因慢病毒质粒)，各个CAR慢病毒质粒中CAR基因由延长因子-1α(EF-1α)启动子调控表达。
4.1慢病毒包装
1×106的293T细胞总数悬于2mL含10％FBS的DMEM培养基中，于6孔板中过夜培养；约20-24h后，观察细胞融合度约80-90％，将辅助质粒gag/pol、Rev和VSV-G与重组慢病毒质粒，按相应比例加至50x质粒总质量体积的无血清培养基Opti-MEM中，充分混合均匀，按质粒总量:转染试剂为1:3的比例，加入转染试剂FUGENEHD(Promega，E2311)，混合均匀，室温静置孵育10min；吸去6孔细胞培养板中适量培养基，将质粒/FUGENEHD转染复合物逐滴加入培养板中摇晃混匀，37℃，5％CO2培养过夜；转染约20h后去除含质粒培养基，更换为2mL含5％FBS的DMEM培养基，培养48小时；收集2mL病毒上清，3000rpm，10min离心，分装冻存-80℃。
4.2慢病毒滴度检测
快速解冻4.1中制备的病毒液，5×105的293T细胞总数悬于2.7mL含10％FBS的DMEM培养基中，接种于6孔细胞培养板中；加入终浓度为10μg/mL的Polybrene试剂；加2μL，5μL和10μL含病毒原液，设置1-2个不加入病毒液的空白孔作为流式检测时的阴性对照，充分混匀，37℃，5％CO2静置培养；48h后吸去原培养基，1xPBS洗涤，胰酶消化，取适量 细胞悬液到流式管中，1×PBS(含2％FBS)洗涤，离心去上清；加入检测抗体4℃孵育60min，BD流式细胞仪检测细胞阳性率，采用FlowJo-V10软件数据处理，仅当各测试管阳性率-阴性对照阳性率为5％-20％时，结果可用；病毒滴度计算公式如下：
经检测，上述CAR慢病毒滴度范围在1-10×106TU/mL。
实施例5.CAR-T细胞的制备及慢病毒感染
作为实例，各组合的双特异性嵌合抗原受体CAR结构及单特异性嵌合抗原受体BCMA CAR、GPRC5D CAR的CAR-T细胞制备方法如下：
5.1 CD3+T细胞分选激活
本实施例采用CD3MicroBeads试剂盒(Miltenyi Biotec)分选PBMC中CD3+T细胞，具体而言，PBMC(Ficoll密度梯度离心分离处理)，500g，5min离心去冻存液；按1×107细胞总数加入80μL预冷分选液重悬细胞；按1×107细胞总数加入20μL CD3MicroBeads充分混匀，4℃孵育15min；孵育结束后，按1×107细胞总数加入1.5mL预冷分选液，500g，5min离心去上清；按1×107细胞总数加入0.1mL室温分选液重悬，吹打混匀，将细胞悬液加至润洗后的LS分离柱(置于磁场中)，收集CD3+T细胞，充分混匀，500g，5min离心去上清，X-VIVO完全培养基调整细胞密度为1.25×10 6cells/mL，按细胞数:磁珠数为1:3的比例，加入CD3/CD28 DynaBeads对T细胞进行激活，37℃，5％CO2培养，PBMC经CD3阳选磁珠分选后得到纯度>95％的CD3+T细胞。
5.2慢病毒转导T淋巴细胞
将激活24h的T淋巴细胞收集于50mL离心管中，500g，5min离心去培养上清，X-VIVO完全培养基重悬计数，按7x105细胞总数，接种到12孔板中，感染复数MOI＝3加入所需慢病毒体积，按终浓度为10μg/mL加入相应量的Polybrene试剂，充分混匀，1200rpm，离心60min，37℃，5％CO2维持培养；24h后，收集细胞，500g，5min离心去上清，X-VIVO完全培养基重悬调整细胞密度为5-7×105cells/mL，混匀接种至12孔细胞培养板中，37℃，5％CO2培养。
实施例6.T细胞CAR基因表达效率及体外扩增检测
6.1 CAR基因表达检测
CD3/CD28 DynaBeads激活的T细胞，慢病毒感染后，培养72h进行CAR-T细胞CAR 基因表达效率检测，具体如下：
(1)混匀细胞，AO/PI计数，检测CAR-T细胞密度，取4×105细胞总数至流式管内，加入2mL的1xPBS(含2％FBS)buffer，涡旋混匀，1600rpm，4min；
(2)去上清，加50μL 1×PBS(含2％FBS)buffer重悬细胞；
(3)加入1μL BCMA-His，1μL Biotin-Anti-Human Fab抗体，充分混匀，4℃孵育1h；
(4)加入1mL的1×PBS(含2％FBS)buffer洗涤，涡旋混匀，1600rpm，4min；
(5)去上清，加50μL1×PBS(含2％FBS)buffer重悬细胞，加1μL PE-Anti-6×His，加1μL APC-Streptavidin protein，涡旋仪充分混匀后，4℃孵育30min；
(6)重复步骤4，洗涤3次；
(7)每管300μL的1×PBS(含2％FBS)buffer重悬，充分混匀后上机检测。
本实施例结果如图5所示，在慢病毒感染复数MOI＝3时，双特异性嵌合抗原受体CAR基因稳定表达，且T细胞中72h的转导效率均在75-95％之间；如图6所示，示例性双特异性嵌合抗原受体OriC321 CAR基因在不同供体T细胞的表达效率与单特异性嵌合抗原受体BCMA CAR和GPRC5D CAR不具有显著性差异。
6.2 CAR-T细胞体外扩增
CD3/CD28 DynaBeads激活的CAR-T细胞，慢病毒感染，按培养时间D5、D7、D9、D13进行细胞传代；充分混匀CAR-T细胞，取20μL细胞悬液于1.5mL EP管中，加入等体积AO/PI试剂，混匀取20μL混合液加入计数板中，细胞计数仪(Auto2000)荧光背景下计数，根据所得细胞密度，记录细胞生长情况并绘制生长曲线，同时调整细胞传代密度为7×105cells/mL，接种于6孔板中，培养周期为10-13天；所得数据使用GraphPad 6.0进行数据处理分析。
在本申请的实施例中，OriCAR001的结构为图2中的OriCAR-dual06，OriCAR002的结构为图2中的OriCAR-dual02，OriCAR003的结构为图2中的OriCAR-dual02的linker替换为GGGGSEAAAK，OriCAR004的结构为图2中的OriCAR-dual14，OriCAR005的结构为图2中的OriCAR-dual10，OriCAR006的结构为图2中的OriCAR-dual10的linker替换为GGGGSEAAAK，OriCAR007的结构为图2中的OriCAR-dual17，OriCAR008的结构为图2中的OriCAR-dual21。其中，OriC321的结构为图2中的OriCAR-dual02。本实施例结果如图7所示，经激活培养13天的双特异性嵌合抗原受体CAR-T细胞总扩增倍数在100-350倍之间；如图8所示，经激活培养13天的双特异性嵌合抗原受体CAR-T细胞活率随着培养时间延长而逐渐升高，细胞活率维持在90％左右；如图9所示，示例性双特异性嵌合抗原受体 OriC321 CAR-T细胞总扩增倍数在400倍左右；与Mock T对照细胞相比，体外扩增倍数无显著性差异，可以用于细胞学功能实验。
实施例7.双特异性CAR-T细胞体外效应杀伤活性评估
CD3/CD28 DynaBeads激活的CAR-T细胞，慢病毒感染，按培养时间D9进行CAR-T细胞毒性实验，靶细胞为过表达BCMA的CHO-BCMA细胞和过表达GPRC5D的CHO-GPRC5D细胞；阴性对照细胞为空白CHO细胞。采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)检测，靶细胞体系按CHO-BCMA:CHO-GPRC5D为1:1的比例混合均匀；检测CAR-T细胞杀伤效率，如下：
(1)靶细胞总数量为2×104cells，铺于96孔V底板中，设置不同效靶比；
(2)设置只含与实验组等量效应细胞孔作为效应细胞自释放LDH背景组；
(3)设置只含与实验组等量空白T细胞孔为空白T细胞自释放LDH对照组；
(4)设置只含与实验组相同效靶比空白T细胞孔作为T+靶细胞阴性对照组；
(5)设置只含与实验组等量靶细胞孔作为靶细胞自释放LDH背景组；
(6)设置只含与实验组等量靶细胞孔作为靶细胞最大释放LDH背景组；
(7)设置只含200μL细胞培养基单独孔为培养基背景LDH对照孔；
(8)设置只含200μL细胞培养基单独孔为体积校正对照孔；
(9)相应效应细胞和靶细胞共孵育于200μLX-VIVOTM15空白培养基中；
(10)37℃、5％CO2培养箱培养20-24h；
(11)共孵育结束提前约45min，于体积校正对照孔及靶细胞最大释放LDH背景孔加入20μL裂解液(10×)，37℃、5％CO2培养箱，静置45min；
(12)250g，离心4min；取50μL上清液于96孔平底板中，每孔加入50μL已配制底物(检测缓冲液配置)，室温避光孵育20min；
(13)每孔加入50μL终止液；
(14)酶标仪测定OD490值。
细胞杀伤活性计算公式如下：
所得数据使用GraphPad 6.0进行数据处理分析，采用unpaired t test判断有无统计学差异，P<0.05为有显著性差异。本实施例结果如图10-11所示，和空白效应细胞T细胞相比，结果显示，双特异性CAR-T细胞特异性杀伤具有两个不同靶点的靶细胞，BCMA CAR-T和 GPRC5D CAR-T细胞只特异性杀伤对应靶细胞，且对CHO-negative细胞没有杀伤活性，揭示不同双特异性CAR-T的靶向特异性；如图12所示，示例性OriC321 CAR-T细胞对靶细胞杀伤效率显著优于BCMA CAR与GPRC5D CAR，在效靶比为5:1，杀伤效率达70％。
实施例8.双特异性CAR-T细胞脱颗粒分子CD107a表达水平测定
本实施例以检测靶向BCMA和GPRC5D双特异性CAR-T在阳性靶细胞CHO-BCMA和CHO-GPRC5D刺激下CD107a的表达水平。CD3/CD28 DynaBeads激活的双特异性CAR-T细胞及对照Mock T细胞，培养D9进行细胞脱颗粒水平CD107a检测，对应阳性率的CAR-T细胞与靶细胞以1:1的效靶比，共培养于X-VIVO空白培养基中，靶细胞与阳性CAR-T细胞均为5×106cells/mL，0.6mL/孔，接种到12孔细胞培养板中，混匀后37℃，5％CO2培养，共培养4-6h，收集细胞，1×PBS(2％FBS)洗涤，离心去上清；加入PE标记的小鼠抗人CD107a抗体、APC/Cy7标记的小鼠抗人CD3抗体，4℃避光孵育30min，BD流式细胞仪检测CD3+T细胞中CD107a的比例。
本实施例结果如图13所示，双特异性CAR-T细胞与靶细胞CHO-BCMA、CHO-GPRC5D共培养CD107a表达在15-40％左右，其中，dCAR02，dCAR09的CD107a表达阳性率接近40％；Mock T对照细胞几乎没有CD107a表达。
实施例9.双特异性CAR-T细胞的免疫突触机制研究
免疫突触(Immune synapse,IS)是T细胞与抗原提呈细胞结合的接触区，包含T细胞抗原受体与相应抗原及一些辅助性分子。免疫突触的形成也是CAR-T细胞识别抗原，增殖和活化的关键步骤。免疫突触的形成与CAR-T细胞的效应功能相关，在CAR-T水平上，测量免疫突触结构微管组织中心(MTOC)的极化，以进一步评估CAR-T细胞的功能优势。CAR-T细胞与靶细胞按1：1比例共培养5分钟后转移至Nunc chamber slides中，孵育20分钟，4％PFA室温固定，PBS清洗3次后，免疫染色通透液(Triton X-100)，室温孵育；10％马血清/免疫染色通透液(Triton X-100)，室温孵育；加入2％马血清/免疫染色通透液(Triton X-100)稀释的一抗室温孵育；加入2％马血清/免疫染色通透液(Triton X-100)稀释的二抗室温孵育；分离载玻片，ProLong Diamond封片，干燥凝固后激光共聚焦100倍油镜，紫外、488nm、543nm，1024x1024，Z轴扫描0.4μm，叠加得到3D图像和影片。
本实施例结果如图14所示，BCMA CAR、GPRC5D CAR与OriC321 CAR T细胞共聚焦成像；其中，细胞核染色为蓝色，γ-微管蛋白染色为绿色，T细胞染色为红色，白色箭头表示IS处微管组织中心MTOC的极化位置，MTOC与IS的距离越近，则IS结构越稳定。如 图15所示，与BCMA CAR和GPRC5D CAR-T细胞相比，OriC321 CAR T细胞对IS的MTOC极化水平明显增加，表现为MTOC与IS的距离减小，从而获得了更优越、更稳定的IS结构，因而具有更优越的功能优势，表明OriC321 CAR T细胞介导的细胞毒性优于单靶向的BCMA CAR和GPRC5D CAR-T细胞。
实施例10.双特异性CAR-T细胞因子分泌检测
双特异性CAR-T细胞培养D9进行CAR-T细胞释放因子IL-2、IFN-γ检测，采用R&D公司IL-2、IFN-γ细胞因子ELISA检测试剂盒。CAR-T细胞预先检测阳性率及细胞密度，以空白T细胞调整慢病毒感染后的CAR-T细胞，使组间细胞总数一致；将CAR-T细胞和MM.1S细胞，以效靶比1:1的比例进行共孵育，即CAR阳性细胞总数为2×104，MM1S细胞总数为2×104，共孵育24h后，250g离心5min，收集上清，对应的IL-2、IFN-γELISA试剂盒检测细胞因子分泌量；如下：在96孔ELISA板上包被稀释的捕获抗体，浓度1μg/ml，4℃过夜。然后用10％脱脂奶粉封闭非特异性结合位点，充分洗涤后，3倍稀释的样本上清加入96孔板，室温孵育2h；充分洗涤后，加入生物素化检测抗体工作液(100μL/孔)，室温孵育2h；充分洗涤后，加入酶结合物工作液(100μL/孔)，避光室温孵育20min；充分洗涤后，加入TMB显色剂(100μL/孔)，避光室温孵育20min；加入硫酸终止液(50μL/孔)，于450nm处测定各孔OD值。所得数据使用GraphPad 6.0进行数据处理分析，采用unpaired t test判断有无统计学差异，P<0.05为有显著性差异。
本实施例结果如图16和图17所示，双特异性CAR-T的IL-2、IFN-γ因子分泌量与BCMA CAR-T、GPRC5D CAR-T相比，释放量显著增多，单效应细胞和不转导CAR的Mock T细胞基本不分泌IL-2或分泌少量的IFN-γ，说明了双特异性CAR-T细胞相对单靶向CAR-T细胞诱导产生细胞因子表达的效应能力较强。
实施例11.双特异性CAR-T细胞靶向增殖能力
本实施例CAR-T细胞的靶向增殖能力检测选用未经处理的MM.1S细胞作为激活靶细胞。CD3/CD28 DynaBeads激活的CAR-T细胞及对照Mock T细胞，慢病毒感染，按培养时间D9或D13左右进行CAR-T细胞靶向增殖能力检测。CAR-T细胞和靶细胞，以效靶比1:1的比例进行第一轮靶向刺激，37℃，5％CO2培养；在共孵育5天后，收集所有细胞，AO/PI试剂细胞计数，记录细胞增殖情况，含细胞密度、细胞活率等，根据计数所得细胞密度，按照1:3(Total CAR-T细胞数:MM.1S细胞数)的比例，进行第二轮靶向刺激，再次共孵育5天后， 进行第三轮靶向刺激。三轮靶向刺激期间，按照细胞生长情况每1-2天补加适量CAR-T细胞基础培养基X-VIVO，统计每一轮计数结果并绘制靶向增殖曲线，所得数据使用GraphPad 6.0进行数据处理分析，采用unpaired t test判断有无统计学差异，P<0.05为有显著性差异。
本实施例结果如图18所示，双特异性CAR-T细胞接受靶细胞刺激后存在不同程度的靶向增殖能力，与BCMA CAR-T、GPRC5D CAR-T相比没有显著性差异，增殖倍数在200-800倍之间，与对照Mock T细胞相比具有显著性差异，预示了双特异性CAR-T细胞在生物体内针对特异性肿瘤可有效迅速扩增。
实施例12.双特异性CAR-T细胞NSG小鼠体内药效验证
12.1体内回输的CAR-T细胞冻存制备
参照实施例5中双特异性CAR-T细胞制备，CD3/CD28 DynaBeads激活的CAR-T细胞，慢病毒感染，培养D9进行CAR-T细胞冻存。CAR-T细胞预先检测阳性率及细胞密度(AO/PI)，以空白T细胞调整使组间细胞总数一致；按所需回输CAR-T细胞密度冻存相应CAR-T细胞，收集所需细胞，300g，5min离心，去上清，按1:2体积比加入冻存液A液(原启生物)重悬细胞，缓慢加入冻存液B液(原启生物)，混匀后分装于2mL冻存管中放置在程序降温盒中，于-80℃冻存，24h后将冻存细胞转移液氮保存。
12.2 MM.1S肿瘤移植小鼠模型抗肿瘤活性
NSG小鼠(百奥赛图)皮下接种MM.1S细胞，剂量为1×107cells/200μL/只，荷瘤14天后，测得小鼠成瘤体积约为50-150mm3，将小鼠随机分为5组，每组5只，分别为对照Mock T组、双特异性CAR-T组、BCMA CAR-T组；GPRC5D CAR-T组；尾静脉注射方式，向Mock T组注射T细胞(冻存剂量为6×106cells/200μL/只)，向双特异性CAR-T组注射对应CAR+T细胞(冻存剂量为6×106cells/200μL/只)，向BCMA CAR-T组注射对应CAR+T细胞(冻存剂量为6×106cells/200μL/只)，向GPRC5D CAR-T组注射对应CAR+T细胞(冻存剂量为6×106cells/200μL/只)。CAR-T回输后每周一、周三、周五分别测量肿瘤的体积，观察小鼠毛色、排泄物、饮食进水、躯体运动等生命体征，以及小鼠死亡情况；CAR-T细胞回输后的D7、D14、D21分别尾静脉采血，CBA人Th1/Th2/Th17细胞因子试剂盒(BD Biosciences)检测血清中IL2、IL4、IL6、IL10、TNF-α、IFN-γ和IL17A等细胞因子表达，BD流式检测CD3+CD8+T细胞比例。
本实施例中NSG小鼠体内抑瘤实验流程如图19所示；如图20所示，示例性双特异性嵌合抗原受体OriC321 CAR-T细胞与对照组BCMA CAR-T，GPRC5D CAR-T在D14天产生的IFN-γ无显著性差异，相对于Mock T组，具有显著性差异(图20A)；OriC321 CAR-T细胞组 小鼠体重随着时间呈稳定增长趋势，与Mock T组的小鼠体重曲线趋势一致，此外，OriC321 CAR-T细胞组注射后小鼠毛色光亮，小鼠行为等生命体征正常，说明OriC321 CAR-T细胞在小鼠体内的良好安全性(图20B)；相对于Mock T细胞组，OriC321 CAR-T细胞具有显著消瘤作用，抑瘤率可接近100％(图20C)；示例性双特异性嵌合抗原受体OriC321 CAR-T细胞可明显延长小鼠生存率(图20C)；阐述了OriC321 CAR-T细胞在小鼠模型中治疗MM时提供更大的疗效和生存效益，在临床应用上可能表现出更优越的抗肿瘤活性。
实施例13.基因敲除的MM.1S靶细胞验证OriC321 CAR-T细胞体内抗肿瘤活性
进一步模拟检测CAR-T细胞在人体环境下的靶细胞效应杀伤活性，采用CRISPR-Cas9技术，将靶细胞中的BCMA和GPRC5D基因分别敲除，流式分选BCMAKO和GPRC5DKO的阳性细胞，得到BCMA基因敲除的MM.1S-BCMAKO细胞，GPRC5D基因敲除的MM.1S-GPRC5DKO细胞，同时表达BCMA和GPRC5D基因的MM.1S-WT细胞(如图21)，将MM.1S-BCMAKO、MM.1S-GPRC5DKO、MM.1S-WT细胞按1:1:1等比例混合均匀，为动物实验小鼠荷瘤细胞，NSG小鼠体内验证示例性双特异性嵌合抗原受体OriC321 CAR-T细胞的消瘤、抑瘤活性，具体步骤参照本实施例12MM.1S肿瘤移植小鼠模型抗肿瘤活性。
本实施例中基于基因敲除的MM.1S靶细胞造模的NSG小鼠体内抑瘤实验流程如图22所示；如图23所示，Mock T无抑瘤效果，BCMA CAR-T及GPRC5D CAR-T在发挥轻微抗肿瘤活性后，随着时间延长，最终无法抑制肿瘤生长，示例性双特异性OriC321 CAR-T细胞完全抑制肿瘤生长，具有显著消瘤效果，抑瘤率可接近100％(图23B)；示例性双特异性嵌合抗原受体OriC321 CAR-T细胞可明显延长小鼠生存率(图23C)，阐述了示例性双特异性嵌合抗原受体OriC321 CAR-T细胞在基因敲除的MM.1S靶细胞小鼠模型中治疗MM时的明显疗效和生存效益。
实施例14.GPRC5D抗体序列与不同种属靶抗原的交叉结合研究
使用表达人GPRC5D，食蟹猴GPRC5D(SEQ.A)，小鼠GPRC5D(SEQ.B)，人GPRC5D对照亚型GPRC5A(SEQ.C)的稳定转染CHO细胞，通过流式鉴定评估不同抗GPRC5D抗体对人，食蟹猴和鼠GPRC5D的结合。简而言之，使用PBS缓冲液配置浓度为1x106细胞/mL的CHO-HuGPRC5D、CHO-CyGPRC5D、CHO-MuGPRC5D、CHO-HuGPRC5A靶细胞，加入96孔V底板，每孔30μL；使用PBS缓冲液配置检测抗体GPRC5D VHH-hFc浓度为30μg/mL，并按3倍比稀释抗体，形成8个浓度梯度(最后浓度为0)；将配置好的不同浓度的抗体按30μL/孔加入已铺好的靶细胞中混匀；4℃冰箱孵育1小时；每孔加150μL缓冲液，300g离心5分钟弃 上清；重复洗涤一次；使用缓冲液按1:200配比配置Dyna650-山羊抗人IgG(Fc特异性，ab98593)荧光二抗，每孔30μL加入细胞中混匀，4℃冰箱孵育30分钟；每孔加170μL缓冲液，300g离心5分钟弃上清；重复洗涤一次；每孔加35μL缓冲液混匀后使用iQue Screener流式仪(购自IntelliCyt公司)检测。
本实施例流式结合活性检测结果如图24和图25所示，结合特性汇总如表3所示，可知GPRC5D VHH-hFc抗体显示与人GPRC5D、小鼠GPRC5D、食蟹猴GPRC5D的交叉结合活性，其中，GPRC5D VHH-hFc抗体与人GPRC5D和食蟹猴GPRC5D存在强结合活性，与小鼠GPRC5D存在相对较弱的结合活性；未检测到GPRC5D VHH-hFc抗体与空白的CHO细胞的结合，未检测到GPRC5D VHH-hFc抗体与表达人GPRC5D同一家族不同亚型的GPRC5A的CHO细胞的结合，说明了应用于本产品的GPRC5D VHH-hFc抗体的高度特异性，可以减少各种脱靶副作用的发生风险。
表3 GPRC5D VHH-hFc抗体与不同种属靶抗原的交叉结合特性汇总
注：-不存在结合活性；+存在相对较弱结合活性；+++存在较强结合活性。
SEQ ID NO:73.Cynomolgus monkey GPRC5D
SEQ ID NO:74.Mouse GPRC5D
SEQ ID NO:75.Human GPRC5A
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (176)

  1. 嵌合抗原受体，其包含第一抗原结合结构域和第二抗原结合结构域，所述第一抗原结合结构域靶向B细胞成熟抗原(BCMA)，且所述第二抗原结合结构域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述第一抗原结合结构域包含抗体或其抗原结合片段。
  3. 根据权利要求2所述的嵌合抗原受体，其中所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  4. 根据权利要求2-3中任一项所述的嵌合抗原受体，其中所述抗原结合片段包含scFv。
  5. 根据权利要求1-4中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:9所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含SEQ ID NO:3所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含VH，且所述VH包含SEQ ID NO:9所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含抗体轻链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含SEQ ID NO:11(LGS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含VL，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含VH和VL，所述VH和VL通过连接子相连，所述连接子包含SEQ ID NO:18所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:19所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含抗体或其抗原结合片段。
  14. 根据权利要求13所述的嵌合抗原受体，其中所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  15. 根据权利要求13-14中任一项所述的嵌合抗原受体，其中所述抗原结合片段包括VHH。
  16. 根据权利要求1-15中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含抗体重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
  17. 根据权利要求16所述的嵌合抗原受体，其中所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下述任一组的氨基酸序列：
    (1)HCDR1：SEQ ID NO:21；HCDR2：SEQ ID NO:22；和HCDR3：SEQ ID NO:23；
    (2)HCDR1：SEQ ID NO:30；HCDR2：SEQ ID NO:31；和HCDR3：SEQ ID NO:32；
    (3)HCDR1：SEQ ID NO:35；HCDR2：SEQ ID NO:36；和HCDR3：SEQ ID NO:37；以及
    (4)HCDR1：SEQ ID NO:41；HCDR2：SEQ ID NO:42；和HCDR3：SEQ ID NO: 43。
  21. 根据权利要求1-20中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含VH，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
  22. 根据权利要求21所述的嵌合抗原受体，其中所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的嵌合抗原受体，其中所述第二抗原结合结构域包含VHH，且所述VHH包含SEQ ID NO:72所示的氨基酸序列。
  24. 根据权利要求23所述的嵌合抗原受体，其中所述VHH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
  25. 根据权利要求1-24中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域和所述第二抗原结合结构域分别包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3，所述第一抗原结合结构域和所述第二抗原结合结构域的HCDR1，HCDR2和HCDR3的序列选自下述任一组氨基酸序列：
    (1)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:22，和HCDR3：SEQ ID NO:23；
    (2)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:31，和HCDR3：SEQ ID NO:32；
    (3)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:35，HCDR2：SEQ ID NO:36，和HCDR3：SEQ ID NO:37；以及
    (4)第一抗原结合结构域：HCDR1：SEQ ID NO:2，HCDR2：SEQ ID NO:3，HCDR3：SEQ ID NO:4，LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:11(LGS)，和LCDR3：SEQ ID NO:12；第二抗原结合结构域：HCDR1：SEQ ID NO:41，HCDR2：SEQ ID NO:42，和HCDR3：SEQ ID NO:43。
  26. 根据权利要求1-25中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域的C端和所述第二抗原结合结构域的N端直接或间接相连。
  27. 根据权利要求1-26中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域的N端与所述第二抗原结合结构域的C端直接或间接相连。
  28. 根据权利要求1-27中任一项所述的嵌合抗原受体，其中所述第一抗原结合结构域和所述第二抗原结合结构域通过连接子相连。
  29. 根据权利要求28所述的嵌合抗原受体，其中所述连接子包含肽接头。
  30. 根据权利要求28-29中任一项所述的嵌合抗原受体，其中所述连接子包含(GGGGS)n的氨基酸序列，其中所述n为1-10中的任意正整数。
  31. 根据权利要求28-30中任一项所述的嵌合抗体受体，其中所述连接子包含(EAAAK)n的氨基酸序列，其中所述n为1-10中的任意正整数。
  32. 根据权利要求1-31中任一项所述的嵌合抗原受体，其包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  33. 根据权利要求32所述的嵌合抗原受体，其中所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
  34. 根据权利要求32-33中任一项所述的嵌合抗原受体，其中所述共刺激信号区域包含SEQ ID NO:56所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的嵌合抗原受体，其包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
  36. 根据权利要求35所述的嵌合抗原受体，其中所述胞内信号传导域为源自CD3ζ的信号传导结构域。
  37. 根据权利要求35-36中任一项所述的嵌合抗原受体，其中所述胞内信号传导域包含SEQ ID NO:60所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的嵌合抗原受体，其包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、 NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  39. 根据权利要求38所述的嵌合抗原受体，其中所述跨膜区为源自CD8α的跨膜区。
  40. 根据权利要求38-39中任一项所述的嵌合抗原受体，其中所述跨膜区包含SEQ ID NO:52所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的嵌合抗原受体，其还包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  42. 根据权利要求41所述的嵌合抗原受体，其中所述铰链区为源自CD8α的铰链区。
  43. 根据权利要求41-42中任一项所述的嵌合抗原受体，其中所述铰链区包含SEQ ID NO:50所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的嵌合抗原受体，其还包含低密度脂蛋白受体相关蛋白或其片段。
  45. 根据权利要求44所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段位于所述胞内信号区域的C端。
  46. 根据权利要求44-45中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  47. 根据权利要求44-46中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
  48. 根据权利要求44-47中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
  49. 根据权利要求1-48中任一项所述的嵌合抗原受体，其还包含信号肽。
  50. 根据权利要求49所述的嵌合抗原受体，其中所述信号肽为源自CD8α蛋白的信号肽。
  51. 根据权利要求49-50中任一项所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:48所示的氨基酸序列。
  52. 分离的一种或多种核酸分子，其编码权利要求1-51中任一项所述的嵌合抗原受体。
  53. 根据权利要求52所述的核酸分子，其还包含编码剪切肽的序列。
  54. 根据权利要求53所述的核酸分子，其中所述剪切肽包含2A肽。
  55. 根据权利要求53-54中任一项所述的核酸分子，其中所述剪切肽选自下组：P2A、T2A、F2A和E2A。
  56. 根据权利要求53-55中任一项所述的核酸分子，其中所述剪切肽的序列包含SEQ ID NO:62所示的氨基酸序列。
  57. 根据权利要求53-56中任一项所述的核酸分子，其中编码所述剪切肽的序列包含SEQ ID NO:63所示的核酸序列。
  58. 根据权利要求52-57中任一项所述的核酸分子，其还包含编码低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  59. 根据权利要求58所述的核酸分子，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  60. 根据权利要求58-59中任一项所述的核酸分子，其中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
  61. 根据权利要求58-60中任一项所述的核酸分子，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
  62. 根据权利要求58-61中任一项所述的核酸分子，其中编码所述低密度脂蛋白受体相关蛋白或其片段的序列包含SEQ ID NO:65所示的核酸序列。
  63. 表达载体，其包含权利要求52-62中任一项所述的核酸分子。
  64. 表达载体，其包含编码权利要求1-51中任一项所述的靶向BCMA的嵌合抗原受体的核酸序列和编码权利要求1-51中任一项所述的靶向GPRC5D的嵌合抗原受体的核酸序列。
  65. 表达载体，其包括编码靶向BCMA的嵌合抗原受体的核酸序列和编码靶向GPRC5D的嵌合抗原受体的核酸序列。
  66. 根据权利要求65所述的表达载体，其中所述编码靶向BCMA的嵌合抗原受体的核酸序列包含编码靶向BCMA的抗原结合结构域的核酸序列。
  67. 根据权利要求65-66中任一项所述的表达载体，其中所述靶向BCMA的抗原结合结构域包含重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:9所示的氨基酸序列。
  68. 根据权利要求65-67中任一项所述的表达载体，其中所述靶向BCMA的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含SEQ ID NO:3所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4所示的氨基酸序列。
  69. 根据权利要求65-68中任一项所述的表达载体，其中所述靶向BCMA的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:9所示的氨基酸序列。
  70. 根据权利要求65-69中任一项所述的表达载体，其中所述靶向BCMA的抗原结合结构域包含重链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
  71. 根据权利要求65-70中任一项所述的表达载体，其中所述靶向BCMA的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含SEQ ID NO:11(LGS)所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
  72. 根据权利要求65-71中任一项所述的表达载体，其中所述靶向BCMA的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:17所示的氨基酸序列。
  73. 根据权利要求65-72中任一项所述的表达载体，其中所述靶向BCMA的抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:19所示的氨基酸序列。
  74. 根据权利要求73所述的表达载体，其中编码所述scFv的序列包含SEQ ID NO:20所示的核酸序列。
  75. 根据权利要求65-74中任一项所述的表达载体，其中所述靶向BCMA的嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  76. 根据权利要求75所述的表达载体，其中所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
  77. 根据权利要求75-76中任一项所述的表达载体，其中所述共刺激信号区域为源自CD28的胞内共刺激信号区域。
  78. 根据权利要求75-77中任一项所述的表达载体，其中编码所述共刺激信号区域的序列包含SEQ ID NO:57或SEQ ID NO:59所示的核酸序列。
  79. 根据权利要求65-78中任一项所述的表达载体，其中所述靶向BCMA的嵌合抗原受体包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、 卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
  80. 根据权利要求79所述的表达载体，其中所述胞内信号传导域为源自CD3ζ的信号传导结构域。
  81. 根据权利要求79-80中任一项所述的表达载体，其中编码所述胞内信号传导域的序列包含SEQ ID NO:61所示的核酸序列。
  82. 根据权利要求65-81中任一项所述的表达载体，其中所述靶向BCMA的嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  83. 根据权利要求82所述的表达载体，其中所述跨膜区为源自CD8α的跨膜区。
  84. 根据权利要求82-83中任一项所述的表达载体，其中所述跨膜区为源自CD28的跨膜区。
  85. 根据权利要求82-84中任一项所述的表达载体，其中编码所述跨膜区的序列包含SEQ ID NO:53或SEQ ID NO:55所示的核酸序列。
  86. 根据权利要求65-85中任一项所述的表达载体，其中所述靶向BCMA的嵌合抗原受体包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  87. 根据权利要求86所述的表达载体，其中所述铰链区为源自CD8α的铰链区。
  88. 根据权利要求86-87中任一项所述的表达载体，其中编码所述铰链区的序列包含SEQ ID NO:51所示的核酸序列。
  89. 根据权利要求65-88中任一项所述的表达载体，其中所述靶向BCMA的嵌合抗原受体包括信号肽。
  90. 根据权利要求89所述的表达载体，其中所述信号肽包含为源自CD8α蛋白的信号肽。
  91. 根据权利要求89-90中任一项所述的表达载体，其中编码所述信号肽的序列包含SEQ ID NO:49所示的核酸序列。
  92. 根据权利要求65-91中任一项所述的表达载体，其中所述靶向GPRC5D的抗原结合结构 域包含抗体或其抗原结合片段。
  93. 根据权利要求92所述的表达载体，其中所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  94. 根据权利要求92-93中任一项所述的表达载体，其中所述抗原结合片段包括VHH。
  95. 根据权利要求65-94中任一项所述的表达载体，其中所述靶向GPRC5D的抗原结合结构域包含重链可变区VH中的至少一个CDR，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
  96. 根据权利要求65-95中任一项所述的表达载体，其中所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
  97. 根据权利要求96所述的表达载体，其中编码所述VH的序列包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
  98. 根据权利要求65-97中任一项所述的表达载体，其中所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所述HCDR2包含SEQ ID NO:68(IX1X2X3X4GX5T，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
  99. 根据权利要求65-98中任一项所述的表达载体，其中所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:21、SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:41中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22、SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:42中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:43中任一项所示的氨基酸序列。
  100. 根据权利要求65-99中任一项所述的表达载体，其中所述靶向GPRC5D的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下述任一组的氨基酸序列：
    (1)HCDR1：SEQ ID NO:21；HCDR2：SEQ ID NO:22；和HCDR3：SEQ ID NO:23；
    (2)HCDR1：SEQ ID NO:30；HCDR2：SEQ ID NO:31；和HCDR3：SEQ ID NO:32；
    (3)HCDR1：SEQ ID NO:35；HCDR2：SEQ ID NO:36；和HCDR3：SEQ ID NO:37；以及
    (4)HCDR1：SEQ ID NO:41；HCDR2：SEQ ID NO:42；和HCDR3：SEQ ID NO:43。
  101. 根据权利要求65-100中任一项所述的表达载体，其中所述靶向GPRC5D的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:72所示的氨基酸序列。
  102. 根据权利要求101所述的表达载体，其中所述VH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
  103. 根据权利要求101-102中任一项所述的表达载体，其中编码所述VH的序列包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
  104. 根据权利要求65-100中任一项所述的表达载体，其中所述靶向GPRC5D的抗原结合结构域包含VHH，且所述VHH包含SEQ ID NO:72所示的氨基酸序列。
  105. 根据权利要求104所述的表达载体，其中所述VHH包含SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:39和SEQ ID NO:46中任一项所示的氨基酸序列。
  106. 根据权利要求104-105中任一项所述的表达载体，其中编码所述VHH的序列包含SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:40和SEQ ID NO:47中任一项所示的核酸序列。
  107. 根据权利要求65-106中任一项所述的表达载体，其中所述靶向GPRC5D的嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  108. 根据权利要求107所述的表达载体，其中所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
  109. 根据权利要求107-108中任一项所述的表达载体，其中所述共刺激信号区域为源自CD28的胞内共刺激域。
  110. 根据权利要求107-109中任一项所述的表达载体，其中所述共刺激信号区域包含SEQ ID NO:56或SEQ ID NO:58所示的氨基酸序列。
  111. 根据权利要求107-110中任一项所述的表达载体，其中编码所述共刺激信号区域的序列包含SEQ ID NO:57或SEQ ID NO:59所示的核酸序列。
  112. 根据权利要求65-111中任一项所述的表达载体，其中所述靶向GPRC5D的嵌合抗原受体包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的 胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
  113. 根据权利要求112所述的表达载体，其中所述胞内信号传导域为源自CD3ζ的信号传导结构域。
  114. 根据权利要求112-113中任一项所述的表达载体，其中所述胞内信号传导域包含SEQ ID NO:60所示的氨基酸序列。
  115. 根据权利要求112-114中任一项所述的表达载体，其中编码所述胞内信号传导域的序列包含SEQ ID NO:61所示的核酸序列。
  116. 根据权利要求65-115中任一项所述的表达载体，其中所述靶向GPRC5D的嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  117. 根据权利要求116所述的表达载体，其中所述跨膜区为源自CD8α的跨膜区。
  118. 根据权利要求116-117中任一项所述的表达载体，其中所述跨膜区为源自CD28的跨膜区。
  119. 根据权利要求116-118中任一项所述的表达载体，其中所述跨膜区包含SEQ ID NO:52或SEQ ID NO:54所示的氨基酸序列。
  120. 根据权利要求116-119中任一项所述的表达载体，其中编码所述跨膜区的序列包含SEQ ID NO:53或SEQ ID NO:55中任一项所示的核酸序列。
  121. 根据权利要求65-120中任一项所述的表达载体，其中所述靶向GPRC5D的嵌合抗原受体包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、CD8α、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  122. 根据权利要求121所述的表达载体，其中所述铰链区为源自CD8α的铰链区。
  123. 根据权利要求121-122中任一项所述的表达载体，其中所述铰链区包含SEQ ID NO:50 所示的氨基酸序列。
  124. 根据权利要求121-123中任一项所述的表达载体，其中编码所述铰链区的序列包含SEQ ID NO:51所示的核酸序列。
  125. 根据权利要求65-124中任一项所述的表达载体，其中所述靶向GPRC5D的嵌合抗原受体包括信号肽。
  126. 根据权利要求125所述的表达载体，其中所述信号肽包含为源自CD8α蛋白的信号肽。
  127. 根据权利要求125-126中任一项所述的表达载体，其中所述信号肽包含SEQ ID NO:48所示的氨基酸序列。
  128. 根据权利要求125-127中任一项所述的表达载体，其中编码所述信号肽的序列包含SEQ ID NO:49所示的核酸序列。
  129. 根据权利要求65-128中任一项所述的表达载体，其中所述编码靶向BCMA的嵌合抗原受体的核酸序列和所述编码靶向GPRC5D的嵌合抗原受体的核酸序列通过编码剪切肽的序列连接。
  130. 根据权利要求129所述的表达载体，其中所述剪切肽为2A肽。
  131. 根据权利要求129-130中任一项所述的表达载体，其中所述剪切肽选自下组：P2A、T2A、F2A和E2A。
  132. 根据权利要求129-131中任一项所述的表达载体，其中所述剪切肽包含SEQ ID NO:62所示的氨基酸序列。
  133. 根据权利要求129-132中任一项所述的表达载体，其中编码所述剪切肽的序列包含SEQ ID NO:63所示的氨基酸序列。
  134. 根据权利要求65-133中任一项所述的表达载体，其中所述编码靶向BCMA的嵌合抗原受体的核酸序列的3’端和所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的5’端通过剪切肽相连。
  135. 根据权利要求65-134中任一项所述的表达载体，其中所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端和所述编码靶向BCMA的嵌合抗原受体的核酸序列的5’端通过剪切肽相连。
  136. 根据权利要求65-135中任一项所述的表达载体，其还包含编码低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  137. 根据权利要求136所述的表达载体，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  138. 根据权利要求136-137中任一项所述的表达载体，其中所述低密度脂蛋白受体相关蛋白 或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
  139. 根据权利要求136-138中任一项所述的表达载体，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
  140. 根据权利要求136-139中任一项所述的表达载体，其中编码所述低密度脂蛋白受体相关蛋白或其片段的序列包含SEQ ID NO:65所示的核酸序列。
  141. 根据权利要求136-140中任一项所述的表达载体，其中所述编码低密度脂蛋白受体相关蛋白或其片段的核酸序列的5’端与所述编码靶向GPRC5D的嵌合抗原受体的核酸序列的3’端通过剪切肽相连。
  142. 根据权利要求136-141中任一项所述的表达载体，其中所述编码低密度脂蛋白受体相关蛋白或其片段的核酸序列的5’端与所述编码靶向BCMA的嵌合抗原受体的核酸序列的3’端通过剪切肽相连。
  143. 根据权利要求65-142中任一项所述的表达载体，其从5’端至3’端分别依次包含编码信号肽的序列、编码靶向BCMA的嵌合抗原受体的序列、编码剪切肽的序列、编码靶向GPRC5D的嵌合抗原受体的序列、编码剪切肽的序列，以及编码低密度脂蛋白受体相关蛋白或其片段的序列。
  144. 根据权利要求65-143中任一项所述的表达载体，其从5’端至3’端分别依次包含编码信号肽的序列、编码靶向GPRC5D的嵌合抗原受体的序列、编码剪切肽的序列、编码靶向BCMA的嵌合抗原受体的序列、编码剪切肽的序列，以及编码低密度脂蛋白受体相关蛋白或其片段的序列。
  145. 细胞，其包含权利要求1-51中任一项所述的嵌合抗原受体，权利要求52-62中任一项所述的核酸分子，或权利要求63-144中任一项所述的表达载体。
  146. 根据权利要求145所述的细胞，其还包含和/或表达低密度脂蛋白受体相关蛋白或其片段。
  147. 根据权利要求146所述的细胞，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  148. 根据权利要求146-147中任一项所述的细胞，其中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
  149. 根据权利要求146-148中任一项所述的细胞，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:64所示的氨基酸序列。
  150. 根据权利要求145-149中任一项所述的细胞，其包含免疫效应细胞。
  151. 根据权利要求145-150中任一项所述的细胞，其包括T细胞、B细胞、天然杀伤细胞 (NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
  152. 根据权利要求145-151中任一项所述的细胞，其为T细胞。
  153. 制备权利要求1-51中任一项所述的嵌合抗原受体的方法，所述方法包括在使得权利要求1-51中任一项所述的嵌合抗原受体表达的条件下，培养根据权利要求145-152中任一项所述的细胞。
  154. 制备经修饰的免疫效应细胞的方法，其包括向免疫效应细胞中引入权利要求63-144中任一项所述的表达载体。
  155. 药物组合物，其包含权利要求1-51中任一项所述的嵌合抗原受体，权利要求52-62中任一项所述的核酸分子，权利要求63-144中任一项所述的表达载体，和/或权利要求145-152中任一项所述的细胞，以及任选地药学上可接受的载体。
  156. 权利要求1-51中任一项所述的嵌合抗原受体，权利要求52-62中任一项所述的核酸分子，权利要求63-144中任一项所述的表达载体，权利要求145-152中任一项所述的细胞，和/或权利要求155所述的药物组合物在制备药物中的用途，所述药物用于预防、治疗和/或缓解疾病和/或病症。
  157. 根据权利要求156所述的用途，其中所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症和/或与BCMA异常表达相关的疾病和/或病症。
  158. 根据权利要求156-157中任一项所述的用途，其中所述疾病和/或病症包括肿瘤。
  159. 根据权利要求158所述的用途，其中所述肿瘤包括实体瘤和/或非实体瘤。
  160. 根据权利要求158-159中任一项所述的用途，其中所述肿瘤包括血液瘤和/或淋巴瘤。
  161. 根据权利要求158-160中任一项所述的用途，其中所述肿瘤包括骨髓瘤。
  162. 根据权利要求161所述的用途，其中所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
  163. 一种预防、治疗和/或缓解疾病和/或病症的方法，所述方法包括向有需要的受试者施用权利要求145-152中任一项所述的细胞，和/或权利要求155所述的药物组合物。
  164. 根据权利要求163所述的方法，其中所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症，和/或与BCMA异常表达相关的疾病和/或病症。
  165. 根据权利要求162-164中任一项所述的方法，其中所述疾病和/或病症包括肿瘤。
  166. 根据权利要求165所述的方法，其中所述肿瘤包括实体瘤和/或非实体瘤。
  167. 根据权利要求165-166中任一项所述的方法，其中所述肿瘤包括血液瘤和/或淋巴瘤。
  168. 根据权利要求165-167中任一项所述的方法，其中所述肿瘤包括骨髓瘤。
  169. 根据权利要求168所述的方法，其中所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
  170. 权利要求1-51中任一项所述的嵌合抗原受体，权利要求52-62中任一项所述的核酸分子，权利要求63-144中任一项所述的表达载体，权利要求145-152中任一项所述的细胞，和/或权利要求155所述的药物组合物，其用于预防、治疗和/或缓解疾病和/或病症。
  171. 根据权利要求170所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其中所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症，和/或与BCMA异常表达相关的疾病和/或病症。
  172. 根据权利要求170-171中任一项所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其中所述疾病和/或病症包括肿瘤。
  173. 根据权利要求172所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其中所述肿瘤包括实体瘤和/或非实体瘤。
  174. 根据权利要求172-173中任一项所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其中所述肿瘤包括血液瘤和/或淋巴瘤。
  175. 根据权利要求172-174中任一项所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其中所述肿瘤包括骨髓瘤。
  176. 根据权利要求175所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其中所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
PCT/CN2023/080741 2022-03-10 2023-03-10 靶向bcma和gprc5d的嵌合抗原受体及其应用 WO2023169555A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210238010 2022-03-10
CN202210238010.3 2022-03-10

Publications (2)

Publication Number Publication Date
WO2023169555A1 WO2023169555A1 (zh) 2023-09-14
WO2023169555A9 true WO2023169555A9 (zh) 2023-10-12

Family

ID=87936153

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/080741 WO2023169555A1 (zh) 2022-03-10 2023-03-10 靶向bcma和gprc5d的嵌合抗原受体及其应用

Country Status (2)

Country Link
TW (1) TW202342550A (zh)
WO (1) WO2023169555A1 (zh)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113784733B (zh) * 2019-05-07 2024-09-06 亘喜生物科技（上海）有限公司 靶向bcma的工程化免疫细胞及其用途
CN112778417B (zh) * 2019-11-07 2024-04-12 原启生物科技(上海)有限责任公司 一种分离的结合抗原bcma的蛋白及其用途

Also Published As

Publication number Publication date
TW202342550A (zh) 2023-11-01
WO2023169555A1 (zh) 2023-09-14

Similar Documents

Publication Publication Date Title
JP7280828B2 (ja) Ｂｃｍａを標的とする抗体およびその使用
EP3875484A1 (en) Cll1-targeting antibody and application thereof
JP2019530431A (ja) キメラ抗原受容体
WO2020020359A1 (en) Nef-containing t cells and methods of producing thereof
WO2021057866A1 (zh) 一种单域抗体及包含抗体结构的嵌合抗原受体
WO2023030272A1 (zh) 抗gprc5d抗原结合蛋白及其用途
WO2023093811A1 (zh) 分子开关调控型嵌合抗原受体细胞和抗体的组合及其应用
WO2018103734A1 (zh) 嵌合抗原受体及其用途和制备方法
EP4257617A1 (en) Antigen-binding protein targeting cd70 and use thereof
KR20240016246A (ko) 융합 단백질 및 이의 용도
KR20220150918A (ko) γδ T 세포 및 이의 용도
WO2023131285A1 (zh) 靶向cldn18.2和msln的嵌合抗原受体及其用途
WO2023202280A1 (zh) 抗B7H6的scFv抗体、其编码基因及其应用
TWI713843B (zh) 標靶bcma的抗體及其應用
WO2023024084A1 (zh) 一种嵌合抗原受体及其用途
WO2023169555A9 (zh) 靶向bcma和gprc5d的嵌合抗原受体及其应用
WO2023134718A1 (zh) 靶向gprc5d的嵌合抗原受体及其用途
US20230242666A1 (en) Methods and Compositions for the Reduction of Chimeric Antigen Receptor Tonic Signaling
WO2023016554A1 (zh) 靶向cd22的抗原结合蛋白及其用途
WO2024082178A1 (zh) 靶向cd19和cd22的双特异性嵌合抗原受体
WO2022228429A1 (zh) 靶向bcma的嵌合抗原受体及其应用
WO2023217062A1 (zh) 一种嵌合抗原受体及其应用
WO2023131276A1 (zh) 靶向msln的抗原结合蛋白及其应用
CN118652340A (zh) 人源化抗体b09及其在制备car-t细胞中的应用
JP2024528038A (ja) 調節性ｔ細胞を標的として自己免疫疾患を処置するための滑膜細胞外マトリックス特異的キメラ抗原受容体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23766132

Country of ref document: EP

Kind code of ref document: A1