WO2023131285A1 - 靶向cldn18.2和msln的嵌合抗原受体及其用途 - Google Patents

靶向cldn18.2和msln的嵌合抗原受体及其用途 Download PDF

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WO2023131285A1
WO2023131285A1 PCT/CN2023/070968 CN2023070968W WO2023131285A1 WO 2023131285 A1 WO2023131285 A1 WO 2023131285A1 CN 2023070968 W CN2023070968 W CN 2023070968W WO 2023131285 A1 WO2023131285 A1 WO 2023131285A1
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acid sequence
seq
nucleic acid
amino acid
sequence shown
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PCT/CN2023/070968
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French (fr)
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杨沙沙
吴凯
王华菁
杨焕凤
何晓文
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原启生物科技(上海)有限责任公司
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    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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, in particular to a chimeric antigen receptor targeting CLDN18.2 and MSLN and its application.
  • MSLN Mesothelin
  • MSLN is a glycoprotein located on the cell surface, anchored to the cell membrane by glycosylphosphatidylinositol.
  • the mesothelin gene encodes a 69kDa precursor protein, which is hydrolyzed into two chains by furin (paired basic amino acid protease, furin)-like invertase, and the membrane-bound protein of about 40KD at the C-terminal is the mature mesothelial protein
  • furin paired basic amino acid protease, furin
  • MPF megakaryocyte-promoting factor
  • MPF and membrane anchor MSLN are N-glycosylated, MPF can promote the formation of megakaryocyte clones in vitro, membrane anchor MSLN can interact with MUC16, and plays an important role in the process of cell adhesion, so currently targeted therapy is Membrane-anchored MSLN is selected as the target, so currently MSLN specifically refers to the C-terminal 40KD fragment of MSLN, that is, membrane-anchored MSLN.
  • CLDN18.2 (Claudin 18.2) is only expressed in differentiated gastric parietal cells, but not in normal tissues. The latest research shows that CLDN18.2 is overexpressed in more than 77% of gastric cancer patients and more than 80% of pancreatic cancer patients. In addition, it is overexpressed in solid tumors such as lung cancer, esophageal cancer, and ovarian cancer. CLDN18.2 belongs to a family of tight junction proteins that control the flow of molecules between layer cells. In tumors, however, the tight junctions are disrupted and the CLDN protein loses its main role. Because CLDN18.2 is abundantly present in gastric tumors, the researchers estimated that half of all patients with advanced gastric cancer could be candidates for new therapies targeting CLDN18.2 antibodies.
  • This application provides a new chimeric antigen receptor structure and cells containing the chimeric antigen receptor structure.
  • the chimeric antigen receptors and/or cells provided in this application can bind to antigens with higher affinity.
  • this application combines two or more targets to provide chimeric CARs that can express different targets. cells with antigen receptors. The cells described in this application not only significantly improve the safety and effectiveness, but also reduce the toxic and side effects of CAR-T cells.
  • the application provides a cell comprising and/or expressing a chimeric antigen receptor targeting tight junction protein 18.2 (CLDN18.2), and a chimeric antigen receptor targeting mesothelin (MSLN) protein body.
  • CLDN18.2 chimeric antigen receptor targeting tight junction protein 18.2
  • MSLN mesothelin
  • the CLDN18.2-targeting chimeric antigen receptor comprises an antigen binding domain.
  • the antigen-binding domain targeting CLDN18.2 comprises at least one CDR in the VH of the heavy chain variable region, and the VH comprises the amino acid sequence shown in SEQ ID NO:147.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 144.
  • the antigen binding domain targeting CLDN18.2 comprises HCDR3, and the HCDR3 comprises any one of SEQ ID NO:24, SEQ ID NO:59 and SEQ ID NO:73 amino acid sequence.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:145.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR2, and the HCDR2 comprises any one of SEQ ID NO:22, SEQ ID NO:57 and SEQ ID NO:71 amino acid sequence.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR1
  • the HCDR1 comprises SEQ ID NO: 146 (X 1 YX 2 X 3 X 4 , wherein X 1 is N or R , X2 is G, I or V, X3 is I or M, X4 is the amino acid sequence shown in H, N or S).
  • the antigen binding domain targeting CLDN18.2 comprises HCDR1
  • the HCDR1 comprises any one of SEQ ID NO:20, SEQ ID NO:55 and SEQ ID NO:69 amino acid sequence.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:57
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73.
  • the antigen-binding domain targeting CLDN18.2 comprises VH comprising the amino acid sequence shown in SEQ ID NO: 147.
  • the antigen binding domain targeting CLDN18.2 comprises a VH comprising SEQ ID NO:26, SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:75 and The amino acid sequence shown in any one of SEQ ID NO:79.
  • the antigen-binding domain targeting CLDN18.2 comprises at least one CDR in the VL of the light chain variable region, and the VL comprises the amino acid sequence shown in SEQ ID NO:151.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 148.
  • the antigen binding domain targeting CLDN18.2 comprises LCDR3, and said LCDR3 comprises SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:85 and SEQ ID NO:91 The amino acid sequence shown in any one.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30 or SEQ ID NO:83.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:150.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR1, and the LCDR1 comprises any one of SEQ ID NO:28, SEQ ID NO:45 and SEQ ID NO:81 of amino acids.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from any one of the following groups:
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:91;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:81
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:83
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:85.
  • the antigen-binding domain targeting CLDN18.2 comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:151.
  • the antigen binding domain targeting CLDN18.2 comprises a VL
  • the VL comprises SEQ ID NO:34, SEQ ID NO:49, SEQ ID NO:65, SEQ ID NO:87 and the amino acid sequence shown in any one of SEQ ID NO:93.
  • the antigen binding domain targeting CLDN18.2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising Any set of amino acid sequences:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:57
  • said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59
  • said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:91;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:81
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:83
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:85.
  • the antigen-binding domain targeting CLDN18.2 comprises VH and VL, and the VH and VL comprise amino acid sequences selected from any one of the following groups:
  • VH comprises the amino acid sequence shown in SEQ ID NO:26
  • VL comprises the amino acid sequence shown in SEQ ID NO:34
  • VH comprises the amino acid sequence shown in SEQ ID NO:43
  • VL comprises the amino acid sequence shown in SEQ ID NO:49
  • VH comprises the amino acid sequence shown in SEQ ID NO:75
  • VL comprises the amino acid sequence shown in SEQ ID NO:49
  • VH comprises the amino acid sequence shown in SEQ ID NO:61
  • VL comprises the amino acid sequence shown in SEQ ID NO:65;
  • VH comprises the amino acid sequence shown in SEQ ID NO:79
  • VL comprises the amino acid sequence shown in SEQ ID NO:93
  • VH comprises the amino acid sequence shown in SEQ ID NO:79
  • VL comprises the amino acid sequence shown in SEQ ID NO:87.
  • the antigen binding domain targeting CLDN18.2 comprises a scFv.
  • the scFv targeting CLDN18.2 comprises SEQ ID NO:38, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:89 and SEQ ID The amino acid sequence shown in any one of NO:95.
  • the CLDN18.2-targeting chimeric antigen receptor comprises a co-stimulatory domain comprising a co-stimulatory 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 for CD83, CD40 and MyD88.
  • a co-stimulatory domain comprising a co-stimulatory 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
  • the co-stimulatory domain is an intracellular co-stimulatory signaling region derived from 4-1BB.
  • the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:8.
  • the chimeric antigen receptor targeting CLDN18.2 comprises a transmembrane region comprising a transmembrane region derived from 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 is a transmembrane region derived from CD8.
  • the transmembrane region is a transmembrane region derived from human CD8.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:6.
  • the chimeric antigen receptor targeting CLDN18.2 comprises 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, 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, 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 is a hinge region derived from human CD8.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:4.
  • the CLDN18.2-targeting chimeric antigen receptor comprises a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:2.
  • the CLDN18.2-targeting chimeric antigen receptor does not comprise an intracellular signaling domain.
  • the MSLN-targeting chimeric antigen receptor comprises an antigen binding domain.
  • the antigen binding domain of the MSLN-targeted chimeric antigen receptor comprises at least one CDR in a heavy chain variable region VH comprising SEQ ID NO: 103, SEQ ID NO: 107.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises HCDR3, and the HCDR3 comprises SEQ ID NO: 101, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 110, SEQ ID NO: The amino acid sequence shown in any one of ID NO:119 and SEQ ID NO:154.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises HCDR2, and the HCDR2 comprises SEQ ID NO: 99, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 109, SEQ ID NO: The amino acid sequence shown in any one of ID NO:118 and SEQ ID NO:153.
  • the antigen binding domain of the MSLN-targeting chimeric antigen receptor comprises HCDR1, and the HCDR1 comprises SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:108 , the amino acid sequence shown in any one of SEQ ID NO:117 and SEQ ID NO:152.
  • the antigen-binding domain of the chimeric antigen receptor targeting MSLN comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:97
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:99
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:101;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:104
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:105
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:106;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:108
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:109
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:110;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:117
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:118
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:119;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:152
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:153
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:154.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises a VH
  • the VH comprises SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 111, SEQ ID NO: The amino acid sequence shown in any one of ID NO:120 and SEQ ID NO:155.
  • the antigen binding domain of the MSLN-targeting chimeric antigen receptor comprises a VHH.
  • VHH comprises the amino acid sequence shown in any one of SEQ ID NO: 103, SEQ ID NO: 107 and SEQ ID NO: 155.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123 or SEQ ID NO: 114.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:113 or SEQ ID NO:122.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:112 or SEQ ID NO:121.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR1, LCDR2 and LCDR3 comprising an amino acid sequence selected from any of the following groups:
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:112
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:113
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:114;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:121
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:122
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:123.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:115 or SEQ ID NO:124.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises a scFv, and the scFv comprises the amino acid sequence shown in SEQ ID NO: 116 or SEQ ID NO: 125.
  • the MSLN-targeting chimeric antigen receptor comprises an intracellular signaling domain comprising an intracellular protein derived from one or more proteins selected from the group consisting of Signaling domains: 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 herpes virus ( HSKV), DAP10, DAP-12 and domains containing at least one ITAM.
  • Signaling domains 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
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the MSLN-targeting chimeric antigen receptor comprises a transmembrane region comprising a transmembrane region derived from 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 is a transmembrane region derived from CD28.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:14.
  • the MSLN-targeting chimeric antigen receptor comprises 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, 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, 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 CD28.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO: 12.
  • the MSLN-targeting chimeric antigen receptor comprises a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:2.
  • the MSLN-targeting chimeric antigen receptor further comprises low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises 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-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the MSLN-targeting chimeric antigen receptor does not comprise a co-stimulatory domain.
  • the expression level of the chimeric antigen receptor targeting CLDN18.2 and the expression level of the chimeric antigen receptor targeting MSLN are about 1:1.
  • the expression level of the chimeric antigen receptor targeting CLDN18.2 and the expression level of the chimeric antigen receptor targeting MSLN are about 2:1.
  • the cells further comprise and/or express low-density lipoprotein receptor-associated protein or fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises 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-associated protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 18.
  • 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 dendritic cells
  • granulocytes lymphocytes
  • leukocytes granulocytes
  • peripheral Blood mononuclear cells embryonic stem cells
  • lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells are T cells.
  • the present application provides an expression vector, which includes a nucleic acid sequence encoding a chimeric antigen receptor targeting CLDN18.2 and a nucleic acid sequence encoding a chimeric antigen receptor targeting MSLN.
  • the present application provides an expression vector, which includes the nucleic acid sequence encoding the CLDN18.2-targeting chimeric antigen receptor described in the present application and the nucleic acid sequence encoding the MSLN-targeting chimeric antigen receptor described in the present application. nucleic acid sequence.
  • the nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 includes the nucleic acid sequence encoding the antigen binding domain.
  • the antigen-binding domain targeting CLDN18.2 comprises at least one CDR in VH
  • the nucleic acid sequence encoding said VH comprises SEQ ID NO:25, SEQ ID NO:42, SEQ ID NO :60, the nucleic acid sequence shown in any one of SEQ ID NO:74 and SEQ ID NO:78.
  • the antigen binding domain targeting CLDN18.2 comprises HCDR3, and the nucleic acid sequence encoding the HCDR3 comprises SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:58 and SEQ ID NO:58 and SEQ ID NO:58 The nucleic acid sequence shown in any one of ID NO:72.
  • the antigen binding domain targeting CLDN18.2 comprises HCDR2
  • the nucleic acid sequence encoding the HCDR2 comprises SEQ ID NO:21, SEQ ID NO:40, SEQ ID NO:56 and SEQ ID NO:56 and SEQ ID NO: The nucleic acid sequence shown in any one of ID NO:70.
  • the antigen binding domain targeting CLDN18.2 comprises HCDR1
  • the nucleic acid sequence encoding the HCDR1 comprises SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:54 and SEQ ID NO:54 and SEQ ID NO: The nucleic acid sequence shown in any one of ID NO:68.
  • the antigen binding domain targeting CLDN18.2 comprises VH
  • the nucleic acid sequence encoding the VH comprises SEQ ID NO:25, SEQ ID NO:42, SEQ ID NO:60, SEQ ID NO:60, SEQ ID NO: The nucleic acid sequence shown in any one of ID NO:74 and SEQ ID NO:78.
  • the antigen binding domain targeting CLDN18.2 comprises LCDR3, and the nucleic acid sequence encoding the LCDR3 comprises SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:62, SEQ ID NO:62, SEQ The nucleic acid sequence shown in any one of ID NO:84 and SEQ ID NO:90.
  • the antigen binding domain targeting CLDN18.2 comprises LCDR2, and the nucleic acid sequence encoding the LCDR2 comprises any of SEQ ID NO:29, SEQ ID NO:46 and SEQ ID NO:82 Nucleic acid sequence indicated in one item.
  • the antigen binding domain targeting CLDN18.2 comprises LCDR1, and the nucleic acid sequence encoding the LCDR1 comprises any of SEQ ID NO:27, SEQ ID NO:44 and SEQ ID NO:80 Nucleic acid sequence indicated in one item.
  • the antigen binding domain targeting CLDN18.2 comprises VL
  • the nucleic acid sequence encoding the VL comprises SEQ ID NO:33, SEQ ID NO:48, SEQ ID NO:64, SEQ ID NO:64, SEQ ID NO: The nucleic acid sequence shown in any one of ID NO:86 and SEQ ID NO:92.
  • the antigen binding domain targeting CLDN18.2 comprises a scFv.
  • the nucleic acid sequence encoding the scFv comprises SEQ ID NO:37, SEQ ID NO:52, SEQ ID NO:66, SEQ ID NO:76, SEQ ID NO:88 and SEQ ID NO:94 The nucleic acid sequence shown in any one.
  • the nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 comprises a nucleic acid sequence encoding a co-stimulatory domain
  • the co-stimulatory domain comprises a nucleic acid sequence derived from one or more of the following group Costimulatory domains of various proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, Ligands for GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40, and MyD88.
  • the co-stimulatory domain is an intracellular co-stimulatory signaling region derived from 4-1BB.
  • the nucleic acid sequence encoding the co-stimulatory domain comprises the nucleic acid sequence shown in SEQ ID NO:7.
  • the nucleic acid sequence encoding the CLDN18.2-targeting chimeric antigen receptor comprises a nucleic acid sequence encoding a transmembrane region comprising one or more genes selected from the group consisting of Transmembrane regions of various proteins: 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 is a transmembrane region derived from CD8.
  • the nucleic acid sequence encoding the transmembrane region comprises the nucleic acid sequence shown in SEQ ID NO:5.
  • the nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 comprises a nucleic acid sequence encoding a hinge region comprising one or more Hinge region of protein: 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.
  • a hinge region comprising one or more Hinge region of protein: 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 is a hinge region derived from CD8.
  • the nucleic acid sequence encoding the hinge region comprises the nucleic acid sequence shown in SEQ ID NO:5.
  • the nucleic acid sequence encoding the CLDN18.2-targeting chimeric antigen receptor comprises a nucleic acid sequence encoding a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the nucleic acid sequence encoding the signal peptide comprises the nucleic acid sequence shown in SEQ ID NO:1.
  • the nucleic acid sequence encoding a chimeric antigen receptor targeting CLDN18.2 does not comprise a nucleic acid sequence encoding an intracellular signaling domain.
  • the nucleic acid sequence encoding the MSLN-targeting chimeric antigen receptor comprises a nucleic acid sequence encoding an antigen binding domain.
  • the MSLN-targeted antigen binding domain comprises at least one CDR in a heavy chain variable region VH comprising SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111.
  • the nucleic acid sequence encoding the VH comprises the nucleic acid sequence shown in SEQ ID NO: 102 or SEQ ID NO: 156.
  • the MSLN-targeted antigen binding domain comprises HCDR3 comprising SEQ ID NO: 101, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 119 and SEQ ID The amino acid sequence shown in any one of NO:154.
  • the nucleic acid sequence encoding the HCDR3 comprises the nucleic acid sequence shown in SEQ ID NO:100.
  • the MSLN-targeted antigen binding domain comprises HCDR2, and the HCDR2 comprises SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:118, and SEQ ID NO:118 The amino acid sequence shown in any one of ID NO:153.
  • the nucleic acid sequence encoding the HCDR2 comprises the nucleic acid sequence shown in SEQ ID NO:98.
  • the MSLN-targeted antigen binding domain comprises HCDR1 comprising SEQ ID NO: 97, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 117, and SEQ ID The amino acid sequence shown in any one of NO:152.
  • the nucleic acid sequence encoding the HCDR1 comprises the nucleic acid sequence shown in SEQ ID NO:96.
  • the MSLN-targeted antigen binding domain comprises a VH comprising SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 120, and SEQ ID The amino acid sequence shown in any one of NO:155.
  • the nucleic acid sequence encoding the VH comprises the nucleic acid sequence shown in SEQ ID NO: 102 or SEQ ID NO: 156.
  • the MSLN-targeted antigen-binding domain comprises a VHH comprising the amino acid sequences shown in SEQ ID NO: 103, SEQ ID NO: 107 and SEQ ID NO: 155.
  • the nucleic acid sequence encoding the VHH comprises the nucleic acid sequence shown in SEQ ID NO: 102 or SEQ ID NO: 156.
  • the MSLN-targeted antigen-binding domain comprises LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 114 and SEQ ID NO: 123.
  • the nucleic acid sequence encoding the LCDR3 comprises the nucleic acid sequence shown in SEQ ID NO: 123 or SEQ ID NO: 114.
  • the antigen-binding domain targeting MSLN comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 113 or SEQ ID NO: 122.
  • the MSLN-targeted antigen-binding domain comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 112 or SEQ ID NO: 121.
  • the MSLN-targeted antigen-binding domain comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 115 or SEQ ID NO: 124.
  • the antigen-binding domain targeting MSLN comprises a scFv
  • the scFv comprises the amino acid sequence shown in SEQ ID NO: 116 or SEQ ID NO: 125.
  • the nucleic acid sequence encoding the MSLN-targeting chimeric antigen receptor comprises a nucleic acid sequence encoding an intracellular signaling domain comprising a protein derived from a group selected from the group consisting of Intracellular signaling domains of one or more 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's sarcoma herpesvirus (HSKV), DAP10, DAP-12 and a domain comprising at least one ITAM.
  • Intracellular signaling domains of one or more proteins CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine le
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the nucleic acid sequence encoding the intracellular signaling domain comprises the nucleic acid sequence shown in SEQ ID NO:15.
  • the nucleic acid sequence encoding the MSLN-targeted chimeric antigen receptor comprises a nucleic acid sequence encoding a transmembrane region comprising one or more Transmembrane regions of proteins: 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 is a transmembrane region derived from CD28.
  • the nucleic acid sequence encoding the transmembrane region comprises the nucleic acid sequence shown in SEQ ID NO:13.
  • the nucleic acid sequence encoding the MSLN-targeted chimeric antigen receptor comprises a nucleic acid sequence encoding a hinge region comprising a protein derived from one or more proteins selected from the group consisting of Hinge region: 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.
  • Hinge region 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 is a hinge region derived from CD28.
  • the nucleic acid sequence encoding the hinge region comprises the nucleic acid sequence shown in SEQ ID NO:11.
  • the nucleic acid sequence encoding the CLDN18.2-targeting chimeric antigen receptor comprises a nucleic acid sequence encoding a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the nucleic acid sequence encoding the signal peptide comprises the nucleic acid sequence shown in SEQ ID NO:1.
  • the nucleic acid sequence encoding the MSLN-targeting chimeric antigen receptor does not comprise a nucleic acid sequence encoding the co-stimulatory domain.
  • nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 and the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN are linked by a sequence encoding a cleavage peptide.
  • the cleavage peptide is selected from the group consisting of P2A, T2A, F2A and E2A.
  • the cleaved peptide comprises the amino acid sequence shown in SEQ ID NO:10.
  • the nucleic acid sequence encoding the cleaved peptide comprises the nucleic acid sequence shown in SEQ ID NO:9.
  • the expression vector comprises at least one nucleic acid sequence encoding a chimeric antigen receptor targeting CLDN18.2.
  • the expression vector comprises two nucleic acid sequences encoding the chimeric antigen receptor targeting CLDN18.2.
  • the two nucleic acid sequences encoding the CLDN18.2-targeting chimeric antigen receptor are linked by a nucleic acid sequence encoding a cleavage peptide.
  • the cleavage peptide is selected from the group consisting of P2A, T2A, F2A and E2A.
  • nucleic acid sequence encoding the cleaved peptide comprises the nucleic acid sequence shown in SEQ ID NO:9.
  • the expression vector further comprises a nucleic acid sequence encoding low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises 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 encoding or a fragment thereof comprises the nucleic acid sequence shown in SEQ ID NO:17.
  • the nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof is directly linked to the nucleic acid sequence encoding the intracellular signaling domain of the chimeric antigen receptor targeting MSLN.
  • nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof and the nucleic acid sequence encoding the MSLN-targeting chimeric antigen receptor are linked by a cleavage peptide.
  • nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 and the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN are located in the same nucleic acid molecule.
  • nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN, and the low-density lipoprotein receptor-related
  • the nucleic acid sequences of proteins or fragments thereof are in the same nucleic acid molecule.
  • the nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 in the first item, and the chimeric antigen receptor encoding targeting CLDN18.2 in the second item are located in the same nucleic acid molecule.
  • the expression vector comprises sequentially from the 5' end to the 3' end: a nucleic acid sequence encoding a chimeric antigen receptor targeting CLDN18.2, a nucleic acid sequence encoding a cleaved peptide, a nucleic acid sequence encoding a targeting The nucleic acid sequence of the chimeric antigen receptor of MSLN, the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the expression vector comprises sequentially from the 5' end to the 3' end: a nucleic acid sequence encoding a chimeric antigen receptor targeting CLDN18.2, a nucleic acid sequence encoding a cleaved peptide, a nucleic acid sequence encoding a targeting The nucleic acid sequence of the chimeric antigen receptor of CLDN18.2, the nucleic acid sequence encoding the cleavage peptide, the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN, the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or its fragment nucleic acid sequence.
  • the expression vector comprises sequentially from the 5' end to the 3' end: a nucleic acid sequence encoding a signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a hinge region, a nucleic acid sequence encoding a transmembrane region Nucleic acid sequence encoding co-stimulatory domain, nucleic acid sequence encoding splice peptide, nucleic acid sequence encoding signal peptide, nucleic acid sequence encoding MSLN VHH, nucleic acid sequence encoding hinge region, nucleic acid sequence encoding transmembrane region, encoding The nucleic acid sequence of the intracellular signaling domain and the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the expression vector comprises sequentially from the 5' end to the 3' end: a nucleic acid sequence encoding a CD8 signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a CD8 hinge region, a nucleic acid sequence encoding a CD8
  • the expression vector comprises sequentially from the 5' end to the 3' end: a nucleic acid sequence encoding a signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a hinge region, a nucleic acid sequence encoding a transmembrane region Nucleic acid sequence encoding co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding signal peptide, nucleic acid sequence encoding CLDN18.2 scFv, nucleic acid sequence encoding hinge region, nucleic acid sequence encoding transmembrane region , nucleic acid sequence encoding co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding signal peptide, nucleic acid sequence encoding MSLN VHH, nucleic acid sequence encoding hinge region, nucleic acid sequence
  • the expression vector comprises sequentially from the 5' end to the 3' end: a nucleic acid sequence encoding a CD8 signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a CD8 hinge region, a nucleic acid sequence encoding a CD8 Nucleic acid sequence of transmembrane region, nucleic acid sequence encoding 4-1BB co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding CD8 signal peptide, nucleic acid sequence encoding CLDN18.2 scFv, nucleic acid sequence encoding CD8 hinge region , nucleic acid sequence encoding CD8 transmembrane region, nucleic acid sequence encoding 4-1BB co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding CD8 signal peptide, nucleic
  • the present application also provides cells comprising the expression vector described in the present application.
  • the cells are 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 dendritic cells
  • granulocytes lymphocytes
  • leukocytes granulocytes
  • 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 the use of the cells and/or the expression vectors in the preparation of medicines for preventing and/or treating diseases and/or conditions.
  • the present application also provides the use of the chimeric antigen receptor targeting CLDN18.2 and the chimeric antigen receptor targeting MSLN in the preparation of medicines, and the medicines are used for preventing and/or Treat diseases and/or conditions.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises solid tumors and/or non-solid tumors.
  • the tumor includes a tumor that expresses both CLDN18.2 and MSLN antigens.
  • the tumor comprises gastric cancer, pancreatic cancer, and/or gastroesophageal junction cancer.
  • the present application also provides a method for preventing and/or treating diseases and/or conditions, which comprises administering the cells described in the present application to a subject in need.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises solid tumors and/or non-solid tumors.
  • the tumor includes a tumor that expresses both CLDN18.2 and MSLN antigens.
  • the tumor comprises gastric cancer, pancreatic cancer, and/or gastroesophageal junction cancer.
  • Figure 1 shows the antigen-binding activity of CLDN18.2 scFv.
  • Figure 2 shows the identification of the binding activity of CLDN18.2 single chain antibody to non-target cells.
  • FIGS 3A-3D show the stable expression of CLDN18.2-specific CAR in T cells.
  • FIG. 4 shows the experimental results of CLDN18.2-specific CAR specifically killing target cells in vitro.
  • FIGS 5A-5B show the factor secretion of CLDN18.2-specific CAR.
  • Figures 6A-6D show the tumor-suppressive effect of CLDN18.2-specific CAR-T cells in a mouse colon cancer CDX model.
  • Figure 7A-7B shows a schematic diagram of the construction of the dual CAR described in this application.
  • FIGS 8A-8B show the expression and proliferation of the CAR-T cells described in this application.
  • Figure 9 shows the in vitro killing activity of CAR-T cells.
  • Figures 10A-10B show the secretion of cytokines by the CAR-T cells described in this application.
  • FIGS 11A-11C show the tumor-suppressive effect of the CAR-T cells described in this application in the CDX model of human gastric cancer.
  • Figures 12A-12E show the toxicity experiments of the CAR-T cells described in this application in mice.
  • Figure 13A shows a schematic diagram of the construction of the CAR described in this application
  • Figures 13B-13C show the expression of CAR described in this application in cells and the proliferation of CAR-T cells.
  • Figure 14 shows the proliferation of CAR-T cells described in this application under repeated stimulation of target cells.
  • Figure 15 shows the in vitro cell killing of the CAR-T cells described in this application.
  • Figure 16 shows the detection of in vitro cytokine secretion of the CAR-T cells described in this application.
  • Figures 17A-17D show the tumor-suppressive effect of the CAR-T cells described in this application in the mouse CDX model of human gastric cancer.
  • Figure 18 shows the detection of the flow cytometry binding activity between the targeting part of CLDN18.2 described in the present application and the cells with high expression of human CLDN18.2.
  • Figure 19 shows the detection of the flow cytometry binding activity between the targeting part of CLDN18.2 described in the present application and high-expressing human CLDN18.1 cells.
  • Figure 20 shows the detection of the flow cytometry binding activity of the targeting moiety of CLDN18.2 described in the present application to tumor cell lines.
  • CLDN18.2 or “Claudin18.2” are used interchangeably and generally refer to subtype 2 of the cell junction claudin Claudin18.
  • the term encompasses "full length", unprocessed CLDN18.2 as well as any form of CLDN18.2 produced by cellular processing.
  • CLDN18.2 may include intact CLDN18.2 and fragments thereof, functional variants, isoforms, species homologues, derivatives, analogs and analogs having at least one common epitope with CLDN18.2.
  • the amino acid sequence of CLDN18.2 (eg, human CLDN18.2) may be known in the art.
  • the human CLDN18.2 nucleotide sequence can be shown under GeneBank accession number NM_001002026.3.
  • the mouse CLDN18.2 nucleotide sequence can be shown under GeneBank Accession No. NM_001194921.1.
  • the cynomolgus monkey CLDN18.2 nucleotide sequence can be shown under GeneBank accession number XM_001114708.4.
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of the heavy and light chains may be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and comprise the antigen binding site.
  • variable generally means that some segments of the variable domain may have large differences in sequence between antibodies.
  • the variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed throughout the variable domains. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved portions of variable domains are called the framework regions (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of the ⁇ -sheet structure .
  • the CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • CDR also referred to as “complementarity determining region” generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structure-defining loop.
  • antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • naturally occurring camelid antibodies consisting only of heavy chains are capable of functioning and stabilizing in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, CLDN18.2).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region ( CH 1 ) of the H chain ( VH ).
  • Fab' fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment.
  • a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • F(ab')2 generally refers to antibody fragments produced by pepsin digestion of intact antibodies.
  • the F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds.
  • F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.
  • Fv fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions.
  • the heavy chain variable region and the light chain variable region include, for example, the CDRs.
  • an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains are variable
  • the regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv can have the VL and VH variable regions described in any order (eg, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include a VL-linker-VH Or VH-linker-VL can be included.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see e.g. Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and to other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
  • the term “directly connected” is opposite to the term “indirectly connected”, and the term “directly connected” generally refers to a direct connection.
  • the direct connection may be a case where substances are directly connected without a spacer.
  • the spacer may be a linker.
  • the linker can be a peptide linker.
  • the term “indirectly linked” generally refers to the situation where substances are not directly linked.
  • the indirect connection may be through a spacer.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 Phages and animal viruses, etc.
  • Animal virus species used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, and papillomaviruses.
  • vascular viruses such as SV40.
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes or protein coats, but not only.
  • the term "cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule as described herein or a nucleic acid molecule as described herein. Carrier.
  • Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • Cells may include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (e.g., E.
  • yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells , NSO cells or stem cells (eg ES cells, iPS cells, mesenchymal stem cells) or immune cells (eg T cells, NK cells, NKT cells, macrophages).
  • the term "pharmaceutical composition” generally refers to a composition for preventing/treating a disease or condition.
  • the pharmaceutical composition may comprise the chimeric antigen receptor described in the application, the nucleic acid molecule described in the application, the carrier described in the application and/or the cell described in the application, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the term "pharmaceutically acceptable carrier” generally includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is effective in the dosage and concentration employed to the cell or mammal to which it is exposed. non-toxic.
  • Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
  • the term "specifically binds" or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. Presence determines the presence of a target.
  • an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets .
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • tumor generally refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • tumors may be solid tumors or hematological tumors.
  • the tumor may be a CLDN18.2 positive tumor.
  • cancer generally refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Cancer in this application includes but not limited to gastric cancer and/or colon cancer and the like.
  • tumor and cancer are used interchangeably herein, e.g., both terms encompass solid tumors and liquid tumors, e.g., diffuse or circulating tumors.
  • cancer or “tumor” can include premalignant as well as malignant cancers and tumors.
  • protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, substituted, deleted or added to the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to the CLDN18.2 protein). or a protein or polypeptide of multiple amino acids.
  • the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion.
  • Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration.
  • the substitutions may be conservative substitutions.
  • the homologue may be at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence Homologous proteins or polypeptides.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • Perfectage of sequence homology can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity.
  • Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA FASTA and BLAST.
  • a description of the FASTA algorithm can be found in "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J.
  • the term "about” generally refers to a range of 0.5%-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%.
  • the application provides a cell comprising and/or expressing a chimeric antigen receptor targeting tight junction protein 18.2 (CLDN18.2), and a chimeric antigen receptor targeting mesothelin (MSLN) protein .
  • CLDN18.2 chimeric antigen receptor targeting tight junction protein 18.2
  • MSLN mesothelin
  • the present application provides an expression vector, which includes the nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 and the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN.
  • the present application also provides a cell comprising the expression vector described in the present application.
  • the chimeric antigen receptor targeting CLDN18.2 comprises an antigen-binding domain targeting CLDN18.2.
  • the antigen-binding domain targeting CLDN18.2 comprises at least one CDR in the VH of the heavy chain variable region, and the VH comprises the amino acid sequence shown in SEQ ID NO:147.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 144.
  • the antigen binding domain targeting CLDN18.2 comprises HCDR3, and the HCDR3 comprises any one of SEQ ID NO:24, SEQ ID NO:59 and SEQ ID NO:73 amino acid sequence.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:145.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR2, and the HCDR2 comprises any one of SEQ ID NO:22, SEQ ID NO:57 and SEQ ID NO:71 amino acid sequence.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR1
  • the HCDR1 comprises SEQ ID NO: 146 (X 1 YX 2 X 3 X 4 , wherein X 1 is N or R , X2 is G, I or V, X3 is I or M, X4 is the amino acid sequence shown in H, N or S).
  • the antigen binding domain targeting CLDN18.2 comprises HCDR1
  • the HCDR1 comprises any one of SEQ ID NO:20, SEQ ID NO:55 and SEQ ID NO:69 amino acid sequence.
  • the antigen-binding domain targeting CLDN18.2 comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:57
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73.
  • the antigen-binding domain targeting CLDN18.2 comprises VH comprising the amino acid sequence shown in SEQ ID NO: 147.
  • the antigen binding domain targeting CLDN18.2 comprises a VH comprising SEQ ID NO:26, SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:75 and The amino acid sequence shown in any one of SEQ ID NO:79.
  • the antigen-binding domain targeting CLDN18.2 comprises at least one CDR in the VL of the light chain variable region, and the VL comprises the amino acid sequence shown in SEQ ID NO:151.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 148.
  • the antigen binding domain targeting CLDN18.2 comprises LCDR3, and said LCDR3 comprises SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:85 and SEQ ID NO:91 The amino acid sequence shown in any one.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:30 and SEQ ID NO:83.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:150.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR1, and the LCDR1 comprises any one of SEQ ID NO:28, SEQ ID NO:45 and SEQ ID NO:81 of amino acids.
  • the antigen-binding domain targeting CLDN18.2 comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from any one of the following groups:
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:91;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:81
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:83
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:85.
  • the antigen-binding domain targeting CLDN18.2 comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:151.
  • the antigen binding domain targeting CLDN18.2 comprises a VL
  • the VL comprises SEQ ID NO:34, SEQ ID NO:49, SEQ ID NO:65, SEQ ID NO:87 and the amino acid sequence shown in any one of SEQ ID NO:93.
  • the antigen binding domain targeting CLDN18.2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising Any set of amino acid sequences:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:57
  • said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59
  • said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:91;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:73
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:81
  • said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:83
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:85.
  • the antigen-binding domain targeting CLDN18.2 comprises VH and VL, and the VH and VL comprise amino acid sequences selected from any one of the following groups:
  • VH comprises the amino acid sequence shown in SEQ ID NO:26
  • VL comprises the amino acid sequence shown in SEQ ID NO:34
  • VH comprises the amino acid sequence shown in SEQ ID NO:43
  • VL comprises the amino acid sequence shown in SEQ ID NO:49
  • VH comprises the amino acid sequence shown in SEQ ID NO:75
  • VL comprises the amino acid sequence shown in SEQ ID NO:49
  • VH comprises the amino acid sequence shown in SEQ ID NO:61
  • VL comprises the amino acid sequence shown in SEQ ID NO:65;
  • VH comprises the amino acid sequence shown in SEQ ID NO:79
  • VL comprises the amino acid sequence shown in SEQ ID NO:93
  • VH comprises the amino acid sequence shown in SEQ ID NO:79
  • VL comprises the amino acid sequence shown in SEQ ID NO:87.
  • the antigen binding domain targeting CLDN18.2 comprises a scFv.
  • the scFv targeting CLDN18.2 comprises SEQ ID NO:38, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:89 and SEQ ID The amino acid sequence shown in any one of NO:95.
  • the CLDN18.2-targeting chimeric antigen receptor comprises a co-stimulatory domain comprising a co-stimulatory 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 for CD83, CD40 and MyD88.
  • a co-stimulatory domain comprising a co-stimulatory 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
  • the co-stimulatory domain is an intracellular co-stimulatory signaling region derived from 4-1BB.
  • the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:8.
  • the chimeric antigen receptor targeting CLDN18.2 comprises a transmembrane region comprising a transmembrane region derived from 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 is a transmembrane region derived from CD8.
  • the transmembrane region is a transmembrane region derived from human CD8.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:6.
  • the chimeric antigen receptor targeting CLDN18.2 comprises 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, 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, 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 is a hinge region derived from human CD8.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:4.
  • the CLDN18.2-targeting chimeric antigen receptor comprises a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:2.
  • the CLDN18.2-targeting chimeric antigen receptor does not comprise an intracellular signaling domain.
  • nucleic acid molecules encoding each part of the chimeric antigen receptor targeting CLDN18.2 are also provided.
  • the chimeric antigen receptor targeting MSLN comprises an antigen-binding domain targeting MSLN.
  • the antigen binding domain of the MSLN-targeted chimeric antigen receptor comprises at least one CDR in a heavy chain variable region VH comprising SEQ ID NO: 103, SEQ ID NO: 107.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises HCDR3, and the HCDR3 comprises SEQ ID NO: 101, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 110, SEQ ID NO: The amino acid sequence shown in any one of ID NO:119 and SEQ ID NO:154.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises HCDR2, and the HCDR2 comprises SEQ ID NO: 99, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 109, SEQ ID NO: The amino acid sequence shown in any one of ID NO:118 and SEQ ID NO:153.
  • the antigen binding domain of the MSLN-targeting chimeric antigen receptor comprises HCDR1, and the HCDR1 comprises SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:108 , the amino acid sequence shown in any one of SEQ ID NO:117 and SEQ ID NO:152.
  • the antigen-binding domain of the chimeric antigen receptor targeting MSLN comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:97
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:99
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:101;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:104
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:105
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:106;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:108
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:109
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:110;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:117
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:118
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:119;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:152
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:153
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:154.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises a VH
  • the VH comprises SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 111, SEQ ID NO: The amino acid sequence shown in any one of ID NO:120 and SEQ ID NO:155.
  • the antigen binding domain of the MSLN-targeting chimeric antigen receptor comprises a VHH.
  • the VHH comprises the amino acid sequence shown in any one of SEQ ID NO: 103, SEQ ID NO: 107 and SEQ ID NO: 155.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123 or SEQ ID NO: 114.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:113 or SEQ ID NO:122.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:112 or SEQ ID NO:121.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises LCDR1, LCDR2 and LCDR3 comprising an amino acid sequence selected from any of the following groups:
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:112
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:113
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:114;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:121
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:122
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:123.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:115 or SEQ ID NO:124.
  • the antigen binding domain of the chimeric antigen receptor targeting MSLN comprises a scFv, and the scFv comprises the amino acid sequence shown in SEQ ID NO: 116 or SEQ ID NO: 125.
  • the MSLN-targeting chimeric antigen receptor comprises an intracellular signaling domain comprising an intracellular protein derived from one or more proteins selected from the group consisting of Signaling domains: 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 herpes virus ( HSKV), DAP10, DAP-12 and domains containing at least one ITAM.
  • Signaling domains 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
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the MSLN-targeting chimeric antigen receptor comprises a transmembrane region comprising a transmembrane region derived from 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 is a transmembrane region derived from CD28.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:14.
  • the MSLN-targeting chimeric antigen receptor comprises 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, 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, 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 CD28.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO: 12.
  • the MSLN-targeting chimeric antigen receptor comprises a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:2.
  • the MSLN-targeting chimeric antigen receptor further comprises low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises 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-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the MSLN-targeting chimeric antigen receptor does not comprise a co-stimulatory domain.
  • nucleic acid molecules encoding each part of the MSLN-targeting chimeric antigen receptor are also provided.
  • the expression level of the chimeric antigen receptor targeting CLDN18.2 and the expression level of the chimeric antigen receptor targeting MSLN contained and/or expressed in the cells may be about 1:1.
  • the expression level of the chimeric antigen receptor targeting CLDN18.2 and the expression level of the chimeric antigen receptor targeting MSLN contained and/or expressed in the cells may be about 2:1
  • the cells may also contain 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 following group: low-density lipoprotein receptor-associated protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-associated protein or a fragment thereof may be low-density lipoprotein receptor-associated protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragments thereof may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the cells may comprise 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.
  • NK cells natural killer cells
  • 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 can be T cells.
  • the expression vector may further comprise a nucleic acid sequence encoding the 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 following group: low-density lipoprotein receptor-associated protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-associated protein or a fragment thereof may be low-density lipoprotein receptor-associated protein 5 and/or 6 or a fragment thereof.
  • encoding the low-density lipoprotein receptor-associated protein or a fragment thereof may comprise the nucleic acid sequence shown in SEQ ID NO: 18.
  • nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 and the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN may be linked by a cleavage peptide.
  • the cleavage peptide may be selected from the group consisting of P2A, T2A, F2A and E2A.
  • the cleaved peptide comprises the amino acid sequence shown in SEQ ID NO:10.
  • the nucleic acid sequence encoding the cleavage peptide may comprise the nucleic acid sequence shown in SEQ ID NO:9.
  • the expression vector may comprise at least one nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2.
  • the expression vector may contain two nucleic acid sequences encoding the chimeric antigen receptor targeting CLDN18.2.
  • nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 and the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN may be located in the same nucleic acid molecule.
  • nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN, and the low-density lipoprotein receptor-related protein or The nucleic acid sequences of its fragments are located in the same nucleic acid molecule.
  • the nucleic acid sequence encoding the chimeric antigen receptor targeting CLDN18.2 described in the first article, and the chimeric antigen receptor encoding targeting CLDN18.2 described in the second The nucleic acid sequence of the body, the nucleic acid sequence encoding the chimeric antigen receptor targeting MSLN, and the nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof may be located in the same nucleic acid molecule.
  • the expression vector may respectively comprise from the 5' end to the 3' end: a nucleic acid sequence encoding a chimeric antigen receptor targeting CLDN18.2, a nucleic acid sequence encoding a cleaved peptide, a nucleic acid sequence encoding a The nucleic acid sequence of the chimeric antigen receptor, the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the expression vector may respectively comprise from the 5' end to the 3' end: a nucleic acid sequence encoding a chimeric antigen receptor targeting CLDN18.2, a nucleic acid sequence encoding a cleaved peptide, a nucleic acid sequence encoding a .2
  • the expression vectors respectively comprise from the 5' end to the 3' end: a nucleic acid sequence encoding a signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a hinge region, and a nucleic acid sequence encoding a transmembrane region sequence, nucleic acid sequence encoding co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding signal peptide, nucleic acid sequence encoding MSLN VHH, nucleic acid sequence encoding hinge region, nucleic acid sequence encoding transmembrane region, encoding intracellular
  • the expression vectors respectively comprise from the 5' end to the 3' end: a nucleic acid sequence encoding a CD8 signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a CD8 hinge region, a nucleic acid sequence encoding a CD8 transmembrane Nucleic acid sequence encoding 4-1BB co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding CD8 signal peptide, nucleic acid sequence encoding MSLN VHH, nucleic acid sequence encoding CD28 hinge region, encoding CD28 span The nucleic acid sequence of the membrane region, the nucleic acid sequence encoding the CD3 ⁇ intracellular signaling domain, and the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or its fragments.
  • the expression vectors respectively comprise from the 5' end to the 3' end: a nucleic acid sequence encoding a signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a hinge region, and a nucleic acid sequence encoding a transmembrane region sequence, nucleic acid sequence encoding co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding signal peptide, nucleic acid sequence encoding CLDN18.2 scFv, nucleic acid sequence encoding hinge region, nucleic acid sequence encoding transmembrane region, encoding Nucleic acid sequences encoding co-stimulatory domains, nucleic acid sequences encoding cleavage peptides, nucleic acid sequences encoding signal peptides, nucleic acid sequences encoding MSLN VHH, nucleic acid
  • the expression vectors respectively comprise from the 5' end to the 3' end: a nucleic acid sequence encoding a CD8 signal peptide, a nucleic acid sequence encoding a CLDN18.2 scFv, a nucleic acid sequence encoding a CD8 hinge region, a nucleic acid sequence encoding a CD8 transmembrane Nucleic acid sequence encoding 4-1BB co-stimulatory domain, nucleic acid sequence encoding cleavage peptide, nucleic acid sequence encoding CD8 signal peptide, nucleic acid sequence encoding CLDN18.2 scFv, nucleic acid sequence encoding CD8 hinge region, encoding The nucleic acid sequence of the CD8 transmembrane region, the nucleic acid sequence encoding the 4-1BB co-stimulatory domain, the nucleic acid sequence encoding the cleavage peptide, the nucleic acid sequence encoding the CD8 signal peptide,
  • the present application provides the use of the cell and/or the expression vector in the preparation of a medicament for the prevention and/or treatment of diseases and/or disorders.
  • the present application also provides the use of the chimeric antigen receptor targeting CLDN18.2 and the chimeric antigen receptor targeting MSLN in the preparation of medicines, and the medicines are used for preventing and/or Treat diseases and/or conditions.
  • the present application provides a method for preventing and/or treating diseases and/or conditions, which comprises administering the cells described in the present application to a subject in need.
  • the present application provides the cell and/or the expression vector for preventing and/or treating diseases and/or disorders.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises solid tumors and/or non-solid tumors.
  • the tumor includes a tumor that expresses both CLDN18.2 and MSLN antigens.
  • the tumor comprises gastric cancer, pancreatic cancer, and/or gastroesophageal junction cancer.
  • cells stably expressing human CLDN18.2, that is, SP2/0-hCLDN18.2 cells were obtained by lentivirus infection and flow sorting. Perform cellular immunization on Balb/c mice, the immunogen is SP2/0-hCLDN18.2 cells, use mouse hybridoma technology, flow cytometry screening and subcloning to obtain CLDN18.2 positive monoclonal cells, and in vitro culture to obtain IgG class CLDN18.2 mouse monoclonal antibody.
  • the heavy chain variable region (V H ) and light chain variable region (V L ) sequences were sequenced by degenerate primer amplification method, and the antibody heavy chain variable region (V H ) and the light chain variable region (V L ) are connected into a recombinant gene, and the antibody expressed by the recombinant gene is a CLDN18.2 single-chain antibody, namely 5E6-scFv (SEQ ID NO: 38).
  • Simultaneous lentiviral infection and flow sorting obtained cell SP2/0-hCLDN18.1 stably expressing human CLDN18.1.
  • 5E6-scFv single-chain antibody and Standard antibody SEQ ID NO: 143
  • flow cytometry SP2/0-hCLDN18.2 cells were counted, cells were resuspended in flow buffer and adjusted To 1 ⁇ 10 6 /ml, 30ul/well was added to a V-bottom 96-well plate; 30ul/well was added to a primary antibody, starting at a working concentration of 80ug/ml, and diluted with flow buffer in a 4-fold ratio to form a 7 A gradient, each antibody was set as a PBS negative control; incubated at 4 degrees for 1 hour, washed with flow buffer, and added the secondary antibody goat F(ab')2 anti-human IgG-Fc( 650) (abcam, Cat#ab98593), 30ul/well, incubate at 4°C for 30 minutes; wash with flow buffer twice, shake the cells loose, add 25ul/well flow buffer, and wait for the machine.
  • SP2/0-hCLDN18.1 cells were counted and plated.
  • the working concentration of the primary antibody started from 10ug/ml, and was serially diluted 3 times to form a total of 5 concentration gradients.
  • the secondary antibody was goat F(ab')2 anti-human IgG. -Fc( 650) (abcam, Cat#ab98593), the original data were substituted into GraphPad8.0 software for graphing and calculation, the results are shown in Figure 1: 5E6-scFv single-chain antibody can specifically bind to human CLDN18.2, same as Standard antibody.
  • the binding activity of the 5E6-scFv single-chain antibody to various non-target cells in human tissues was identified using flow cytometric fluorescence sorting (FACS).
  • the cells were human foreskin fibroblasts (HFF), human immortalized epidermal cells (HaCaT), human normal liver cells (LO2), mesenchymal stem cells (MSCs), human normal epidermal cells (KERA), adenocarcinoma human alveolar basal epithelium cells (A549), human breast cancer cells (MCF-7), human skin fibroblasts (BJ).
  • HFF human foreskin fibroblasts
  • HaCaT human immortalized epidermal cells
  • LO2 human normal liver cells
  • MSCs mesenchymal stem cells
  • KERA human normal epidermal cells
  • A549 adenocarcinoma human alveolar basal epithelium cells
  • MCF-7 human breast cancer cells
  • BJ human skin fibroblasts
  • the secondary antibody was goat F(ab')2 anti-human IgG-Fc( 650) (abcam, Cat#ab98593), centrifuged after incubation, resuspended with flow buffer and loaded on the machine, and the results were substituted into GraphPad8.0 software for graphing. The results are shown in Figure 2.
  • the 5E6-scFv single-chain antibody has no non-specific binding to a variety of non-target cells in human tissues. Similar to the Standard antibody, its specificity to tumor targets was preliminarily verified, that is, there is no "off-target effect".
  • Example 3 CLDN18.2-specific CAR is stably expressed in T cells
  • the CLDN18.2-specific CAR structure in this example is composed of human CD8 signal peptide (SEQ ID NO: 2), anti-human CLDN18.2 single-chain antibody (SEQ ID NO: 38), human CD8 Hinge region (SEQ ID NO:4), human CD8 transmembrane region (SEQ ID NO:6), human 4-1BB intracellular co-stimulatory domain (SEQ ID NO:8), human CD3 ⁇ intracellular activation domain (SEQ ID NO : 16) and the added Ori new element (SEQ ID NO: 18).
  • the positive rate of CAR-T cells cultured for 9-14 days the virus used to infect the cells carries Myc-tag, so after the virus infected the cells, the positive rate of Myc was detected by flow cytometry to obtain the positive rate of CAR expression,
  • the direct detection antibody used was Myc-Tag (9B11) Mouse mAb (Alexa 488 Conjugate) (Cell Signaling, 2279S).
  • Example 4 CLDN18.2-specific CAR can specifically kill target cells in vitro
  • CHO-hCLDN18.2 cells stably and highly expressing human CLDN18.2 and CHO-hCLDN18.1 cells stably and highly expressing human CLDN18.1 were obtained by lentivirus infection and flow sorting.
  • a cytotoxicity detection kit Promega, Cat#G1780 was further used to evaluate the specific killing ability of CAR-T cells in vitro by using the LDH method. The steps are as follows:
  • the CAR-T and Mock T cells routinely cultured for 9 days in the above Example 3 were centrifuged and resuspended with blank X-VIVO medium to make the cell density 1 ⁇ 10 5 /ml;
  • the target cells were CHO, Three kinds of CHO-hCLDN18.2 and CHO-hCLDN18.1, three kinds of target cells were digested and counted respectively, and then resuspended with blank X-VIVO medium to make the cell density 5 ⁇ 10 5 /ml; then According to the volume system of 100ul target cell suspension + 100ul CAR-T/Mock T cell suspension per well, mix them into a sterile v-bottom 96-well plate.
  • the control wells were set according to the requirements of the kit.
  • the Human IFN- ⁇ ELISA kit (R&D, DY285B) and the Human IL-2 ELISA kit (R&D, DY202) were used to analyze the secretion of IFN- ⁇ and IL-2 by CAR-T in the process of killing target cells quantity.
  • the target cells with high expression of CLDN18.2 (CHO-hCLDN18.2)
  • the control cells with high expression of CLDN18.1 (CHO-hCLDN18.1)
  • the negative cells CHO were inoculated in sterile 96-well plates
  • Add modified T cells (Mock T cells) and other effector cells.
  • the human CLDN18.2 gene was first introduced into MC38 cells by means of lentivirus, and then the mouse colon cancer cells with high expression of human CLDN18.2 were sorted by flow cytometry, that is, MC38-hCLDN18.2 cells .
  • Female mice were heavily immunized with B-NDG, and a mouse colon cancer tumor model was constructed by subcutaneous injection to verify the tumor-suppressive effect of CLDN18.2-specific CAR-T cells in mice.
  • MC38-hCLDN18.2 cells were inoculated at a dose of 1.5 ⁇ 10 6 /mouse.
  • mice with tumor size 41.68-120.6 mm 3 (average tumor size 80.01 mm 3 ) were randomly divided into 3 groups, 8 mice in each group, the CAR-T (or Mock T) cells that had been cultured for 10 days were reinfused into the tail vein, and the CAR-T reinfusion dose in the 5E6-CART and Standard-CART groups was 3*10 6 cells/mouse , the reinfusion dose of the blank control Mock T cell group was 1*107 cells/mouse.
  • the tumor size and body weight of the mice were measured 3 times a week and observed for 4 weeks, as shown in Figure 6A.
  • mice in the 5E6-CART group lost more than 10% of their body weight on D13, and the relative weight change of a single mouse was -34.51% on D15, and the first The mice in the Standard-CART group lost more than 10% of their body weight on D16, and the relative change in body weight of a single mouse was -36.93% on D16.
  • the first mouse died on D18, due to the weight loss of the mice.
  • PG-D22 ended the experiment, and no obvious tumor inhibitory effect was observed at the end of the experiment, as shown in Figure 6C (the end point is defined as the mouse tumor volume>3000mm 3 ).
  • mice After the mice were euthanized, the mice were dissected, and it was found that the mice in the 5E6-CART group and the Standard-CART group had an abnormal phenomenon of gastric damage and bleeding, as shown in Figure 6D. To sum up, the mice in the 5E6-CART group showed intolerance and obvious side effects in this tumor model, specifically manifested as weight loss, back hair erection, curling up and gastric bleeding, etc.; Mock T group The mice showed tolerance, and the weight of the mice was normal.
  • 5E6-scFv is a single-chain antibody obtained through screening and identification of mouse hybridoma technology.
  • the 5E6-scFv antibody can specifically bind to human CLDN18.2 positive cells and has high specificity for CLDN18.2.
  • the 5E6-scFv single-chain antibody does not have non-specific binding to various non-target cells in human tissues.
  • 5E6-CART is a second-generation CART structure that can be stably expressed and proliferated in T cells.
  • 5E6-CART has CLDN18.2 high specific cell killing activity in vitro, and can specifically induce cytokine secretion.
  • 5E6-CART showed certain toxicity in the drug efficacy experiment of tumor-bearing mice, manifested as weight loss and gastric bleeding in mice, which to some extent explained the poor safety of 5E6-CART in mice. Therefore, in this example, through research, new tumor targets were discovered and combined, and a new CAR molecular structure was designed to improve its in vivo safety while retaining the effectiveness of 5E6, and named it OriCAR-388.
  • the tumor target MSLN is highly expressed in 50-55% of gastric cancer patients and 80-85% of pancreatic cancer patients, and can be used as a gastric cancer target for research.
  • the target MSLN is highly expressed in the trachea, fallopian tube, germinal disc, and tonsil, moderately expressed in the nasopharynx, endometrium, and lowly expressed in the oral mucosa, uterus, and skin.
  • Normal lung, stomach, liver, pancreas, Gallbladder does not express, and basically does not overlap with the expression distribution of CLDN18.2 in normal human tissues, so it is an ideal dual CAR-T target selection.
  • the four-plasmid system was used to package the lentiviral expression vector.
  • virus titer number of infected cells * positive rate / volume of infected virus (ml).
  • MOI virus multiplicity of infection
  • CAR-T and blank T cells The routine culture of CAR-T and blank T cells was carried out according to the method shown in Example 8, and the CAR-T cells (OriCAR-388 T cells and 5E6-CART cells) conventionally cultured in vitro for 12 days and the blank without transfection T cells (Mock T) were centrifuged and resuspended with blank X-VIXO medium (Lonza) after centrifugation to make the cell density 1.33*10 5 /ml.
  • CAR-T cells OriCAR-388 T cells and 5E6-CART cells
  • CHO-NFkB-hCLDN18.2 is a cell that highly expresses human CLDN18.2 and luciferase at the same time obtained by introducing the NFkB luciferase reporter gene through molecular construction on the basis of the above-mentioned Example 4, and then subcloning.
  • NUGC4 cells are human gastric cancer cells, which naturally express human CLDN18.2 and human MSLN under conventional culture conditions in vitro, and obtained NUGC4-NFkB cells by introducing NFkB luciferase reporter gene construction,
  • OVCAR3-NFkB is a human ovarian cancer cell that naturally highly expresses human MSLN protein under conventional culture conditions in vitro, and OVCAR3-NFkB cells were obtained by introducing NFkB luciferase reporter gene construction.
  • Target cells positive for MSLN single target were studied.
  • CHO-hCLDN18.2 was selected as human CLDN18.2 single-positive target cells
  • OVCAR-3 was human MSLN single-positive target cells
  • NUGC-4 was MSLN/CLDN18.2 double-positive target cells
  • the effector cells were cultured in vitro for 8 days.
  • Inoculate wells without target cells i.e., CAR-T cells not co-cultured with target cells
  • wells co-incubated with target cells at a ratio of 1:1 to target cells in a flat-bottomed 96-well plate and after co-incubation for 20 hours , aspirate the supernatant, and use the R&D Cytokine Kit to detect the contents of IL-2 and IFN- ⁇ in the supernatant (R&D, DY285B and R&D, DY202).
  • MSLN-CART secretes factors only when co-incubating with OVCAR-3 or NUGC-4 cells, and the secretion level is low; A large amount of IFN-r and IL-2 cytokines were secreted under co-incubation; while OriCAR-388 T cells basically did not secrete IFN-r and IL-2 cytokines when co-incubated with CHO-hCLDN18.2, and CAR-T cells There is no difference in the secretion level of factors in single incubation wells; OriCAR-388T cells can secrete a small amount of IFN-r and IL-2 cytokines under co-incubation with OVCAR-3, and the secretion level of IL-2 is significantly higher than that in single incubation wells of CAR-T cells The secretion levels of IL-2 and IFN- ⁇ factors were significantly increased when OriCAR-388 T was co-incubated with NUGC-4 (p ⁇ 0.0001).
  • MSLN-VHH is directly connected to the intracellular signal transduction domain of CD3 ⁇ , which can independently transduce the signal of the extracellular MSLN antigen-binding domain and activate the secretion of cytokines
  • CLDN18.2-scFv is directly connected to the 4-1BB co-stimulatory domain, and cannot complete the signal transduction of the extracellular CLDN18.2 antigen-binding domain alone, and thus cannot activate the secretion of cytokines; while the above two The components are connected in parallel.
  • CD3 ⁇ will specifically transduce dual-target signals at the same time, and the 4-1BB domain stimulates the secretion of more cytokines.
  • NUGC-4 cells are human gastric cancer cells, which naturally co-express human CLDN18.2 and human MSLN proteins under conventional culture conditions in vitro, and can be used as target cells for dual-target co-expression to study the drug efficacy of OriCAR-388 T cells in vivo.
  • the mouse strain used was severe immunodeficiency B-NDG mice, and all mice were subcutaneously implanted with tumors (NUGC-4 cells, 3*10 6 cells/mouse) after one week of acclimatization in the mouse cage.
  • mice with an average tumor size of 976mm3 were randomly divided into 3 groups, 5 mice in each group, and OriCAR-388 T cells, 5E6-CART cells and Mock cells that had been cultured for 11 days were reinfused into the tail vein.
  • T cells (denoted as D0 on the day of reinfusion), as shown in Figure 11A, in which Mock T cells were reinfused at a dose of 1*10 7 cells/mouse (marked as G1 group: Mock T cell, 1*10 7 ), OriCAR-388 T cells were reinfused with high dose and low dose respectively, that is, 1*10 7 cells/mouse (marked as G2 group: OriCAR-388 T, 1*10 7 ) and 3*10 6 cells/mouse mice (labeled G3 group: OriCAR-388 T, 3*10 6 ).
  • mice The tumor size and body weight of the mice were measured 3 times a week, and the observation period was about 4 weeks in total, that is, the mice were euthanized after 33 days to end the experiment.
  • the results are shown in Figure 11B.
  • the mice in the 5E6-CART group began to lose body weight on the 8th day after reinfusion of CART, while the Mock T cell group, OriCAR-388 T high-dose group and OriCAR-388 T low-dose group During the entire observation period of the experiment (observation until 33 days after CAR-T reinfusion), the weight of the mice was not abnormal, the coat color of the mice was bright, and the behavior, diet, water intake, and color of urine and stool of the mice were all normal.
  • both the OriCAR-388 T cell high-dose and low-dose groups showed extremely significant tumor inhibitory activity (p ⁇ 0.0001), as shown in Figure 11C (the end point is defined as the mouse tumor volume > 3000mm 3 ), and the reinfusion 33 days after CART, the average tumor volume of the mice in the OriCAR-388 T cell high-dose group decreased to 273.10 mm 3 , and the average tumor volume of the OriCAR-388 T cell low-dose group decreased to 310.44 mm 3 . It is confirmed that OriCAR-388 T cells have a certain anti-tumor effect and good safety in mice.
  • OriCAR-388 can be stably expressed and proliferated in T cells, the CAR expression rate is maintained at about 49%, and the amplification factor of CART cells cultured in vitro for 11 days is about 50 times.
  • OriCAR-388 T cells only have strong killing activity against NUGC-4 double-target positive cells, and there is no or low killing activity against CLDN18.2 single-positive target cells or MSLN single-positive target cells.
  • IFN-r and IL-2 cytokines Under co-incubation with CHO-hCLDN18.2 cells, IFN-r and IL-2 cytokines were basically not secreted, and under co-incubation with OVCAR-3 cells, a small amount of IFN-r and IL-2 cytokines were secreted, while OriCAR-388 When T cells were co-incubated with NUGC-4 cells, the secretion levels of IL-2 and IFN- ⁇ factors were significantly increased. Immunodeficient mice were subcutaneously inoculated with NUGC-4 cells, and CAR-T cells were reinfused when the average tumor size was 976mm3 .
  • the purpose was to use this super-large tumor model to simulate the high tumor burden in the body, and the CAR-T was fully and repeatedly activated possible toxic side effects.
  • the experiment observed that the weight of the mice in the OriCAR-388 T high and low dose groups had no abnormality during the whole experimental observation period, the mouse coat color was bright, and the mouse behavior, diet and water intake, and the color of urine and stool were normal; the OriCAR-388 T cell high and low dose groups had 33 days after reinfusion of CART, the average tumor volume of the mice in the OriCAR-388 T cell high-dose group decreased to 273.10 mm 3 , and the average tumor volume of the mice in the OriCAR-388 T cell low-dose group decreased to The size is 310.44mm 3 , which proves that OriCAR-388 T cells have a certain anti-tumor effect and good safety in mice.
  • the 5E6-CART targeting Claudin18.2 single target showed severe toxicity in the drug efficacy experiment of tumor-bearing
  • this example carried out a single-dose toxicity test in a tumor-bearing mouse model, that is, a mouse toxicity test, as follows: 12 female B-NDG mice Mice were inoculated with NUGC-4 cells subcutaneously, and CAR-T cells were reinfused when the tumor volume reached about 270mm 3 , and were divided into 4 groups on average. T, 5*10 6 per mouse. After reinfusion, the observation continued until PG-D29.
  • the specific grouping scheme is shown in Table 1.
  • the body weight of the mice is shown in Figure 12A, in which the G3/G6/G8/G10 four groups were euthanized 15 days after tail vein reinfusion of CAR-T or Mock T cells (PG-D15) and then grossly dissected Organs were taken, so the weight of the mice was only continuously observed until PG-D15. During the observation period, the weight of the mice remained normal, and there was no significant drop in body weight; After euthanasia, all mice were grossly dissected to collect organs. During the observation period, the weight of the mice also remained normal, and there was no significant weight loss.
  • PG-D15 Mock T cells
  • mice When the average tumor volume of the mice reached 275mm3 , the mice were divided into groups and CAR-T/Mock T cells were reinfused on the same day.
  • the data analysis of the tumor growth of the mice during the observation period was shown in Figure 12B. All OriCAR-388 reinfusion groups were small Compared with the tumor volume of mice in the Mock T group measured at the same time, there is a very significant difference (p ⁇ 0.0001) between the tumor volume of the mice at the end of the tumor monitoring, and has significant tumor inhibitory activity.
  • PG-D15 and PG-D29 the blood biochemical indicators, white blood cell differential count indicators, complete blood cells and reticulocyte count indicators were detected on the mice respectively, and the test results were statistically analyzed.
  • the blood biochemical detection indicators of the mice are shown in Figure 12C , among all the detection indicators of D15 sampling results, the ALT/AST level of OriCAR-388 group was higher than that of Mock T group, but there was no statistical difference (p>0.05). Among all detection indicators of D29 sampling results, the OriCAR-388 group The ALT/AST level of the OriCAR-388 group was still higher than that of the Mock T group, and there was no statistical difference (p>0.05).
  • the CK level of the OriCAR-388 group was significantly lower than that of the Mock T group (p ⁇ 0.01), but this significant difference It has clinical significance; the white blood cell differential count index in the mouse blood is shown in Figure 12D, and the LYMP (lymphocyte) cell count in the D15 sampling result showed that the OriCAR-388 group was significantly higher than the Mock T group (p ⁇ 0.01), and the D29 sampling test At that time, the LYMP (lymphocyte) cell count level of the OriCAR-388 group was still higher than that of the Mock T group, but it slowed down, and there was no statistical difference (p>0.05); the mouse complete blood cell and reticulocyte count indicators The test results are shown in Figure 12E.
  • the PLT level of the OriCAR-388 group was significantly lower than that of the Mock T group when sampling on D15 (p ⁇ 0.0001), while there was still a significant difference in the PLT level of the OriCAR-388 group compared to the Mock T group when sampling on D29 , but the difference is slowly shrinking (p ⁇ 0.05). This example further confirmed the effectiveness and safety of OriCAR-388 T cells in mice.
  • Example 7 and Example 8 Using the operation methods in Example 7 and Example 8 for virus packaging, virus titer detection, CAR-T cell preparation, etc., 5E6-CART (CLDN18.2), MSLN3-CART, MSLN52-CART, OriCAR were prepared -388, OriCAR-387, OriCAR-386, and Mock T cells were counted and statistically analyzed on D1/D5/D7/D9/D13 days of CAR-T cell culture. The results are shown in Figure 13B.
  • the cumulative expansion fold of OriCAR-388 T cells was about 125 times
  • the cumulative expansion fold of OriCAR-387 T cells was about 189 times
  • the cumulative expansion fold of OriCAR-386 T cells was about 185 times.
  • the accumulative amplification factor of the cells is about 155 times
  • OriCAR-387 is better than OriCAR-386 than OriCAR-388, and there is no significant difference compared with Mock T cells.
  • the positive rate of CAR was detected by flow cytometry on D7, D9 and D12 of CAR-T cell culture respectively.
  • the detection antibody used was myc-tag(9B11) mouse mAb(Alexa Fluor 647)(Cell signaling, 2233S).
  • OriCAR-386, OriCAR-387 and OriCAR-388 can all be stably expressed in T cells, and the positive rate of OriCAR-388 CAR is maintained at about 40%. Higher than OriCAR-388CAR positive rate.
  • the conventional expansion multiples of dual-target OriCAR-386 and OriCAR-387 T cells were higher than those of OriCAR-388 T cells; the positive rate of CAR showed that 5E6-CAR, MSLN3-CAR , The positive rate of MSLN52-CAR is higher than that of OriCAR-386 and OriCAR-387, and the positive rate of OriCAR-386 and OriCAR-387 is higher than that of OriCAR-388.
  • Example 14 Comparative analysis of directed proliferation of CAR-T cells under repeated stimulation of target cells in vitro
  • the 5E6-CART, MSLN3-CART, MSLN52-CART, OriCAR-388, OriCAR-387 and OriCAR-386 cells that were expanded and cultured in vitro for 12 days in Example 13 were selected, and after the positive rate was detected by flow cytometry, Blank T cells were used to adjust the proportion of infected CAR-T cells to make the total number of cells consistent in each group.
  • the target cells are human gastric cancer cells NUGC-4 co-expressing human CLDN18.2 and human MSLN proteins
  • NUGC-4 is irradiated with ultraviolet light for 10 min
  • the number of target cells is 1*10 5 /well
  • the stimulation effect in the first round The target ratio is 1:1
  • the second round of stimulation-effect-target ratio is 1:2
  • the third round of stimulation-effect-target ratio is 1:2; set up different control wells, then mix the target cells and effector cells and centrifuge them with X -VIVO blank culture medium was resuspended in a 12-well plate; cultured in a 37-degree incubator for about 3-4 days, counted and statistically analyzed.
  • the results are shown in Figure 14.
  • OriCAR-388 Compared with double-targeted CART cells, after three rounds of repeated stimulation, OriCAR-388 has the best proliferation effect. After three rounds of targeted stimulation of target cells, the cumulative proliferation of T cells is 1996 times; followed by OriCAR-387 T cells, after three rounds of targeted stimulation of target cells, the cumulative proliferation of T cells was 1211 times; OriCAR-386 T cells were relatively poor, and the total cumulative proliferation of T cells was about 299 times after three rounds of targeted stimulation. For single-target CART cells, they can also proliferate after three rounds of co-incubation with irradiated target cells, but the proliferation effect is very poor.
  • the cumulative expansion of 5E6-CART cells is about 40 times in 11 days, and MSLN3-CART cells three rounds of target cells
  • the accumulative amplification to stimulation was about 110 times, and the accumulative amplification of MSLN52-CART cells in three rounds of targeted stimulation was about 45 times. That is, under repeated stimulation of target cells in vitro, the sustained expandability of OriCAR-388 T cells was stronger than that of OriCAR-387 T cells, significantly stronger than that of OriCAR-386 T cells, while single-target 5E6-CART, MSLN3-CART and MSLN52-CART cells had only a small amount of on-target proliferation.
  • the luciferase method was used to detect the killing effect of 5E6-CART, MSLN3-CART, MSLN52-CART, OriCAR-388, OriCAR-387, OriCAR-386 and Mock T cells on NUGC4-NFkB cells in vitro, and the above-mentioned In Example 13, CAR-T or Mock T cells routinely cultured in vitro for 11 days were used as effector cells.
  • All CAR-T cells have killing activity. At the low-efficiency target ratio of 1:1, the killing activity of dual-target OriCAR-388, OriCAR-387 and OriCAR-386 T cells is significantly stronger than that of single-target Point 5E6-CART, MSLN3-CART and MSLN52-CART cells have the killing activity on target cells, among which OriCAR-388 has the strongest killing activity; at the effect-to-target ratio of 3:1 and 9:1, all CAR-T sequences are A kill percentage of about 90% was detected, but no differences between the sequences have been shown. It shows that the unique CAR-T structure of dual-target OriCAR-388 T, OriCAR-387 T and OriCAR-386 T improves the effectiveness of CAR-T cells for specific tumor killing.
  • the CAR-T or Mock T cells conventionally cultured in vitro for 11 days in the above-mentioned Example 13 were selected as effector cells, and CHO-hCLDN18.2, NUGC-4 and OVCAR3 cells were selected as target cells.
  • the ELISA method described in 10 detects the secretion of cytokines after co-incubation for 20 h.
  • the specific detection method and the kit used are the same as in Example 10.
  • the results were statistically analyzed. The results are shown in Figure 16.
  • T cell reinfusion was PG-Day0, and the body weight and tumor volume of the mice were measured three times a week, and the observation continued until 46 days after tail vein reinfusion, namely PG-Day46, as shown in Figure 17A.
  • PG-Day46 46 days after tail vein reinfusion
  • OriCAR-386/OriCAR-387/OriCAR-388 T cells showed extremely significant antitumor efficacy compared with Mock T cells in the human gastric cancer cell transplantation mouse model, and the antitumor activity showed OriCAR-387 is superior to OriCAR-388 and superior to OriCAR-386, as shown in Figure 17C (the end point is defined as the mouse tumor volume > 3000mm 3 ): OriCAR-386/OriCAR-387/OriCAR-388 T cell high-dose group ( 3*10 6 /mouse) mice have very significant anti-tumor activity compared with the Mock T cell group (3*10 6 /mouse) mice, the statistical difference is P ⁇ 0.0001, and OriCAR-386 T cells are highly Three mice in the dose group achieved tumor elimination (3/5), five mice in the high-dose OriCAR-387 T cell group achieved tumor elimination (5/5), and two mice in the high-dose OriCAR-388 T cell group achieved
  • the survival curve of the mice is shown in Figure 17D.
  • the mice in the OriCAR-386/OriCAR-387/OriCAR-388 T cell high-dose group (3*10 6 /mouse) could reach 100%
  • the statistical results show that the median survival period of the mice in the Mock T (3*10 6 /only) group is 26 days, and the median survival period of the mice in the OriCAR-386 (1*10 6 /only) group is At 33 days, the median survival period of mice in OriCAR-387 (1*10 6 /mouse) group was 43 days, and the median survival period of mice in OriCAR-388 (1*10 6 /mouse) group was 43 days.
  • the safety and antitumor efficacy of OriCAR-387 T cells are better than OriCAR-388 T cells, and better than OriCAR-386 T cells.
  • FACS flow cytometry fluorescence sorting technique
  • iQue Screener flow meter purchased from IntelliCyt Company
  • PBS containing 0.1% BSA as buffer to detect the specific binding activity of the above-mentioned chimeric antibody to target cells
  • select Three kinds of target cells a stably transfected cell line expressing human CLDN18.2, a stably transfected cell line expressing human CLDN18.1, and a tumor cell line were tested for their binding activity.
  • the cell lines that obtained stable and high expression of CLDN18 were constructed and marked as 293T-human CLDN18.2, CHO-human CLDN18.2 and SP2/0-human CLDN18.2 cells respectively, the cells were digested and counted, and the cells were resuspended and adjusted in flow buffer To 1 ⁇ 10 6 /ml, add 30ul/well into a V-bottom 96-well plate; add 30ul/well of the primary antibody, starting at a concentration of 30ug/ml, dilute with flow buffer in a two- or three-fold gradient to form a 7
  • Each antibody was set as a PBS negative control, and the positive control antibody was zolbetuximab purified in Example 1; incubated at 4°C for 1 hour, washed once with flow buffer, and added secondary antibody (abcam, Cat#ab98593), 30ul/ Well, incubate at 4°C for 30 minutes; wash the flow buffer twice, shake the cells loose, add 25ul/well flow buffer, and wait
  • the positive control antibody is a commercially available anti-CLDN18 antibody (Anti-Claudin18 antibody) (abcam, Cat#ab203563), and its antigen binding site is located in the intracellular part of the CLDN18.2 quadruple crotch protein, which needs to be analyzed by flow cytometry intracellular staining .
  • the cells are constructed 293T-human CLDN18.1 and SP2/0-human CLDN18.1 cells.
  • the cells After the cells are digested and counted, they are fixed and ruptured, and the treated cells are resuspended to 1 ⁇ 10 6 in flow buffer /ml, 30ul/well was added to a V-bottom 96-well plate; 30ul/well was added to the primary antibody, the antibody was initially diluted at a concentration of 30ug/ml, and was diluted with flow buffer in a three-fold ratio to form 7 gradients.
  • PBS negative control, positive control antibody dilution conditions are the same as above; incubate at 4°C for 1 hour, wash with flow buffer, add secondary antibody (abcam, Cat#ab98593 and Cat#ab150079), 30ul/well, incubate at 4°C for 30 minutes; Wash twice with flow buffer, shake the cells loose, add 25ul/well flow buffer, and wait for the machine. Substituting the original data into GraphPad8.0 software for drawing and calculation, the results are shown in Figure 19.

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Abstract

提供了一种细胞，其包含和/或表达靶向紧密连接蛋白18.2(CLDN18.2)的嵌合抗原受体，以及靶向间皮素(MSLN)蛋白的嵌合抗原受体。还提供了一种表达载体，其包括编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列。还提供了所述细胞和/或所述表达载体的制备方法和用途。

Description

靶向CLDN18.2和MSLN的嵌合抗原受体及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向CLDN18.2和MSLN的嵌合抗原受体及其用途。
背景技术
间皮素(Mesothelin，MSLN)是位于细胞表面的糖蛋白，通过糖基磷脂酰肌醇锚定于细胞膜上。间皮素基因编码一种69kDa的前体蛋白，被弗林蛋白酶(成对碱性氨基酸蛋白酶，furin)样转化酶水解为两条链，C端约40KD的膜结合蛋白即为成熟的间皮素，N端约30KD称之为巨核细胞促进因子(MPF)的片断脱落并释放出细胞外。MPF和膜锚MSLN均为N-糖基化，MPF可在体外促进巨核细胞克隆的形成，膜锚MSLN可以与MUC16互作，在细胞粘附过程中起重要作用，因此目前靶向治疗中均选择膜锚MSLN做为靶点，故目前MSLN专指MSLN的C端40KD片段，即膜锚MSLN。
CLDN18.2(Claudin 18.2)只表达于分化型胃壁细胞，在正常组织中无表达。最新研究表明，CLDN18.2在77％以上胃癌患者以及80％以上胰腺癌患者中都过表达，此外在肺癌，食管癌以及卵巢癌等实体瘤中过表达。CLDN18.2属于紧密连接蛋白质家族，可以控制层细胞之间的分子流动。然而，在肿瘤中，紧密连接被破坏，CLDN蛋白质失去了其主要作用。因为CLDN18.2大量存在于胃部肿瘤中，研究者估计所有晚期胃癌患者的一半可以作为CLDN18.2抗体靶点新疗法的候选人。
然而，现有针对CLDN18.2的CAR-T细胞仍面临很多问题，主要集中在安全性及有效性上，因此，仍需探索更高效及更安全的嵌合抗原受体，以解决目前CLDN18.2CAR-T细胞中存在的问题。
发明内容
本申请提供了一种新的嵌合抗原受体结构，以及包含该嵌合抗原受体结构的细胞。一方面，本申请提供的嵌合抗原受体和/或细胞能够以较高的亲和力与抗原相结合。另一方面，为解决现有CAR产品中存在的安全性不好，有效性有限的问题，本申请将两个或两个以上的靶点结合起来，提供能够表达靶向不同靶点的嵌合抗原受体的细胞。本申请所述的细胞不仅在安全性和有效性上显著提高，还能够减少CAR-T细胞的毒副作用。
一方面，本申请提供了一种细胞，其包含和/或表达靶向紧密连接蛋白18.2 (CLDN18.2)的嵌合抗原受体，以及靶向间皮素(MSLN)蛋白的嵌合抗原受体。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含抗原结合结构域。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:147所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:144所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:24、SEQ ID NO:59和SEQ ID NO:73中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:145所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:22、SEQ ID NO:57和SEQ ID NO:71中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:146(X 1YX 2X 3X 4，其中，X 1为N或R，X 2为G、I或V，X 3为I或M，X 4为H、N或S)所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:20、SEQ ID NO:55和SEQ ID NO:69中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2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氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:24所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:55所示的氨基酸序列，所示HCDR2包含SEQ ID NO:57所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:59所示的氨基酸序列；以及
(3)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH，所述VH包含SEQ ID NO:147所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH，所述VH包含SEQ ID NO:26、SEQ ID NO:43、SEQ ID NO:61、SEQ ID NO:75和SEQ ID NO:79中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含轻链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:151所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:148所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:32、SEQ ID NO:63、SEQ ID NO:85和SEQ ID NO:91中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:149所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:30或SEQ ID NO:83所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:150所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:28、SEQ ID NO:45和SEQ ID NO:81中任一项所示的氨基酸。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1，LCDR2和LCDR3包含选自下述任一组氨基酸序列：
(1)所述LCDR1包含SEQ ID NO:28所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(2)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(3)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:63所示的氨基酸序列；
(4)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:91所示的氨基酸序列；以及
(5)所述LCDR1包含SEQ ID NO:81所示的氨基酸序列，所述LCDR2包含SEQ ID NO:83所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:85所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:151所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:34、SEQ ID NO:49、SEQ ID NO:65、SEQ ID NO:87和SEQ ID NO:93中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3，所述HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3包含选自下述任一组氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，所述HCDR3包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1包含SEQ ID NO:28所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，所述HCDR3包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(3)所述HCDR1包含SEQ ID NO:55所示的氨基酸序列，所示HCDR2包含SEQ ID NO:57所示的氨基酸序列，所述HCDR3包含SEQ ID NO:59所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:63所示的氨基酸序列；
(4)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序 列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(5)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:91所示的氨基酸序列；以及
(6)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:81所示的氨基酸序列，所述LCDR2包含SEQ ID NO:83所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:85所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH和VL，所述VH和VL包含选自下述任一组氨基酸序列：
(1)所述VH包含SEQ ID NO:26所示的氨基酸序列，且所述VL包含SEQ ID NO:34所示的氨基酸序列；
(2)所述VH包含SEQ ID NO:43所示的氨基酸序列，且所述VL包含SEQ ID NO:49所示的氨基酸序列；
(3)所述VH包含SEQ ID NO:75所示的氨基酸序列，且所述VL包含SEQ ID NO:49所示的氨基酸序列；
(4)所述VH包含SEQ ID NO:61所示的氨基酸序列，且所述VL包含SEQ ID NO:65所示的氨基酸序列；
(5)所述VH包含SEQ ID NO:79所示的氨基酸序列，且所述VL包含SEQ ID NO:93所示的氨基酸序列；以及
(6)所述VH包含SEQ ID NO:79所示的氨基酸序列，且所述VL包含SEQ ID NO:87所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含scFv。
在某些实施方式中，所述靶向CLDN18.2所述scFv包含SEQ ID NO:38、SEQ ID NO:53、SEQ ID NO:67、SEQ ID NO:77、SEQ ID NO:89和SEQ ID NO:95中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含共刺激域，所述共刺激域包含源自选自下组中的一种或多种蛋白的共刺激域：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，所述共刺激域包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含跨膜区，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区：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。
在某些实施方式中，所述跨膜区为源自CD8的跨膜区。
在某些实施方式中，所述跨膜区为源自人CD8的跨膜区。
在某些实施方式中，所述跨膜区包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含铰链区，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、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CD8的铰链区。
在某些实施方式中，所述铰链区为源自人CD8的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含信号肽。
在某些实施方式中，所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体不包含胞内信号传导域。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体包含抗原结合结构域。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:101、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:119和SEQ ID NO:154中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:99、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:118和SEQ ID NO:153中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的所述抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:108、SEQ ID NO:117和SEQ ID NO:152中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体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氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:97所示的氨基酸序列，所述HCDR2包含SEQ ID NO:99所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:101所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:104所示的氨基酸序列，所述HCDR2包含SEQ ID NO:105所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:106所示的氨基酸序列；
(3)所述HCDR1包含SEQ ID NO:108所示的氨基酸序列，所述HCDR2包含SEQ ID NO:109所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:110所示的氨基酸序列；
(4)所述HCDR1包含SEQ ID NO:117所示的氨基酸序列，所述HCDR2包含SEQ ID NO:118所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:119所示的氨基酸序列；以及
(5)所述HCDR1包含SEQ ID NO:152所示的氨基酸序列，所述HCDR2包含SEQ ID NO:153所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:154所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含VHH。
根据权利要求49所述的细胞，其中所述VHH包含SEQ ID NO:103、SEQ ID NO:107和 SEQ ID NO:155中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:123或SEQ ID NO:114所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:113或SEQ ID NO:122所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:112或SEQ ID NO:121所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1，LCDR2和LCDR3包含选自下述任一组的氨基酸序列：
(1)所述LCDR1包含SEQ ID NO:112所示的氨基酸序列，所述LCDR2包含SEQ ID NO:113所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:114所示的氨基酸序列；以及
(2)所述LCDR1包含SEQ ID NO:121所示的氨基酸序列，所述LCDR2包含SEQ ID NO:122所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:123所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:115或SEQ ID NO:124所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:116或SEQ ID NO:125所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的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传导域：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的信号传导域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体包含跨膜区，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区：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。
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:14所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体包含铰链区，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、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CD28的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体包含信号肽。
在某些实施方式中，所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的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:18所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体不包含共刺激域。
在某些实施方式中，在所述细胞中，所述靶向CLDN18.2的嵌合抗原受体的表达量与靶向MSLN的嵌合抗原受体的表达量为约1：1。
在某些实施方式中，在所述细胞中，所述靶向CLDN18.2的嵌合抗原受体的表达量与靶向MSLN的嵌合抗原受体的表达量为约2：1。
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，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:18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，本申请提供了表达载体，其包括编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列。
另一方面，本申请提供了表达载体，其包括编码本申请所述的靶向CLDN18.2的嵌合抗原受体的核酸序列和编码本申请所述的靶向MSLN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，编码靶向CLDN18.2的嵌合抗原受体的核酸序列包括编码抗原结合结构域的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH中的至少一个CDR，编码所述VH的核酸序列包含SEQ ID NO:25、SEQ ID NO:42、SEQ ID NO:60、SEQ ID NO:74和SEQ ID NO:78中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且编码所述HCDR3的核酸序列包含SEQ ID NO:23、SEQ ID NO:41、SEQ ID NO:58和SEQ ID NO:72中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且编码所述HCDR2的核酸序列包含SEQ ID NO:21、SEQ ID NO:40、SEQ ID NO:56和SEQ ID NO:70中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且编码所述HCDR1的核酸序列包含SEQ ID NO:19、SEQ ID NO:39、SEQ ID NO:54和SEQ ID NO:68中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH，且编码所述VH的核酸序列包含SEQ ID NO:25、SEQ ID NO:42、SEQ ID NO:60、SEQ ID NO:74和SEQ ID NO:78中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且编码所述LCDR3的核酸序列包含SEQ ID NO:31、SEQ ID NO:47、SEQ ID NO:62、SEQ ID NO:84和SEQ ID NO:90中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR2，且编码所述 LCDR2的核酸序列包含SEQ ID NO:29、SEQ ID NO:46和SEQ ID NO:82中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且编码所述LCDR1的核酸序列包含SEQ ID NO:27、SEQ ID NO:44和SEQ ID NO:80中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VL，且编码所述VL的核酸序列包含SEQ ID NO:33、SEQ ID NO:48、SEQ ID NO:64、SEQ ID NO:86和SEQ ID NO:92中任一项所示的核酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含scFv。
在某些实施方式中，编码所述scFv的核酸序列包含SEQ ID NO:37、SEQ ID NO:52、SEQ ID NO:66、SEQ ID NO:76、SEQ ID NO:88和SEQ ID NO:94中任一项所示的核酸序列。
在某些实施方式中，编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码共刺激域的核酸序列，所述共刺激域包含源自选自下组中的一种或多种蛋白的共刺激域：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，所述编码共刺激域的核酸序列包含SEQ ID NO:7所示的核酸序列。
在某些实施方式中，编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码跨膜区的核酸序列，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区：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。
在某些实施方式中，所述跨膜区为源自CD8的跨膜区。
在某些实施方式中，所述编码跨膜区的核酸序列包含SEQ ID NO:5所示的核酸序列。
在某些实施方式中，编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码铰链 区的核酸序列，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、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CD8的铰链区。
在某些实施方式中，所述编码铰链区的核酸序列包含SEQ ID NO:5所示的核酸序列。
在某些实施方式中，编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码信号肽的核酸序列。
在某些实施方式中，所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述编码信号肽的核酸序列包含SEQ ID NO:1所示的核酸序列。
在某些实施方式中，所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列不包含编码胞内信号传导域的核酸序列。
在某些实施方式中，所述编码靶向MSLN的嵌合抗原受体的核酸序列包含编码抗原结合结构域的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
在某些实施方式中，编码所述VH的核酸序列包含SEQ ID NO:102或SEQ ID NO:156所示的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含HCDR3，所述HCDR3包含SEQ ID NO:101、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:119和SEQ ID NO:154中任一项所示的氨基酸序列。在某些实施方式中，编码所述HCDR3的核酸序列包含SEQ ID NO:100所示的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:99、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:118和SEQ ID NO:153中任一项所示的氨基酸序列。在某些实施方式中，编码所述HCDR2的核酸序列包含SEQ ID NO:98所示的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含HCDR1，所述HCDR1包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:108、SEQ ID NO:117和SEQ ID NO:152中任一项所示的氨基酸序列。在某些实施方式中，编码所述HCDR1的核酸序列包含SEQ ID NO:96所示的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含VH，所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。在某些实施方式中，编码所述VH的核酸序列包含SEQ ID NO:102或SEQ ID NO:156所示的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含VHH，所述VHH包含SEQ ID NO:103、SEQ ID NO:107和SEQ ID NO:155所示的氨基酸序列。在某些实施方式中，编码所述VHH的核酸序列包含SEQ ID NO:102或SEQ ID NO:156所示的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含LCDR3，所述LCDR3包含SEQ ID NO:114和SEQ ID NO:123中任一项所示的氨基酸序列。在某些实施方式中，编码所述LCDR3的核酸序列包含SEQ ID NO:123或SEQ ID NO:114所示的核酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:113或SEQ ID NO:122所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含LCDR1，所述LCDR1包含SEQ ID NO:112或SEQ ID NO:121所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含VL，所述VL包含SEQ ID NO:115或SEQ ID NO:124所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:116或SEQ ID NO:125所示的氨基酸序列。
在某些实施方式中，编码所述靶向MSLN的嵌合抗原受体的核酸序列包含编码胞内信号传导域的核酸序列，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传导域：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的信号传导域。
在某些实施方式中，所述编码胞内信号传导域的核酸序列包含SEQ ID NO:15所示的核酸序列。
在某些实施方式中，编码所述靶向MSLN的嵌合抗原受体的核酸序列包含编码跨膜区的核酸序列，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区：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。
在某些实施方式中，跨膜区为源自CD28的跨膜区。
在某些实施方式中，所述编码跨膜区的核酸序列包含SEQ ID NO:13所示的核酸序列。
在某些实施方式中，编码所述靶向MSLN的嵌合抗原受体的核酸序列包含编码铰链区的核酸序列，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、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CD28的铰链区。
在某些实施方式中，所述编码铰链区的核酸序列包含SEQ ID NO:11所示的核酸序列。
在某些实施方式中，编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码信号肽的核酸序列。
在某些实施方式中，所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述编码信号肽的核酸序列包含SEQ ID NO:1所示的核酸序列。
在某些实施方式中，编码所述靶向MSLN的嵌合抗原受体的核酸序列不包含编码所述共刺激域的核酸序列。
在某些实施方式中，编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN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:10所示的氨基酸序列。
在某些实施方式中，编码所述剪切肽的核酸序列包含SEQ ID NO:9所示的核酸序列。
在某些实施方式中，所述表达载体包含至少一条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列。
在某些实施方式中，所述表达载体包含两条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列。
在某些实施方式中，所述两条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列通过编码剪切肽的核酸序列连接。
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，在所述表达载体载体中，其中所述编码剪切肽的核酸序列包含SEQ ID NO:9所示的核酸序列。
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，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。
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，所述编码低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:17所示的核酸序列。
在某些实施方式中，所述编码低密度脂蛋白受体相关蛋白或其片段与所述编码靶向MSLN的嵌合抗原受体的胞内信号传导域的核酸序列直接相连。
在某些实施方式中，所述编码低密度脂蛋白受体相关蛋白或其片段与所述编码靶向MSLN的嵌合抗原受体的核酸序列通过剪切肽相连。
在某些实施方式中，所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列位于同一条核酸分子中。
在某些实施方式中，所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列位于同一条核酸分子中。
在某些实施方式中，在所述表达载体中，其中第一条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、第二条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列位于同一条核酸分子中。
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：编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、编码所述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，所述表达载体从5’端至3’端分别依次包含：编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、编码所述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，所述表达载体从5’端至3’端分别依次包含：编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码MSLN VHH的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码胞内信号传导域的核酸序列以及编码所述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，所述表达载体从5’端至3’端分别依次包含：编码CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码MSLN VHH的核酸序列、编码CD28铰链区的核酸序列、编码CD28跨膜区的核酸序列、编码CD3ζ胞内信号传导域的核酸序列以及编码所述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，所述表达载体从5’端至3’端分别依次包含：编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码MSLN VHH的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码胞内信号传导域的核酸序列以及编码所述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，所述表达载体从5’端至3’端分别依次包含：编码CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码MSLN VHH的核酸序列、编码CD28铰链区的核酸序列、编码CD28跨膜区的核酸序列、编码CD3ζ胞内信号传导域的核酸序列以及编码所述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，本申请还提供了细胞，其包含本申请所述的表达载体。
在某些实施方式中，所述细胞为免疫效应细胞。
在某些实施方式中，所述细胞包括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，本申请还提供了所述细胞、和/或所述表达载体在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了所述靶向CLDN18.2的嵌合抗原受体和所述靶向MSLN的嵌合抗原受体在制备药物中的用途，所述药物用于预防和/或治疗疾病和/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和/或非实体瘤。
在某些实施方式中，所述肿瘤包括同时表达CLDN18.2和MSLN两种抗原的肿瘤。
在某些实施方式中，所述肿瘤包括胃癌、胰腺癌和/或胃食管交界处癌。
另一方面，本申请还提供了一种预防和/或治疗疾病和/或病症的方法，其包括向有需要的受试者施用本申请所述的细胞。
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和/或非实体瘤。
在某些实施方式中，所述肿瘤包括同时表达CLDN18.2和MSLN两种抗原的肿瘤。
在某些实施方式中，所述肿瘤包括胃癌、胰腺癌和/或胃食管交界处癌。
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显示的是CLDN18.2单链抗体的抗原结合活性。
图2显示的是CLDN18.2单链抗体与非靶细胞的结合活性鉴定。
图3A-3D显示的是CLDN18.2特异性CAR在T细胞中稳定表达。
图4显示的是CLDN18.2特异性CAR在体外特异性杀伤靶细胞的实验结果。
图5A-5B显示的是CLDN18.2特异性CAR的因子分泌情况。
图6A-6D显示的是CLDN18.2特异性CAR-T细胞在小鼠结肠癌CDX模型中的抑瘤作用。
图7A-7B显示的是本申请所述dual CAR的构建示意图。
图8A-8B显示的是本申请所述CAR-T细胞的表达及增殖情况。
图9显示的是CAR-T细胞的体外杀伤活性。
图10A-10B显示的是本申请所述CAR-T细胞分泌细胞因子的情况。
图11A-11C显示的是本申请所述CAR-T细胞在人胃癌CDX模型中的抑瘤作用。
图12A-12E显示的是本申请所述CAR-T细胞在小鼠体内的毒理实验。
图13A显示的是本申请所述CAR的构建示意图；图13B-13C显示的是本申请所述CAR在细胞中的表达和CAR-T细胞增殖情况。
图14显示的是在靶细胞反复刺激下，本申请所述CAR-T细胞的增殖情况。
图15显示的是本申请所述CAR-T细胞的体外细胞杀伤情况。
图16显示的是本申请所述CAR-T细胞的体外细胞因子分泌情况检测。
图17A-17D显示的是本申请所述CAR-T细胞在人胃癌小鼠CDX模型中的抑瘤作用。
图18显示的是本申请所述的CLDN18.2的靶向部分与高表达人CLDN18.2细胞的流式结合活性检测。
图19显示的是本申请所述的CLDN18.2的靶向部分与高表达人CLDN18.1细胞的流式结合活性检测。
图20显示的是本申请所述的CLDN18.2的靶向部分与肿瘤细胞系的流式结合活性检测。
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，本领域技术人员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
在本申请中，术语“CLDN18.2”、或“Claudin18.2”可以互换使用，通常指细胞连接密蛋白Claudin18的亚型2。所述术语涵盖“全长”、未加工的CLDN18.2以及由细胞加工所产生的任何形式的CLDN18.2。CLDN18.2可以包括完整的CLDN18.2及其片段，其功能性变体、同工型、物种同源物、衍生物、类似物以及具有至少一个与CLDN18.2共同表位的类似物。CLDN18.2(例如人CLDN18.2)的氨基酸序列可以是本领域已知的。例如，人CLDN18.2核苷酸序列可以显示于GeneBank登录号NM_001002026.3下。例如，小鼠CLDN18.2核苷酸序列可以显示于GeneBank登录号NM_001194921.1下。例如，食蟹猴CLDN18.2核苷酸序列可以显示于GeneBank登录号XM_001114708.4下。
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“V H”和“V L”(或者分别称为“VH”和“VL”)。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“可变”通常指在抗体之间可变结构域的某些区段在序列上可能存在较大差异。可变结构域介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性并非在整个可变结构域范围内均匀分布。它通常集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991))。
在本申请中，术语“CDR”也称“互补决定区”，通常指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在某些实施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。参见，例如,Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，CLDN18.2)能力的一个或多个片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv，VHH和/或dAb。
在本申请中，术语“Fab”通常指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(V H)的第一恒定区(C H1)组成。
在本申请中，术语“Fab′片段”通常指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。
在本申请中，术语“F(ab')2”通常指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够交联抗原。
在本申请中，术语“Fv片段”通常指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区 包括例如CDR。例如，Fv片段包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。
在本申请中，术语“scFv”通常指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
在本申请中，术语“dAb”通常是指具有VH域、VL域或具有VH域或VL域的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490；以及参考例如WO 06/030220、WO 06/003388和DomantisLtd的其它公布的专利申请。
在本申请中，术语“直接相连”与术语“间接相连”相对，术语“直接相连”通常是指直接连接。例如，所述直接相连可以为物质间没有间隔子而直接相连的情况。所述间隔子可以是连接子。例如，所述连接子可以为肽连接子。术语“间接相连”通常是指物质间不直接相连的情况。例如，所述间接相连可以为通过间隔子而连接的情况。
在本申请中，术语“分离的核酸分子”通常指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、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得以表达。举例来说，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类可以包括逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。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)。细胞可包括用本申请所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞或干细胞(例如ES细胞、iPS细胞、间充质干细胞)或免疫细胞(例如T细胞、NK细胞、NKT细胞、巨噬细胞)。
在本申请中，术语“药物组合物”通常指用于预防/治疗疾病或病症的组合物。所述药物组合物可以包含本申请所述的嵌合抗原受体、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，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包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载剂”通常包括药剂学可接受的载剂、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。生理学可接受的载体可包括例如缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，例如钠；和/或非离子表面活性剂。
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“肿瘤”通常指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或血液瘤。例如，在本申请中，肿瘤可以是CLDN18.2阳性的肿瘤。
术语“癌症”通常指以异常细胞快速且失控生长为特征的疾病。癌细胞可以局部地或通过血流和淋巴系统扩散到身体其他部分。本申请中的癌症包括但不限于胃癌和/或结肠癌等。术语“肿瘤”和“癌症”在本文中互换地使用，例如，两种术语涵盖实体瘤和液体肿瘤，例 如，弥散型或循环型肿瘤。如本文所用，术语“癌症”或“肿瘤”可以包括恶变前以及恶性癌症和肿瘤。
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合CLDN18.2蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合CLDN18.2蛋白的抗体或其片段)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)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的 局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
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
一方面，本申请提供一种细胞，其包含和/或表达靶向紧密连接蛋白18.2(CLDN18.2)的嵌合抗原受体，以及靶向间皮素(MSLN)蛋白的嵌合抗原受体。
另一方面，本申请提供一种表达载体，其包括编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列和编码所述靶向MSLN的嵌合抗原受体的核酸序列。
另一方面，本申请还提供一种细胞，其包含本申请所述的表达载体。
靶向CLDN18.2的嵌合抗原受体
在本申请中，所述靶向CLDN18.2的嵌合抗原受体包含靶向CLDN18.2的抗原结合结构域。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:147所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:144所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:24、SEQ ID NO:59和SEQ ID NO:73中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:145所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:22、SEQ ID NO:57和SEQ ID NO:71中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:146(X 1YX 2X 3X 4，其中，X 1为N或R，X 2为G、I或V，X 3为I或M，X 4为H、N或S)所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:20、SEQ ID NO:55和SEQ ID NO:69中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2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氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:24所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:55所示的氨基酸序列，所示HCDR2包含SEQ ID NO:57所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:59所示的氨基酸序列；以及
(3)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH，所述VH包含SEQ ID NO:147所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH，所述VH包含SEQ ID NO:26、SEQ ID NO:43、SEQ ID NO:61、SEQ ID NO:75和SEQ ID NO:79中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含轻链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:151所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:148所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:32、SEQ ID NO:63、SEQ ID NO:85和SEQ ID NO:91中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:149所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:30和SEQ ID NO:83中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且所述 LCDR1包含SEQ ID NO:150所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:28、SEQ ID NO:45和SEQ ID NO:81中任一项所示的氨基酸。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1，LCDR2和LCDR3包含选自下述任一组氨基酸序列：
(1)所述LCDR1包含SEQ ID NO:28所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(2)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(3)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:63所示的氨基酸序列；
(4)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:91所示的氨基酸序列；以及
(5)所述LCDR1包含SEQ ID NO:81所示的氨基酸序列，所述LCDR2包含SEQ ID NO:83所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:85所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:151所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:34、SEQ ID NO:49、SEQ ID NO:65、SEQ ID NO:87和SEQ ID NO:93中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3，所述HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3包含选自下述任一组氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，所述HCDR3包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1包含SEQ ID NO:28所示的氨基酸序列，所述LCDR2包含SEQ  ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，所述HCDR3包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(3)所述HCDR1包含SEQ ID NO:55所示的氨基酸序列，所示HCDR2包含SEQ ID NO:57所示的氨基酸序列，所述HCDR3包含SEQ ID NO:59所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:63所示的氨基酸序列；
(4)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
(5)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:91所示的氨基酸序列；以及
(6)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:81所示的氨基酸序列，所述LCDR2包含SEQ ID NO:83所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:85所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含VH和VL，所述VH和VL包含选自下述任一组氨基酸序列：
(1)所述VH包含SEQ ID NO:26所示的氨基酸序列，且所述VL包含SEQ ID NO:34所示的氨基酸序列；
(2)所述VH包含SEQ ID NO:43所示的氨基酸序列，且所述VL包含SEQ ID NO:49所示的氨基酸序列；
(3)所述VH包含SEQ ID NO:75所示的氨基酸序列，且所述VL包含SEQ ID NO:49所示的氨基酸序列；
(4)所述VH包含SEQ ID NO:61所示的氨基酸序列，且所述VL包含SEQ ID NO:65所示的氨基酸序列；
(5)所述VH包含SEQ ID NO:79所示的氨基酸序列，且所述VL包含SEQ ID NO:93所示的氨基酸序列；以及
(6)所述VH包含SEQ ID NO:79所示的氨基酸序列，且所述VL包含SEQ ID NO:87所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的抗原结合结构域包含scFv。
在某些实施方式中，所述靶向CLDN18.2所述scFv包含SEQ ID NO:38、SEQ ID NO:53、SEQ ID NO:67、SEQ ID NO:77、SEQ ID NO:89和SEQ ID NO:95中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含共刺激域，所述共刺激域包含源自选自下组中的一种或多种蛋白的共刺激域：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，所述共刺激域包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含跨膜区，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区：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。
在某些实施方式中，所述跨膜区为源自CD8的跨膜区。
在某些实施方式中，所述跨膜区为源自人CD8的跨膜区。
在某些实施方式中，所述跨膜区包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含铰链区，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、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CD8的铰链区。
在某些实施方式中，所述铰链区为源自人CD8的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体包含信号肽。
在某些实施方式中，所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述靶向CLDN18.2的嵌合抗原受体不包含胞内信号传导域。
在本申请中，还提供了编码所述靶向CLDN18.2的嵌合抗原受体各部分的核酸分子。
靶向MSLN的嵌合抗原受体
在本申请中，所述靶向MSLN的嵌合抗原受体包含靶向MSLN的抗原结合结构域。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:101、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:119和SEQ ID NO:154中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:99、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:118和SEQ ID NO:153中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的所述抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:108、SEQ ID NO:117和SEQ ID NO:152中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体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氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:97所示的氨基酸序列，所述HCDR2包含SEQ ID NO:99所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:101所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:104所示的氨基酸序列，所述HCDR2包含SEQ  ID NO:105所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:106所示的氨基酸序列；
(3)所述HCDR1包含SEQ ID NO:108所示的氨基酸序列，所述HCDR2包含SEQ ID NO:109所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:110所示的氨基酸序列；
(4)所述HCDR1包含SEQ ID NO:117所示的氨基酸序列，所述HCDR2包含SEQ ID NO:118所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:119所示的氨基酸序列；以及
(5)所述HCDR1包含SEQ ID NO:152所示的氨基酸序列，所述HCDR2包含SEQ ID NO:153所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:154所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含VHH。
在某些实施方式中，所述VHH包含SEQ ID NO:103、SEQ ID NO:107和SEQ ID NO:155中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:123或SEQ ID NO:114所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:113或SEQ ID NO:122所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:112或SEQ ID NO:121所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1，LCDR2和LCDR3包含选自下述任一组的氨基酸序列：
(1)所述LCDR1包含SEQ ID NO:112所示的氨基酸序列，所述LCDR2包含SEQ ID NO:113所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:114所示的氨基酸序列；以及
(2)所述LCDR1包含SEQ ID NO:121所示的氨基酸序列，所述LCDR2包含SEQ ID NO:122所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:123所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:115或SEQ ID NO:124所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体的抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:116或SEQ ID NO:125所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的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传导域：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的信号传导域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体包含跨膜区，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区：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。
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:14所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体包含铰链区，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、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CD28的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体包含信号肽。
在某些实施方式中，所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的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:18所示的氨基酸序列。
在某些实施方式中，所述靶向MSLN的嵌合抗原受体不包含共刺激域。
在本申请中，还提供了编码所述靶向MSLN的嵌合抗原受体各部分的核酸分子。
细胞
在本申请中，所述细胞中包含和/或表达靶向CLDN18.2的嵌合抗原受体的表达量与靶向MSLN的嵌合抗原受体的表达量可以为约1：1。
在本申请中，所述细胞中包含和/或表达靶向CLDN18.2的嵌合抗原受体的表达量与靶向MSLN的嵌合抗原受体的表达量可以为约2：1
在本申请中，所述细胞还可以包含和/或表达低密度脂蛋白受体相关蛋白或其片段。
在本申请中，所述低密度脂蛋白受体相关蛋白或其片段包含可以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。
在本申请中，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和/或6或其片段。
在本申请中，所述低密度脂蛋白受体相关蛋白或其片段可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述细胞可以包含免疫效应细胞。
在本申请中，所述细胞可以包括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，所述细胞可以为T细胞。
表达载体
在本申请中，所述表达载体还可以包含编码所述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，所述低密度脂蛋白受体相关蛋白或其片段包含可以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。
在本申请中，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和/或6或其片段。
在本申请中，编码所述低密度脂蛋白受体相关蛋白或其片段可以包含SEQ ID NO:18所示的核酸序列。
在本申请中，所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列可以通过剪切肽连接。
例如，所述剪切肽可以选自下组：P2A、T2A、F2A和E2A。
例如，所述剪切肽包含SEQ ID NO:10所示的氨基酸序列。例如，编码所述剪切肽的核酸序列可以包含SEQ ID NO:9所示的核酸序列。
在本申请中，表达载体可以包含至少一条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列。
在本申请中，表达载体可以包含两所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列。
在本申请中，所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列可以位于同一条核酸分子中。
在本申请中，所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列位于同一条核酸分子中。
在本申请中，在所述表达载体中，其中第一条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、第二条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列可以位于同一条核酸分子中。
在本申请中，所述表达载体从5’端至3’端可分别依次包含：编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、编码所述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，所述表达载体从5’端至3’端可分别依次包含：编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、编码所述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，所述表达载体从5’端至3’端分别依次包含：编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码MSLN VHH的核酸序 列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码胞内信号传导域的核酸序列以及编码所述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，所述表达载体从5’端至3’端分别依次包含：编码CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码MSLN VHH的核酸序列、编码CD28铰链区的核酸序列、编码CD28跨膜区的核酸序列、编码CD3ζ胞内信号传导域的核酸序列以及编码所述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，所述表达载体从5’端至3’端分别依次包含：编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码MSLN VHH的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码胞内信号传导域的核酸序列以及编码所述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，所述表达载体从5’端至3’端分别依次包含：编码CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码MSLN VHH的核酸序列、编码CD28铰链区的核酸序列、编码CD28跨膜区的核酸序列、编码CD3ζ胞内信号传导域的核酸序列以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列。
用途
另一方面，本申请提供了所述细胞和/或所述表达载体在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了所述靶向CLDN18.2的嵌合抗原受体和所述靶向MSLN的嵌合抗原受体在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请提供了一种预防和/或治疗疾病和/或病症的方法，其包括向有需要的受试者施用本申请所述的细胞。
另一方面，本申请提供了所述细胞和/或所述表达载体，其用于预防和/或治疗疾病和/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和/或非实体瘤。
在某些实施方式中，所述肿瘤包括同时表达CLDN18.2和MSLN两种抗原的肿瘤。
在某些实施方式中，所述肿瘤包括胃癌、胰腺癌和/或胃食管交界处癌。
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发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1 CLDN18.2单链抗体的生成和抗原结合活性鉴定
本实施例利用慢病毒感染和流式分选获得稳定表达人CLDN18.2的细胞，即SP2/0-hCLDN18.2细胞。对Balb/c小鼠进行细胞免疫，免疫原为SP2/0-hCLDN18.2细胞，利用小鼠杂交瘤技术，流式筛选并亚克隆获得CLDN18.2阳性的单克隆细胞，体外培养获得IgG类CLDN18.2鼠源单抗。采用简并引物扩增法测序获得重链可变区(V H)和轻链可变区(V L)序列，通过人工合成的连接肽(Linker)基因将抗体重链可变区(V H)和轻链可变区(V L)连接成重组基因，由该重组基因表达的抗体为CLDN18.2单链抗体，即5E6-scFv(SEQ ID NO:38)。同时慢病毒感染和流式分选获得稳定表达人CLDN18.1的细胞SP2/0-hCLDN18.1。
采用流式细胞术的方法鉴定5E6-scFv单链抗体和Standard抗体(SEQ ID NO:143)的特异性结合活性：SP2/0-hCLDN18.2细胞计数，使用流式缓冲液重悬细胞并调整至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，一抗以80ug/ml工作浓度起始，用流式缓冲液按4倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照；4度孵育1小时，流式缓冲液清洗一遍，加入二抗山羊F(ab')2抗人IgG-Fc(
Figure PCTCN2023070968-appb-000001
650)(abcam,Cat#ab98593)，30ul/孔，4度孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。同时SP2/0-hCLDN18.1细胞计数进行铺板，一抗工作浓度从10ug/ml起始，3倍比梯度稀释，共形成5个浓度梯度，二抗为山羊F(ab')2抗人IgG-Fc(
Figure PCTCN2023070968-appb-000002
650)(abcam,Cat#ab98593)，将原始数据代入GraphPad8.0软件作图并计算，结果如图1：5E6-scFv单链抗体能特异性结合人CLDN18.2，同Standard抗体。
实施例2 CLDN18.2单链抗体与非靶细胞的结合活性鉴定
本实施例利用流式细胞荧光分选技术(FACS)，鉴定5E6-scFv单链抗体与人体组织中多 种非靶细胞的结合活性。细胞分别为人包皮成纤维细胞(HFF)、人类永生化表皮细胞(HaCaT)、人正常肝细胞(LO2)、间充质干细胞(MSC)、人正常表皮细胞(KERA)、腺癌人类肺泡基底上皮细胞(A549)、人乳腺癌细胞(MCF-7)、人皮肤成纤维细胞(BJ)。抗体从30ug/ml的工作浓度起始，3倍比梯度稀释，共形成7个浓度梯度，二抗为山羊F(ab')2抗人IgG-Fc(
Figure PCTCN2023070968-appb-000003
650)(abcam,Cat#ab98593)，孵育后离心，用流式缓冲液重悬后上机，结果代入GraphPad8.0软件作图。结果如图2，5E6-scFv单链抗体与人体组织中多种非靶细胞不存在非特异结合，同Standard抗体，初步验证其对肿瘤靶标的特异性，即不存在“脱靶效应”现象。
实施例3 CLDN18.2特异性CAR在T细胞中稳定表达
如图3A所示，本实施例中的CLDN18.2特异性CAR结构是由人CD8信号肽(SEQ ID NO:2)、抗人CLDN18.2单链抗体(SEQ ID NO:38)、人CD8铰链区(SEQ ID NO:4)、人CD8跨膜区(SEQ ID NO:6)、人4-1BB胞内共刺激域(SEQ ID NO:8)、人CD3ζ胞内激活域(SEQ ID NO:16)以及增加的Ori新元件(SEQ ID NO:18)共同组成。首先，本实施例探究了CLDN18.2特异性CAR在人T细胞中的表达情况和体外常规培养条件下的CART细胞扩增倍数。具体方法如下：
1)37℃水浴复苏液氮冻存的人PBMC细胞，用11mlPBS+1mlPBMC的体系离心重悬润洗三次(500g，5min；400g，5min；300g，5min)，润洗后的人PBMC细胞通过和CD3 MicroBeads,human(美天尼，130-050-101)混合，利用磁力架进行CD3阳选，即分离并保留CD3 +T细胞；用含有(4％FBS+X-VIVO(Lonza公司)+20ng/ml因子1+10ng/ml因子2)培养基重悬CD3 +T细胞至细胞密度为1×10 6cells/ml，按1:3的比例(细胞:磁珠)加入CD3/CD28磁珠(赛默飞40203D，磁珠用培养基清洗两次，用磁力架吸住，静置1min)对T细胞进行激活；T细胞加入磁珠中，混匀，12孔板中每孔补加培养基至700ul，细胞数为7×10 5/孔，密度1×10 6cells/ml，分选当天记为Day0。
2)激活的T细胞静置在37℃CO 2培养箱中培养20小时后，按病毒感染复数(MOI)为4的比例加入对应的病毒上清，加polybrene至终浓度为10ug/ml，吹打混匀后用水平离心机1200rpm离心1小时，将孔板放回37℃CO 2培养箱中培养24小时，感染当天记为Day1。
3)24h后，把12孔板中每孔细胞反复吹打均匀，转移至1.5mlEP管中，400g离心5分钟，去上清，并用新鲜的X-VIVO完全培养基1ml重悬细胞至细胞密度为7×10 5cells/ml，置于37℃ CO 2培养箱中培养，培养基变黄需补加培养基；每2天对细胞进行计数，并补加新鲜的X-VIVO完全培养基将细胞密度调回7×10 5cells/ml；记录统计计数结果，用GraphPad8.0 软件作图并计算常规培养下的CART细胞扩增倍数。
4)培养9-14天的CAR-T细胞检测细胞阳性率：用于感染细胞的病毒中携带Myc-tag，所以病毒感染细胞后通过流式细胞仪检测Myc阳性率从而得到CAR表达阳性率，所用直标检测抗体为Myc-Tag(9B11)Mouse mAb(Alexa
Figure PCTCN2023070968-appb-000004
488 Conjugate)(Cell Signaling,2279S)。
按照MOI＝4分别感染人CLDN18.2序列病毒，包括5E6序列和Standard序列病毒。结果如图3B，CART细胞体外培养第12天时，流式检测CAR阳性率，5E6-CART的阳性率为72.6％，Positive control CAR-T的阳性率为65.4％。本实施例共采用了6个不同的PBMC donar进行CAR阳性率验证，图3C即显示了5E6-CART在6个不同的PBMC donar中的阳性率均保持在60％左右，同Standard-CART。图3D显示了体外常规培养12天的5E6-CART细胞累积扩增倍数约240倍，与Standard-CART的扩增倍数之间无显著性差异。
实施例4 CLDN18.2特异性CAR在体外可以特异性杀伤靶细胞
本实施例首先通过慢病毒感染并流式分选获得稳定高表达人CLDN18.2的CHO-hCLDN18.2细胞，和稳定高表达人CLDN18.1的CHO-hCLDN18.1细胞。本实施例进一步使用细胞毒性检测试剂盒(Promega，Cat#G1780)，利用LDH法评估CAR-T细胞在体外的特异性杀伤能力，步骤如下：
将上述实施例3中常规培养了9天的CAR-T和Mock T细胞，分别离心后用空白的X-VIVO培养基重悬，使细胞密度为1×10 5/ml；靶细胞为CHO、CHO-hCLDN18.2和CHO-hCLDN18.1三种，将三种靶细胞分别进行消化后计数，然后用空白的X-VIVO培养基进行重悬，使细胞密度为5×10 5/ml；然后按照每孔100ul靶细胞悬液+100ulCAR-T/Mock T细胞悬液的体积体系混合加入无菌v底96孔板中。按试剂盒要求设置对照孔，获得的混合物于37℃培养箱共孵育24小时后，酶标仪记录490nM吸光值，利用试剂盒给出的公式计算靶细胞裂解百分比，Graphpad prism 8分析处理数据。结果如图4，5E6-CART在体外特异性诱导CLDN18.2阳性靶细胞的裂解，对阴性及CLDN18.1阳性细胞无裂解作用，同Standard-CART，提示5E6-CART细胞在体外具有人CLDN18.2高特异性靶细胞杀伤活性。
实施例5 CLDN18.2特异性CAR的因子分泌情况
本实施例分别采用Human IFN-γELISA试剂盒(R&D，DY285B)和Human IL-2 ELISA试剂盒(R&D，DY202)分析CAR-T在杀伤靶细胞的过程中对IFN-γ和IL-2的分泌量。具体如下，按照1×10 4个细胞每孔的细胞量将高表达CLDN18.2的靶细胞(CHO-hCLDN18.2)、高表达CLDN18.1的对照细胞(CHO-hCLDN18.1)和阴性细胞CHO分别接种于无菌的96孔板，分别向 靶细胞中按效应细胞(Effector)：靶细胞(Target)＝5:1的比例加入CAR-T细胞(5E6-CART和Standard-CART)和未加修饰的T细胞(Mock T cell)等效应细胞。孵育24h后，按照试剂盒说明书进行操作，取上清用酶联免疫法(ELISA)检测IL-2和IFN-γ含量。结果如图5A和5B所示，5E6-CART与CLDN18.2阳性细胞共孵育时分泌较高的因子水平，与阴性及CLDN18.1阳性细胞共孵育下无显著因子分泌，同Standard-CART。因此5E6-CART细胞对高表达CLDN18.2的肿瘤细胞具有特异细胞因子诱导的作用。
实施例6 CLDN18.2特异性CAR-T细胞在小鼠结肠癌CDX模型中的抑瘤作用
本实施例首先将人CLDN18.2基因通过慢病毒的方式导入MC38细胞中，再通过流式分选仪分选出高表达人CLDN18.2的小鼠结肠癌细胞，即MC38-hCLDN18.2细胞。利用雌性B-NDG重度免疫小鼠，通过皮下注射途径构建小鼠结肠癌肿瘤模型，验证CLDN18.2特异性CAR-T细胞在小鼠体内的抑瘤作用。按照1.5×10 6/只的剂量接种MC38-hCLDN18.2细胞，小鼠接瘤后8天，选取肿瘤大小41.68-120.6mm 3(平均肿瘤大小80.01mm 3)的小鼠随机分为3组，每组8只，尾静脉回输已培养10天的CAR-T(或Mock T)细胞，其中5E6-CART和Standard-CART组的CAR-T回输剂量为3*10 6细胞/只小鼠，空白对照Mock T细胞组的回输剂量为1*107细胞/只小鼠。每周测量小鼠瘤体大小和体重3次，观察4周，如图6A。小鼠尾静脉回输CAR-T(或Mock T)细胞后，5E6-CART组小鼠在D13体重减重10％以上，D15时单只小鼠体重相对变化为-34.51％，D13出现第一只小鼠的死亡；Standard-CART组小鼠在D16体重减重10％以上，D16时单只小鼠体重相对变化为-36.93％，D18出现第一只小鼠的死亡，由于小鼠体重下降严重，所以PG-D22即结束实验，结束实验时暂未观察到明显的抑瘤作用，见图6C(终点定义为小鼠肿瘤体积＞3000mm 3)。对小鼠安乐死后进行解剖，发现5E6-CART组和Standard-CART组的小鼠均出现胃脏受损出血的异常现象，如图6D。综上所述，5E6-CART组小鼠在此肿瘤模型中表现为不耐受问题及明显副作用，具体表现为小鼠体重下降、背毛竖立、蜷缩不动且胃部出血等；Mock T组小鼠表现耐受，小鼠体重等正常。
总结来说，5E6-scFv是通过小鼠杂交瘤技术筛选鉴定获得的单链抗体。5E6-scFv抗体可以特异性结合人CLDN18.2阳性细胞，具有CLDN18.2高特异性。5E6-scFv单链抗体与人体组织中多种非靶细胞不存在非特异结合。5E6-CART为二代CART结构，可以在T细胞中稳定表达和增值。5E6-CART在体外具有CLDN18.2高特异性细胞杀伤活性，同时可以特异性诱导细胞因子分泌。5E6-CART在荷瘤小鼠药效实验中表现出一定毒性，表现为小鼠体重下降和胃部出血，一定程度上说明了5E6-CART在小鼠体内安全性欠佳。所以本实施例通过进行调研，挖掘了新的肿瘤靶点进行组合，设计了新的CAR分子结构，在保留5E6有效性的同 时，提高其体内安全性，并命名为OriCAR-388。
实施例7 OriCAR-388慢病毒载体的构建和病毒包装
肿瘤靶点MSLN高表达于50-55％的胃癌患者和80-85％的胰腺癌患者中，可做为胃癌靶点进行研究。在人体正常组织中，靶点MSLN高表达于气管、输卵管、胚盘、扁桃体，中表达于鼻咽、子宫内膜，低表达于口腔粘膜、子宫和皮肤，正常肺、胃、肝脏、胰腺、胆囊不表达，与CLDN18.2在人体正常组织中的表达分布基本不重叠，是理想的dual CAR-T靶点选择。为了提高5E6-CART的安全性，我们设计了dual CAR-T的分子结构：把人CD8信号肽(SEQ ID NO:2)、实施例1中的抗CLDN18.2的scFv抗原结合域(5E6-scFv，SEQ ID NO:38)、人CD8铰链区(SEQ ID NO:4)、人CD8跨膜区(SEQ ID NO:6)、人4-1BB胞内共刺激结构域(SEQ ID NO:8)以及FurinT2A自切割肽相连构成片段1；把人CD8信号肽(SEQ ID NO:2)、抗人MSLN的VHH抗原结合域(SEQ ID NO:103)、人CD28铰链区(SEQ ID NO:12)、人CD28跨膜区(SEQ ID NO:14)、人CD3ζ胞内信号转导域(SEQ ID NO:16)相连构成片段2，Ori元件命名为片段3，然后进行“片段1+片段1+片段2+片段3”的组合进行相连，结构示意图如图7A。连接后的片段通过同源重组等构建到本公司提供的pCore四质粒慢病毒表达载体上，构建完成的载体经测序无误后进行质粒中抽，中抽后的质粒进行滤膜过滤除菌并测浓度，载体图谱见图7B。
本实施例采用四质粒系统对慢病毒表达载体进行慢病毒包装，具体步骤如下：1)将慢病毒表达载体质粒、辅助质粒PRH1/PMH2/PVH3构成的四质粒系统与FuGENE HD转染试剂(Promega公司，FuGENE HD：质粒＝3：1)混合后，加至一定体积的opti-MEM培养基(Gibco)中，混匀静置15min；2)将上述混合液一滴一滴均匀地加入到铺有293T细胞的6孔板中，轻轻混匀，立即放在CO2培养箱中37℃培养18小时；3)18小时后更换新鲜培养基，轻轻一滴一滴地平缓着滴入1.5ml的含5％FBS的DMEM培养基(提前30min 37℃预热)，过夜继续培养24小时，24h后收集慢病毒培养上清进行病毒滴度检测；4)取10ul的病毒上清去感染293T细胞，在37℃，5％CO 2培养箱中静置培养72小时，72小时后取适量的经病毒感染后的293T细胞，采用流式细胞术的方法进行病毒滴度检测，所用检测抗体为myc-tag(9B11)mouse mAb(Alexa R 647)(Cell signaling,2233S)，病毒滴度计算公式为：滴度＝感染细胞数*阳性率/感染病毒体积(ml)。
实施例8慢病毒转染T细胞、T细胞中CAR的稳定表达及CAR-T细胞稳定增殖
按照上述实施例3所示方法进行PBMC复苏、阳选和激活，得到激活后的CD3阳性的T 细胞；按病毒感染复数(MOI)为3的比例加入对应体积的病毒上清，加polybrene至终浓度为10ug/ml(1：1000)，吹打混匀后用水平离心机1200rpm离心1小时，然后将孔板放回37℃CO 2培养箱中培养18-24小时，病毒感染当天记为Day1；第二天去病毒，更换新鲜的培养基，然后放入37℃CO 2培养箱中继续培养，培养基变黄即补加完全的X-VIVO培养基，培养基配制同实施例3所示；CAR-T细胞常规培养至Day8和Day10时分别测CAR阳性率，检测抗体为myc-tag(9B11)mouse mAb(Alexa R 647)(Cell signaling,2233S)，同时设置Mock T作为阴性对照，结果如图8A，OriCAR-388能在T细胞中稳定表达，CAR表达率维持在49％左右。CAR-T细胞常规培养的Day5、Day7和Day11天分别对CAR-T细胞及Mock T细胞进行计数，统计后用Graphpad prism8.0作图软件进行作图分析，结果如图8B，OriCAR-388 T细胞可以在体外稳定增殖，体外培养11天的累积扩增倍数在50倍左右。
实施例9荧光素酶法测定OriCAR-388 T细胞在体外的杀伤
按照实施例8所示方法进行CAR-T及空白T细胞的常规培养，将体外常规培养了12天的CAR-T细胞(OriCAR-388 T细胞和5E6-CART细胞)以及未经转染的空白T细胞(Mock T)进行离心，离心后用空白的X-VIXO培养基(Lonza)进行重悬，使细胞密度为1.33*10 5/ml。CHO-NFkB-hCLDN18.2为在上述实施例4构建基础上通过分子构建引入了NFkB荧光素酶报告基因，再经过亚克隆后得到的同时高表达人CLDN18.2和荧光素酶的细胞，做为CLDN18.2单靶点靶细胞进行研究；NUGC4细胞为人胃癌细胞，在体外常规培养条件下天然表达人CLDN18.2和人MSLN，通过引入NFkB荧光素酶报告基因构建获得了NUGC4-NFkB细胞，做为双靶点阳性的靶细胞进行研究；OVCAR3-NFkB为人卵巢癌细胞，在体外常规培养条件下天然高表达人MSLN蛋白，通过引入NFkB荧光素酶报告基因构建获得了OVCAR3-NFkB细胞，做为MSLN单靶点阳性的靶细胞进行研究。选择上述三种靶细胞，分别消化计数，用空白的X-VIXO培养基(Lonza)进行重悬，使细胞密度为1.33*10 5/ml；按照“75ul靶细胞悬液+75ul CAR-T/Mock T细胞悬液”的体积体系加入白底不透光的无菌96板中(Nunclon Delta Surface CAT:136101)，即效靶比为E:T＝1:1，同时设置靶细胞单独孵育孔，即“75ul靶细胞悬液+75ul空白X-VIVO培养基”，将96孔板放置在37℃CO 2培养箱中培养18h-24h；然后按照ONE Glo Luciferase Assay System试剂盒的操作要求(promega，E6120)加入等体积的荧光素酶反应底物，室温避光，5min内用TECAN多功能酶标仪Spark(瑞士TECAN)检测96孔板细胞中的荧光素酶含量。根据以下公式计算杀伤效率：杀伤率％＝(1-RLU实验孔/RLU靶细胞单独孔)×100％。
结果如图9，对CHO-NFkB-hCLDN18.2细胞，OriCAR-388 T细胞不存在杀伤活性，5E6- CART细胞存在强杀伤活性；对OVCAR3-NFkB细胞，由于高表达MSLN蛋白，OriCAR-388T细胞存在强杀伤活性，5E6-CART细胞不存在杀伤活性；对NUGC4-NFkB细胞，同时表达MSLN蛋白和CLDN18.2蛋白，OriCAR-388 T细胞存在强杀伤活性，5E6-CART细胞存在中杀伤活性。
实施例10 ELISA法测定OriCAR-388 T细胞分泌细胞因子的情况
本实施例选择CHO-hCLDN18.2为人CLDN18.2单阳靶细胞，OVCAR-3为人MSLN单阳靶细胞，NUGC-4为MSLN/CLDN18.2双阳靶细胞，效应细胞为体外培养了8天的CAR-T细胞和Mock T cell。将效应细胞离心去上清，用空白X-VIVO稀释至2*10 5/ml，每孔取100ul使用。将无靶细胞孔(即不与靶细胞共培养的CAR-T细胞)及与靶细胞按效靶比为1：1的比例共孵育孔分别接种在平底96孔板中，共孵育20小时后，吸取上清，用R&D Cytokine Kit检测上清液中IL-2和IFN-γ的含量(R&D，DY285B和R&D，DY202)。
结果如图10所示，MSLN-CART只有在和OVCAR-3或NUGC-4细胞共孵育时才存在因子分泌，分泌水平较低；5E6-CART细胞在和CHO-hCLDN18.2或NUGC-4细胞共孵育下均分泌大量的IFN-r和IL-2细胞因子；而OriCAR-388 T细胞在和CHO-hCLDN18.2共孵育基本无IFN-r和IL-2细胞因子分泌，与CAR-T细胞单孵育孔因子分泌水平无差异；OriCAR-388T细胞在和OVCAR-3共孵育下可以分泌少量的IFN-r和IL-2细胞因子，IL-2分泌水平显著高于CAR-T细胞单孵育孔因子分泌水平；而OriCAR-388 T在和NUGC-4共孵育下，其IL-2和IFN-γ因子分泌水平即被极显著提高(p＜0.0001)。这也极好的验证了dual CAR-T结构的设计理念，即：MSLN-VHH直接连接CD3ζ胞内信号转导域，可以单独转导胞外MSLN抗原结合域的信号，激活细胞因子的分泌；CLDN18.2-scFv直接连接4-1BB共刺激结构域，无法单独完成胞外CLDN18.2抗原结合域的信号的转导，进而无法激活细胞因子的分泌；而通过自裂解多肽T2A把上述两个元件并联，理论上只有在CLDN18.2和MSLN抗原同时存在情况下，CD3ζ才会特异性地同时转导双靶点信号，同时4-1BB结构域刺激更多细胞因子的分泌。
实施例11 OriCAR-388 T细胞在人胃癌CDX模型中的抑瘤作用
NUGC-4细胞为人胃癌细胞，在体外常规培养条件下天然共表达人CLDN18.2和人MSLN蛋白，可以做为双靶点共表达的靶细胞进行OriCAR-388 T细胞体内药效的研究。具体来说，所用小鼠品系为重度免疫缺陷B-NDG小鼠，小鼠笼内饲养适应一周后对所有小鼠进行皮下接瘤(NUGC-4细胞，3*10 6细胞/只小鼠)，小鼠接瘤后45天，选取平均肿瘤大小976mm 3的 小鼠随机分为3组，每组5只，尾静脉回输已培养11天的OriCAR-388 T细胞、5E6-CART细胞和Mock T细胞(回输当天记为D0)，如图11A，其中Mock T细胞按照1*10 7细胞/只小鼠的剂量进行回输(标记为G1组：Mock T cell，1*10 7)，OriCAR-388 T细胞分别进行高剂量回输和低剂量回输，即1*10 7细胞/只小鼠(标记为G2组：OriCAR-388 T，1*10 7)和3*10 6细胞/只小鼠(标记为G3组：OriCAR-388 T，3*10 6)。每周测量小鼠瘤体大小和体重3次，总共观察了约4周时间，即33天后小鼠安乐死结束实验。结果如图11B，5E6-CART组小鼠在回输CART后的第8天开始出现小鼠体重下降现象，而Mock T cell组、OriCAR-388 T高剂量组以及OriCAR-388 T低剂量组在整个实验观察期间(观察至回输CAR-T后33天)，小鼠体重未见异常，小鼠毛色光亮，小鼠行为、饮食进水以及大小便颜色均正常。相比Mock T cell组，OriCAR-388 T细胞高、低剂量组均表现出极显著的抑瘤活性(p＜0.0001)，如图11C(终点定义为小鼠肿瘤体积＞3000mm 3)，回输CART后33天，OriCAR-388 T细胞高剂量组小鼠平均肿瘤体积下降为273.10mm 3，OriCAR-388 T细胞低剂量组小鼠平均肿瘤体积下降为310.44mm 3大小。证实OriCAR-388 T细胞在小鼠体内具有一定消瘤作用以及较好的安全性。
总结来说，OriCAR-388能在T细胞中稳定表达和增值，CAR表达率维持在49％左右，体外常规培养11天的CART细胞扩增倍数约50倍。OriCAR-388 T细胞只针对NUGC-4双靶点阳性的细胞有强杀伤活性，针对CLDN18.2单阳的靶细胞或MSLN单阳的靶细胞不存在或存在低杀伤活性。在和CHO-hCLDN18.2细胞共孵育下基本不分泌IFN-r和IL-2细胞因子，在和OVCAR-3共孵育下会分泌少量的IFN-r和IL-2细胞因子，而OriCAR-388 T细胞在和NUGC-4细胞共孵育下，其IL-2和IFN-γ因子分泌水平即被极显著提高。免疫缺陷小鼠皮下接瘤NUGC-4细胞，待肿瘤平均大小为976mm 3时进行CAR-T细胞回输，目的是用这个超大肿瘤模型来模拟体内肿瘤高负荷情况下，CAR-T充分反复激活下的可能会出现的毒副作用。实验观察到OriCAR-388 T高低剂量组在整个实验观察期间小鼠体重未见异常，小鼠毛色光亮，小鼠行为、饮食进水以及大小便颜色均正常；OriCAR-388 T细胞高低剂量组均表现出极显著的抑瘤活性，回输CART后33天，OriCAR-388 T细胞高剂量组小鼠平均肿瘤体积下降为273.10mm 3，OriCAR-388 T细胞低剂量组小鼠平均肿瘤体积下降为310.44mm 3大小，证实OriCAR-388 T细胞在小鼠体内具有一定抑瘤作用以及较好的安全性。而针对Claudin18.2单靶点的5E6-CART在荷瘤小鼠药效实验中表现出严重毒性，表现为小鼠体重急剧下降、胃部出血。
实施例12 OriCAR-388 T细胞在小鼠体内的毒理实验
为了进一步验证OriCAR-388 T的有效性和安全性，本实施例进行了荷瘤小鼠模型中的单 次给药毒性试验，即小鼠毒理实验，具体如下：12只雌性B-NDG小鼠皮下接瘤NUGC-4细胞，待肿瘤体积达到270mm 3左右时进行CAR-T细胞回输，平均分为4组，回输剂量分别为：Mock T,5*10 6/只和OriCAR-388 T,5*10 6/只，回输后持续观察至PG-D29，具体分组方案见表1。小鼠的体重见图12A，其中G3/G6/G8/G10四组由于在尾静脉回输CAR-T或Mock T细胞后的15天(PG-D15)时对所有小鼠进行安乐死后大体解剖取脏器，所以小鼠体重只持续观察至PG-D15，观察期间小鼠体重保持正常，不存在体重大幅度下降的现象；G4/G7/G9/G11四组小鼠在PG-D29时对所有小鼠进行安乐死后大体解剖取脏器，观察期间小鼠体重同样保持正常，不存在体重大幅度下降的现象。当小鼠平均肿瘤体积达到275mm 3时对小鼠进行分组并于当天回输CAR-T/Mock T细胞，观察期间小鼠肿瘤生长的数据结果分析如图12B，所有OriCAR-388回输组小鼠在肿瘤监测结束时的肿瘤体积和同时间测量的Mock T组小鼠肿瘤体积相比，均存在极显著差异(p＜0.0001)，具有显著的抑瘤活性。在PG-D15和PG-D29时分别对小鼠进行血生化指标、白细胞分类计数指标、全血细胞和网织红细胞计数指标进行检测，对检测结果进行统计分析，小鼠血生化检测指标见图12C，D15取样结果的所有检测指标中，OriCAR-388组的ALT/AST水平高于Mock T组水平，但无统计学差异(p＞0.05)，D29取样结果的所有检测指标中，OriCAR-388组的ALT/AST水平依然高于Mock T组水平，同样无统计学差异(p＞0.05)，CK水平表现为OriCAR-388组显著低于Mock T组(p＜0.01)，但此显著性差异不存在临床学意义；小鼠血液中白细胞分类计数指标见图12D，D15取样结果中的LYMP(淋巴细胞)细胞计数表现为OriCAR-388组显著高于Mock T组(p＜0.01)，D29取样检测时的OriCAR-388组的LYMP(淋巴细胞)细胞计数水平依然高于Mock T组，但是有所回缓，已经不具有统计学差异(p＞0.05)；小鼠全血细胞和网织红细胞计数指标检测结果如图12E，D15取样时OriCAR-388组的PLT水平极显著低于Mock T组(p＜0.0001)，而D29取样时OriCAR-388组的PLT水平相对于Mock T组依然存在显著性差异，但差异性在慢慢缩小(p＜0.05)。本实施例进一步证实了OriCAR-388 T细胞在小鼠体内的有效性和安全性。
表1
Figure PCTCN2023070968-appb-000005
Figure PCTCN2023070968-appb-000006
实施例13 T细胞中的CAR表达水平和CART细胞增值情况比较分析
本实施例同时构建了OriCAR-388、OriCAR-387和OriCAR-386三个不同结构的MSLN/CLDN18.2双靶点CAR，它们在CAR-T载体结构上的差异见图13A，OriCAR-386相对于OriCAR-388去除了一段double序列，即“CD8ss+CLDN18.2-scFv+CD8hinge+CD8TM+41BB+FurinT2A”的序列，OriCAR-387相较OriCAR-386，将MSLN3-VHH序列替换使用了另外一个名为MSLN52-VHH的人源化序列，其它序列及结构保持一致。采用如实施例7和实施例8中的操作方法进行病毒包装、病毒滴度检测、CAR-T细胞制备等，制备得到了5E6-CART(CLDN18.2)、MSLN3-CART、MSLN52-CART、OriCAR-388、OriCAR-387和OriCAR-386以及Mock T细胞，分别在CAR-T细胞培养的D1/D5/D7/D9/D13天对细胞进行计数并统计分析，结果如图13B，CAR-T细胞培养的第13天，OriCAR-388 T细胞的累积扩增倍数约125倍，OriCAR-387 T细胞的累积扩增倍数约189倍，OriCAR-386 T细胞的累积扩增倍数约185倍，Mock T细胞的累积扩增倍数约155倍，OriCAR-387同OriCAR-386，好于OriCAR-388，和Mock T细胞相比无显著差异。分别在CAR-T细胞培养的D7、D9和D12对CAR阳性率进行流式检测，所用检测抗体为myc-tag(9B11)mouse mAb(Alexa Fluor 647)(Cell signaling,2233S)，结果如图13C，OriCAR-386、OriCAR-387和OriCAR-388均能在T细胞中稳定表达，OriCAR-388CAR阳性率维持在40％左右，OriCAR-386同OriCAR-387，其CAR阳性率维持在55％左右，高于OriCAR-388CAR阳性率。即体外常规培养条件下，双靶点OriCAR-386和OriCAR-387 T细胞的常规扩增倍数高于OriCAR-388 T细胞的常规扩增倍数；CAR阳性率上表现为5E6-CAR、MSLN3-CAR、MSLN52-CAR阳性率高于OriCAR-386和OriCAR-387阳性率，OriCAR-386和OriCAR-387阳性率高于OriCAR-388阳性率。
实施例14体外靶细胞反复刺激下，CAR-T细胞的定向增值情况比较分析
本实施例中选取实施例13中体外扩增培养12天的5E6-CART、MSLN3-CART、MSLN52-CART、OriCAR-388、OriCAR-387和OriCAR-386细胞，流式的方法检测阳性率后，用空白 T细胞调整感染CAR-T细胞比例一致，使各组总细胞数一致。具体为，靶细胞为共表达人CLDN18.2和人MSLN蛋白的人胃癌细胞NUGC-4，NUGC-4进行紫外辐照10min，靶细胞的数量为1*10 5/孔，第一轮刺激效靶比是1：1，第二轮刺激效靶比是1:2，第三轮刺激效靶比是1:2；设置好不同的对照孔，再将靶细胞和效应细胞混合离心，用X-VIVO空白培养基重悬铺于12孔板中；37度培养箱培养3-4天左右，计数并统计分析。结果如图14，双靶点CART细胞进行比较，三轮反复刺激后，OriCAR-388增殖效果最好，靶细胞辐照后三轮靶向刺激T细胞共累计增殖1996倍；其次是OriCAR-387 T细胞，靶细胞辐照后三轮靶向刺激T细胞共累计增殖1211倍；OriCAR-386 T细胞相对较差，在三轮靶向刺激后T细胞共累计增殖约299倍。对于单靶点的CART细胞，与辐照后的靶细胞共孵育三轮后同样能够增殖，但增殖效果很差，5E6-CART细胞11天累计扩增约40倍，MSLN3-CART细胞三轮靶向刺激累计扩增约110倍，MSLN52-CART细胞三轮靶向刺激累计扩增约45倍。即在体外靶细胞反复刺激条件下，OriCAR-388 T细胞的持续可扩增性强于OriCAR-387 T细胞，显著强于OriCAR-386 T细胞，而单靶点5E6-CART、MSLN3-CART和MSLN52-CART细胞仅有少量的靶向增值。该结果反映了CAR-T注射体内后遇到肿瘤负荷较大时，CAR-T受到靶抗原反复刺激后的持续可扩增性，与CAR-T抑制肿瘤生长的疗效密切相关，说明OriCAR-388 T、OriCAR-387 T和OriCAR-386 T所具有的独特的CAR-T结构提高CAR-T细胞可持续扩增的有效性。
实施例15 CAR-T细胞的体外杀伤情况比较分析
本实施例采用荧光素酶的方法检测5E6-CART、MSLN3-CART、MSLN52-CART、OriCAR-388、OriCAR-387、OriCAR-386以及Mock T细胞在体外对NUGC4-NFkB细胞的杀伤作用，选择上述实施例13中体外常规培养了11天的CAR-T或Mock T细胞做为效应细胞，具体检测方法及所用试剂盒同实施例9，设置三个不同的效靶比，即E:T＝1:1，3:1，9:1，共孵育20h后进行检测，对检测结果进行统计分析。结果如图15，所有CAR-T细胞均存在杀伤活性，在低效靶比1：1下，双靶点OriCAR-388、OriCAR-387和OriCAR-386 T细胞的杀伤活性均明显强于单靶点5E6-CART、MSLN3-CART和MSLN52-CART细胞对靶细胞的杀伤活性，其中OriCAR-388的杀伤活性最强；在3：1和9：1的效靶比下，所有CAR-T序列均检测到约90％的杀伤百分比，但序列之间已经显示不出差异。说明双靶点OriCAR-388 T、OriCAR-387 T和OriCAR-386 T所具有的独特的CAR-T结构提高CAR-T细胞对肿瘤特异性杀伤的有效性。
实施例16 CAR-T细胞的体外细胞因子分泌情况比较分析
本实施例选择上述实施例13中体外常规培养了11天的CAR-T或Mock T细胞做为效应细胞，选择CHO-hCLDN18.2、NUGC-4和OVCAR3细胞做为靶细胞，采用如实施例10所描述的ELISA方法检测共孵育20h后的细胞因子分泌情况，具体检测方法及所用试剂盒同实施例10，设置E:T＝1:1的效靶比共孵育20h后进行检测，对检测结果进行统计分析。结果如图16，与CHO-hCLDN18.2细胞共孵育时，只有5E6-CART细胞存在IFN-r和IL-2因子分泌；与OVCAR3细胞共孵育时，除了5E6-CART和Mock T，其它5种CART细胞均存在低水平的IFN-r和IL-2因子分泌，其中MSLN52-CART细胞因子分泌水平高于MSLN3-CART细胞对应水平，OriCAR-387的因子分泌水平高于OriCAR-388、OriCAR-386对应水平；当与NUGC-4细胞共孵育时，OriCAR-388、OriCAR-387和OriCAR-386的因子分泌水平即被显著提高，且OriCAR-387在其中表现最优，优于MSLN3-CART和MSLN52-CART细胞因子分泌。
实施例17 CAR-T细胞在人胃癌小鼠CDX肿瘤模型中的抑瘤作用比较分析
本实施例对三种不同结构的MSLN/CLDN18.2 dual CAR-T细胞的小鼠体内有效性和安全性做了验证，具体如下：体外培养同时表达MSLN和CLDN18.2抗原的靶细胞NUGC-4，按照3*10 6/只小鼠的剂量进行皮下荷瘤，荷瘤小鼠为重度免疫缺陷B-NDG小鼠，荷瘤后按照每周三次的频率测定小鼠体重和肿瘤体积；当所有小鼠肿瘤体积达到平均143mm 3时，进行小鼠分组，分为7组(组别见图17B、17C和17D)，每组5只小鼠，分组当天即进行尾静脉回输CART/Mock T细胞，回输当日记为PG-Day0，每周测定三次小鼠体重和肿瘤体积，持续观察至尾静脉回输后的46天，即PG-Day46，如图17A。在整个观察期间，除了Group2：OriCAR-386,3*10 6/只组中有一只小鼠体重下降率超过15％，于PG-D41进行安乐死，其它所有入组小鼠在实验期间其体重和生理状态均保持正常，如图17B所示。在实验结束的终点，OriCAR-386/OriCAR-387/OriCAR-388 T细胞相对于Mock T细胞在人胃癌细胞小鼠移植模型中都表现出极为显著的抑瘤有效性，且抑瘤活性表现为OriCAR-387优于OriCAR-388优于OriCAR-386，具体如图17C(终点定义为小鼠肿瘤体积＞3000mm 3)所示：OriCAR-386/OriCAR-387/OriCAR-388 T细胞高剂量组(3*10 6/只)小鼠相对于Mock T细胞组(3*10 6/只)小鼠，均具有极显著的抑瘤活性，统计学差异为P＜0.0001，其中OriCAR-386 T细胞高剂量组有3 只小鼠达到消瘤(3/5)，OriCAR-387 T细胞高剂量组有5只小鼠达到消瘤(5/5)，OriCAR-388 T细胞高剂量组有2只小鼠达到消瘤(2/5)；相对于Mock T细胞组，OriCAR-386低剂量组(1*10 6/只)无显著抑瘤活性，不存在统计学差异，OriCAR-387低剂量组(1*10 6/只)具有显 著抑瘤活性(P＜0.01)，OriCAR-388低剂量组(1*10 6/只)无显著抑瘤活性，不存在统计学差异，其中OriCAR-386低剂量组无小鼠消瘤(0/5)，OriCAR-387低剂量组有2只小鼠消瘤(2/5)，OriCAR-388低剂量组有1只小鼠消瘤(1/5)。小鼠生存曲线见图17D，截至实验终点(肿瘤体积＞3000mm 3)，OriCAR-386/OriCAR-387/OriCAR-388 T细胞高剂量组(3*10 6/只)小鼠均可以达到100％的生存率，统计结果表明，Mock T(3*10 6/只)组小鼠的中位生存期为26天，OriCAR-386(1*10 6/只)组小鼠的中位生存期为33天,OriCAR-387(1*10 6/只)组小鼠的中位生存期为43天,OriCAR-388(1*10 6/只)组小鼠的中位生存期为43天。总的来说，人胃癌小鼠CDX肿瘤模型中，OriCAR-387 T细胞的安全性和抑瘤有效性优于OriCAR-388 T细胞，优于OriCAR-386 T细胞。
实施例18 CLDN18.2靶向部分与CLDN18.2特异性结合活性测定
通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液检测上述嵌合抗体与靶细胞的特异性结合活性，选择三种靶细胞：表达人CLDN18.2的稳转细胞株、表达人CLDN18.1的稳转细胞株以及肿瘤细胞系，分别进行结合活性的测定。
1.本申请的CLDN18.2抗原结合蛋白与高表达人CLDN18.2细胞的流式结合活性检测
构建获得稳定高表达CLDN18的细胞株分别标记为293T-人CLDN18.2、CHO-人CLDN18.2和SP2/0-人CLDN18.2细胞，细胞消化计数，使用流式缓冲液重悬细胞并调整至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，抗体以30ug/ml浓度起始，用流式缓冲液按两或三倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照，阳性对照抗体为实施例1中纯化获得的zolbetuximab；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗(abcam,Cat#ab98593)，30ul/孔，4℃孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。将原始数据代入GraphPad8.0软件作图并计算，结果如图18。
2.本申请的CLDN18.2抗原结合蛋白与高表达人CLDN18.1细胞的流式结合活性检测
阳性对照抗体为市售抗CLDN18抗体(Anti-Claudin18 antibody)(abcam,Cat#ab203563)，其抗原结合位点位于CLDN18.2四次胯膜蛋白的胞内部分，需要进行流式胞内染色分析。具体地，细胞为构建的293T-人CLDN18.1和SP2/0-人CLDN18.1细胞，细胞消化计数后进行固定破膜处理，处理后的细胞用流式缓冲液重悬至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，抗体以30ug/ml浓度起始，用流式缓冲液按三倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照，阳性对照抗体稀释条件同上；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗(abcam,Cat#ab98593和Cat#ab150079)，30ul/孔，4℃孵育30分钟；流式 缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。将原始数据代入GraphPad8.0软件作图并计算，结果如图19。
3.本申请的CLDN18.2抗原结合蛋白与肿瘤细胞系的流式结合活性检测
选择构建获得的稳定高表达人CLDN18.2的肿瘤细胞MC38-人CLDN18.2为靶细胞，细胞消化计数，使用流式缓冲液重悬细胞并调整至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，抗体以30ug/ml浓度起始，用流式缓冲液按三倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照，阳性对照抗体为纯化获得的zolbetuximab，稀释条件同上；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗(abcam,Cat#ab98593)，30ul/孔，4℃孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。将原始数据代入GraphPad8.0软件作图并计算，结果如图20。
通过以上操作步骤，表达纯化获得5个CLDN18.2抗原结合蛋白(其中，5E6的VH序列如SEQ ID NO:26所示，VL序列如SEQ ID NO:34所示；7E3的VH序列如SEQ ID NO:75所示，VL序列如SEQ ID NO:49所示；3A6的VH序列如SEQ ID NO:61所示，VL序列如SEQ ID NO:65所示；14E12的VH序列如SEQ ID NO:79所示，VL序列如SEQ ID NO:87所示，17B10的VH序列如SEQ ID NO:79所示，VL序列如SEQ ID NO:93所示)，流式验证其抗原结合活性：如图18所示，5个抗原结合蛋白均呈现和人CLDN18.2浓度依赖性结合活性，且大部分强于zolbetuximab阳性抗体，3种细胞的检测结果保持一致；如图19所示，5个抗原结合蛋白均能特异性与人CLDN18.2结合而不与人CLDN18.1结合；如图20所示，5个抗原结合蛋白均与稳定高表达人CLDN18.2的小鼠结肠癌MC38细胞强结合，结合活性强度呈浓度依赖性，效果与zolbetuximab阳性抗体相当或强于zolbetuximab(重链序列如SEQ ID NO:157所示，轻链序列如SEQ ID NO:158所示)。
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 (180)

  1. 细胞，其包含和/或表达靶向紧密连接蛋白18.2(CLDN18.2)的嵌合抗原受体，以及靶向间皮素(MSLN)蛋白的嵌合抗原受体。
  2. 根据权利要求1所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体包含靶向CLDN18.2的抗原结合结构域。
  3. 根据权利要求2所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:147所示的氨基酸序列。
  4. 根据权利要求2-3中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:144所示的氨基酸序列。
  5. 根据权利要求2-4中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:24、SEQ ID NO:59和SEQ ID NO:73中任一项所示的氨基酸序列。
  6. 根据权利要求2-5中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:145所示的氨基酸序列。
  7. 根据权利要求2-6中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:22、SEQ ID NO:57和SEQ ID NO:71中任一项所示的氨基酸序列。
  8. 根据权利要求2-7中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:146(X 1YX 2X 3X 4，其中，X 1为N或R，X 2为G、I或V，X 3为I或M，X 4为H、N或S)所示的氨基酸序列。
  9. 根据权利要求2-8中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:20、SEQ ID NO:55和SEQ ID NO:69中任一项所示的氨基酸序列。
  10. 根据权利要求2-9中任一项所述的细胞，其中所述靶向CLDN18.2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氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:24所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO:55所示的氨基酸序列，所示HCDR2包含SEQ ID NO:57所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:59所示的氨基酸序列；以及
    (3)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:73所示的氨基酸序列。
  11. 根据权利要求2-10中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含VH，所述VH包含SEQ ID NO:147所示的氨基酸序列。
  12. 根据权利要求2-11中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含VH，所述VH包含SEQ ID NO:26、SEQ ID NO:43、SEQ ID NO:61、SEQ ID NO:75和SEQ ID NO:79中任一项所示的氨基酸序列。
  13. 根据权利要求2-12中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含轻链可变区VL中的至少一个CDR，且所述VL包含SEQ ID NO:151所示的氨基酸序列。
  14. 根据权利要求2-13中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:148所示的氨基酸序列。
  15. 根据权利要求2-14中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:32、SEQ ID NO:63、SEQ ID NO:85和SEQ ID NO:91中任一项所示的氨基酸序列。
  16. 根据权利要求2-15中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:149所示的氨基酸序列。
  17. 根据权利要求2-16中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:30或SEQ ID NO:83所示的氨基酸序列。
  18. 根据权利要求2-17中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:150所示的氨基酸序列。
  19. 根据权利要求2-18中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:28、SEQ ID NO:45和SEQ ID NO:81中任一项所示的氨基酸。
  20. 根据权利要求2-19中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1，LCDR2和LCDR3包含选自下述任一组氨基酸序列：
    (1)所述LCDR1包含SEQ ID NO:28所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
    (2)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
    (3)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:63所示的氨基酸序列；
    (4)所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:91所示的氨基酸序列；以及
    (5)所述LCDR1包含SEQ ID NO:81所示的氨基酸序列，所述LCDR2包含SEQ ID NO:83所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:85所示的氨基酸序列。
  21. 根据权利要求2-20中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:151所示的氨基酸序列。
  22. 根据权利要求2-21中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:34、SEQ ID NO:49、SEQ ID NO:65、SEQ ID NO:87和SEQ ID NO:93中任一项所示的氨基酸序列。
  23. 根据权利要求2-22中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3，所述HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3包含选自下述任一组氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，所述HCDR3包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1包含SEQ ID NO:28所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所示HCDR2包含SEQ ID NO:22所示的氨基酸序列，所述HCDR3包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
    (3)所述HCDR1包含SEQ ID NO:55所示的氨基酸序列，所示HCDR2包含SEQ ID  NO:57所示的氨基酸序列，所述HCDR3包含SEQ ID NO:59所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:63所示的氨基酸序列；
    (4)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；
    (5)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列，所述LCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:91所示的氨基酸序列；以及
    (6)所述HCDR1包含SEQ ID NO:69所示的氨基酸序列，所示HCDR2包含SEQ ID NO:71所示的氨基酸序列，所述HCDR3包含SEQ ID NO:73所示的氨基酸序列，所述LCDR1包含SEQ ID NO:81所示的氨基酸序列，所述LCDR2包含SEQ ID NO:83所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:85所示的氨基酸序列。
  24. 根据权利要求2-23中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含VH和VL，所述VH和VL包含选自下述任一组氨基酸序列：
    (1)所述VH包含SEQ ID NO:26所示的氨基酸序列，且所述VL包含SEQ ID NO:34所示的氨基酸序列；
    (2)所述VH包含SEQ ID NO:43所示的氨基酸序列，且所述VL包含SEQ ID NO:49所示的氨基酸序列；
    (3)所述VH包含SEQ ID NO:75所示的氨基酸序列，且所述VL包含SEQ ID NO:49所示的氨基酸序列；
    (4)所述VH包含SEQ ID NO:61所示的氨基酸序列，且所述VL包含SEQ ID NO:65所示的氨基酸序列；
    (5)所述VH包含SEQ ID NO:79所示的氨基酸序列，且所述VL包含SEQ ID NO:93所示的氨基酸序列；以及
    (6)所述VH包含SEQ ID NO:79所示的氨基酸序列，且所述VL包含SEQ ID NO:87所示的氨基酸序列。
  25. 根据权利要求2-24中任一项所述的细胞，其中所述靶向CLDN18.2的抗原结合结构域包含scFv。
  26. 根据权利要求25所述的细胞，其中所述scFv包含SEQ ID NO:38、SEQ ID NO:53、SEQ ID NO:67、SEQ ID NO:77、SEQ ID NO:89和SEQ ID NO:95中任一项所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体包含共刺激域，所述共刺激域包含源自选自下组中的一种或多种蛋白的共刺激域：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。
  28. 根据权利要求27所述的细胞，其中所述共刺激域为源自4-1BB的胞内共刺激信号区域。
  29. 根据权利要求27-28中任一项所述的细胞，其中所述共刺激域包含SEQ ID NO:8所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体包含跨膜区，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区：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。
  31. 根据权利要求30所述的细胞，其中所述跨膜区为源自CD8的跨膜区。
  32. 根据权利要求31所述的细胞，其中所述跨膜区为源自人CD8的跨膜区。
  33. 根据权利要求30-32中任一项所述的细胞，其中所述跨膜区包含SEQ ID NO:6所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体包含铰链区，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、PD-1、ICOS、OX40、NKG2D、 NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  35. 根据权利要求34所述的细胞，其中所述铰链区为源自CD8的铰链区。
  36. 根据权利要求34-35所述的细胞，其中所述铰链区为源自人CD8的铰链区。
  37. 根据权利要求34-36中任一项所述的细胞，其中所述铰链区包含SEQ ID NO:4所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体包含信号肽。
  39. 根据权利要求38所述的细胞，其中所述信号肽源自CD8蛋白的信号肽。
  40. 根据权利要求38-39所述的细胞，其中所述信号肽包含SEQ ID NO:2所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体不包含胞内信号传导域。
  42. 根据权利要求1-41中任一项所述的细胞，其中所述靶向MSLN的嵌合抗原受体包含靶
    向MSLN的抗原结合结构域。
  43. 根据权利要求42所述的细胞，其中所述靶向MSLN的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
  44. 根据权利要求42-43中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含HCDR3，且所述HCDR3包含SEQ ID NO:101、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:119和SEQ ID NO:154中任一项所示的氨基酸序列。
  45. 根据权利要求42-44中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:99、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:118和SEQ ID NO:153中任一项所示的氨基酸序列。
  46. 根据权利要求42-45中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含HCDR1，且所述HCDR1包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:108、SEQ ID NO:117和SEQ ID NO:152中任一项所示的氨基酸序列。
  47. 根据权利要求42-46中任一项所述的细胞，其中所述靶向MSLN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氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO:97所示的氨基酸序列，所述HCDR2包含SEQ ID  NO:99所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:101所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO:104所示的氨基酸序列，所述HCDR2包含SEQ ID NO:105所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:106所示的氨基酸序列；
    (3)所述HCDR1包含SEQ ID NO:108所示的氨基酸序列，所述HCDR2包含SEQ ID NO:109所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:110所示的氨基酸序列；
    (4)所述HCDR1包含SEQ ID NO:117所示的氨基酸序列，所述HCDR2包含SEQ ID NO:118所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:119所示的氨基酸序列；以及
    (5)所述HCDR1包含SEQ ID NO:152所示的氨基酸序列，所述HCDR2包含SEQ ID NO:153所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:154所示的氨基酸序列。
  48. 根据权利要求42-47中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含VH，且所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
  49. 根据权利要求42-48中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含VHH。
  50. 根据权利要求49所述的细胞，其中所述VHH包含SEQ ID NO:103、SEQ ID NO:107和SEQ ID NO:155中任一项所示的氨基酸序列。
  51. 根据权利要求42-50中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含LCDR3，且所述LCDR3包含SEQ ID NO:123或SEQ ID NO:114所示的氨基酸序列。
  52. 根据权利要求42-51中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:113或SEQ ID NO:122所示的氨基酸序列。
  53. 根据权利要求42-52中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含LCDR1，且所述LCDR1包含SEQ ID NO:112或SEQ ID NO:121所示的氨基酸序列。
  54. 根据权利要求42-53中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含LCDR1，LCDR2和LCDR3，所述LCDR1，LCDR2和LCDR3包含选自下述任一组的氨基酸序列：
    (1)所述LCDR1包含SEQ ID NO:112所示的氨基酸序列，所述LCDR2包含SEQ ID  NO:113所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:114所示的氨基酸序列；
    (2)所述LCDR1包含SEQ ID NO:121所示的氨基酸序列，所述LCDR2包含SEQ ID NO:122所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:123所示的氨基酸序列。
  55. 根据权利要求42-54中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含VL，且所述VL包含SEQ ID NO:115或SEQ ID NO:124所示的氨基酸序列。
  56. 根据权利要求42-55中任一项所述的细胞，其中所述靶向MSLN的抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:116或SEQ ID NO:125所示的氨基酸序列。
  57. 根据权利要求1-56中任一项所述的细胞，其中所述靶向MSLN的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传导域：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。
  58. 根据权利要求57所述的细胞，其中所述胞内信号传导域为源自CD3ζ的信号传导域。
  59. 根据权利要求57-58中任一项所述的细胞，其中所述胞内信号传导域包含SEQ ID NO:16所示的氨基酸序列。
  60. 根据权利要求1-59中任一项所述的细胞，其中所述靶向MSLN的嵌合抗原受体包含跨膜区，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区：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。
  61. 根据权利要求60所述的细胞，其中所述跨膜区为源自CD28的跨膜区。
  62. 根据权利要求60-61中任一项所述的细胞，其中所述跨膜区包含SEQ ID NO:14所示的氨基酸序列。
  63. 根据权利要求1-62中任一项所述的细胞，其中所述靶向MSLN的嵌合抗原受体包含铰链区，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、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和 LIGHT。
  64. 根据权利要求63所述的细胞，其中所述铰链区为源自CD28的铰链区。
  65. 根据权利要求63-64中任一项所述的细胞，其中所述铰链区包含SEQ ID NO:12所示的氨基酸序列。
  66. 根据权利要求1-65中任一项所述的细胞，其中所述靶向MSLN的嵌合抗原受体包含信号肽。
  67. 根据权利要求66所述的细胞，其中所述信号肽源自CD8蛋白的信号肽。
  68. 根据权利要求66-67中任一项所述的细胞，其中所述信号肽包含SEQ ID NO:2所示的氨基酸序列。
  69. 根据权利要求1-68中任一项所述的细胞，其中所述靶向MSLN的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。
  70. 根据权利要求69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。
  71. 根据权利要求69-70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。
  72. 根据权利要求69-71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:18所示的氨基酸序列。
  73. 根据权利要求1-72中任一项所述的细胞，其中所述靶向MSLN的嵌合抗原受体不包含共刺激域。
  74. 根据权利要求1-73中任一项所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体的表达量与靶向MSLN的嵌合抗原受体的表达量为约1：1。
  75. 根据权利要求1-73中任一项所述的细胞，其中所述靶向CLDN18.2的嵌合抗原受体的表达量与靶向MSLN的嵌合抗原受体的表达量为约2：1。
  76. 根据权利要求1-75中任一项所述的细胞，其还包含和/或表达低密度脂蛋白受体相关蛋白或其片段。
  77. 根据权利要求76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。
  78. 根据权利要求76-77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。
  79. 根据权利要求76-78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:18所示的氨基酸序列。
  80. 根据权利要求1-79中任一项所述的细胞，其包含免疫效应细胞。
  81. 根据权利要求1-80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。
  82. 根据权利要求1-81中任一项所述的细胞，其为T细胞。
  83. 表达载体，其包括编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列。
  84. 表达载体，其包括编码权利要求1-82中任一项所述的靶向CLDN18.2的嵌合抗原受体的核酸序列和编码权利要求1-82中任一项所述的靶向MSLN的嵌合抗原受体的核酸序列。
  85. 根据权利要求83-84中任一项所述的表达载体，其中编码靶向CLDN18.2的嵌合抗原受体的核酸序列包括编码靶向CLDN18.2的抗原结合结构域的核酸序列。
  86. 根据权利要求85所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含VH中的至少一个CDR，编码所述VH的核酸序列包含SEQ ID NO:25、SEQ ID NO:42、SEQ ID NO:60、SEQ ID NO:74和SEQ ID NO:78中任一项所示的核酸序列。
  87. 根据权利要求85-86中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR3，且编码所述HCDR3的核酸序列包含SEQ ID NO:23、SEQ ID NO:41、SEQ ID NO:58和SEQ ID NO:72中任一项所示的核酸序列。
  88. 根据权利要求85-87中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR2，且编码所述HCDR2的核酸序列包含SEQ ID NO:21、SEQ ID NO:40、SEQ ID NO:56和SEQ ID NO:70中任一项所示的核酸序列。
  89. 根据权利要求85-88中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含HCDR1，且编码所述HCDR1的核酸序列包含SEQ ID NO:19、SEQ ID NO:39、SEQ ID NO:54和SEQ ID NO:68中任一项所示的核酸序列。
  90. 根据权利要求85-89中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含VH，且编码所述VH的核酸序列包含SEQ ID NO:25、SEQ ID NO:42、SEQ ID NO:60、SEQ ID NO:74和SEQ ID NO:78中任一项所示的核酸序列。
  91. 根据权利要求85-90中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR3，且编码所述LCDR3的核酸序列包含SEQ ID NO:31、SEQ ID NO:47、SEQ ID NO:62、SEQ ID NO:84和SEQ ID NO:90中任一项所示的核酸序列。
  92. 根据权利要求85-91中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结 构域包含LCDR2，且编码所述LCDR2的核酸序列包含SEQ ID NO:29、SEQ ID NO:46和SEQ ID NO:82中任一项所示的核酸序列。
  93. 根据权利要求85-92中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含LCDR1，且编码所述LCDR1的核酸序列包含SEQ ID NO:27、SEQ ID NO:44和SEQ ID NO:80中任一项所示的核酸序列。
  94. 根据权利要求85-93中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含VL，且编码所述VL的核酸序列包含SEQ ID NO:33、SEQ ID NO:48、SEQ ID NO:64、SEQ ID NO:86和SEQ ID NO:92中任一项所示的核酸序列。
  95. 根据权利要求85-94中任一项所述的表达载体，其中所述靶向CLDN18.2的抗原结合结构域包含scFv。
  96. 根据权利要求95所述的表达载体，其中编码所述scFv的核酸序列包含SEQ ID NO:37、SEQ ID NO:52、SEQ ID NO:66、SEQ ID NO:76、SEQ ID NO:88和SEQ ID NO:94中任一项所示的核酸序列。
  97. 根据权利要求83-96中任一项所述的表达载体，其中编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码共刺激域的核酸序列，所述共刺激域包含源自选自下组中的一种或多种蛋白的共刺激域：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。
  98. 根据权利要求97所述的表达载体，其中所述共刺激域为源自4-1BB的胞内共刺激信号区域。
  99. 根据权利要求97-98中任一项所述的表达载体，其中所述编码共刺激域的核酸序列包含SEQ ID NO:7所示的核酸序列。
  100. 根据权利要求83-99中任一项所述的表达载体，其中编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码跨膜区的核酸序列，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区：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。
  101. 根据权利要求100所述的表达载体，其中所述跨膜区为源自CD8的跨膜区。
  102. 根据权利要求100-101中任一项所述的表达载体，其中所述编码跨膜区的核酸序列包含SEQ ID NO:5所示的核酸序列。
  103. 根据权利要求83-102中任一项所述的表达载体，其中编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码铰链区的核酸序列，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、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  104. 根据权利要求103所述的表达载体，其中所述铰链区为源自CD8的铰链区。
  105. 根据权利要求103-104中任一项所述的表达载体，其中所述编码铰链区的核酸序列包含SEQ ID NO:5所示的核酸序列。
  106. 根据权利要求83-105中任一项所述的表达载体，其中编码所述靶向CLDN18.2的嵌合抗原受体的核酸序列包含编码信号肽的核酸序列。
  107. 根据权利要求106所述的表达载体，其中所述信号肽源自CD8蛋白的信号肽。
  108. 根据权利要求106-107中任一项所述的表达载体，其中所述编码信号肽的核酸序列包含SEQ ID NO:1所示的核酸序列。
  109. 根据权利要求83-108中任一项所述的表达载体，其中所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列不包含编码胞内信号传导域的核酸序列。
  110. 根据权利要求83-109中任一项所述的表达载体，其中所述编码靶向MSLN的嵌合抗原受体的核酸序列包含编码靶向MSLN的抗原结合结构域的核酸序列。
  111. 根据权利要求110所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
  112. 根据权利要求111所述的表达载体，其中编码所述VH的核酸序列包含SEQ ID NO:102或SEQ ID NO:156所示的核酸序列。
  113. 根据权利要求110-112中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含HCDR3，所述HCDR3包含SEQ ID NO:101、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:119和SEQ ID NO:154中任一项中任一项所示的氨基酸序列。
  114. 根据权利要求113所述的表达载体，其中编码所述HCDR3的核酸序列包含SEQ ID NO:100所示的核酸序列。
  115. 根据权利要求110-114中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含HCDR2，且所述HCDR2包含SEQ ID NO:99、SEQ ID NO:105、SEQ ID NO: 109、SEQ ID NO:118和SEQ ID NO:153中任一项所示的氨基酸序列。
  116. 根据权利要求115所述的表达载体，其中编码所述HCDR2的核酸序列包含SEQ ID NO:98所示的核酸序列。
  117. 根据权利要求110-116中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含HCDR1，所述HCDR1包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:108、SEQ ID NO:117和SEQ ID NO:152中任一项所示的氨基酸序列。
  118. 根据权利要求117所述的表达载体，其中编码所述HCDR1的核酸序列包含SEQ ID NO:96所示的核酸序列。
  119. 根据权利要求110-118中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含VH，所述VH包含SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:120和SEQ ID NO:155中任一项所示的氨基酸序列。
  120. 根据权利要求119所述的表达载体，其中编码所述VH的核酸序列包含SEQ ID NO:102或SEQ ID NO:156所示的核酸序列。
  121. 根据权利要求110-120中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含VHH，所述VHH包含SEQ ID NO:103、SEQ ID NO:107和SEQ ID NO:155中任一项所示的氨基酸序列。
  122. 根据权利要求121所述的表达载体，其中编码所述VHH的核酸序列包含SEQ ID NO:102或SEQ ID NO:156所示的核酸序列。
  123. 根据权利要求110-122中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含LCDR3，所述LCDR3包含SEQ ID NO:114或SEQ ID NO:123所示的氨基酸序列。
  124. 根据权利要求123所述的表达载体，其中编码所述LCDR3的核酸序列包含SEQ ID NO:123或SEQ ID NO:114所示的核酸序列。
  125. 根据权利要求110-124中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含LCDR2，且所述LCDR2包含SEQ ID NO:113或SEQ ID NO:122所示的氨基酸序列。
  126. 根据权利要求110-125中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含LCDR1，所述LCDR1包含SEQ ID NO:112或SEQ ID NO:121所示的氨基酸序列。
  127. 根据权利要求110-126中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含VL，所述VL包含SEQ ID NO:115或SEQ ID NO:124所示的氨基酸序列。
  128. 根据权利要求110-127中任一项所述的表达载体，其中所述靶向MSLN的抗原结合结构域包含scFv，且所述scFv包含SEQ ID NO:116或SEQ ID NO:125所示的氨基酸序列。
  129. 根据权利要求83-128中任一项所述的表达载体，其中编码所述靶向MSLN的嵌合抗原受体的核酸序列包含编码胞内信号传导域的核酸序列，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传导域：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。
  130. 根据权利要求129所述的表达载体，其中所述胞内信号传导域为源自CD3ζ的信号传导域。
  131. 根据权利要求129-130中任一项所述的表达载体，其中所述编码胞内信号传导域的核酸序列包含SEQ ID NO:15所示的核酸序列。
  132. 根据权利要求83-131中任一项所述的表达载体，其中编码所述靶向MSLN的嵌合抗原受体的核酸序列包含编码跨膜区的核酸序列，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区：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。
  133. 根据权利要求132所述的表达载体，其中所述跨膜区为源自CD28的跨膜区。
  134. 根据权利要求132-133中任一项所述的表达载体，其中所述编码跨膜区的核酸序列包含SEQ ID NO:13所示的核酸序列。
  135. 根据权利要求83-134中任一项所述的表达载体，其中编码所述靶向MSLN的嵌合抗原受体的核酸序列包含编码铰链区的核酸序列，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、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  136. 根据权利要求135所述的表达载体，其中所述铰链区为源自CD28的铰链区。
  137. 根据权利要求135-136中任一项所述的表达载体，其中所述编码铰链区的核酸序列包含SEQ ID NO:11所示的核酸序列。
  138. 根据权利要求83-137中任一项所述的表达载体，其中编码所述靶向CLDN18.2的嵌合抗 原受体的核酸序列包含编码信号肽的核酸序列。
  139. 根据权利要求138所述的表达载体，其中所述信号肽源自CD8蛋白的信号肽。
  140. 根据权利要求138-139中任一项所述的表达载体，其中所述编码信号肽的核酸序列包含SEQ ID NO:1所示的核酸序列。
  141. 根据权利要求83-140中任一项所述的表达载体，其中编码所述靶向MSLN的嵌合抗原受体的核酸序列不包含编码所述共刺激域的核酸序列。
  142. 根据权利要求83-141中任一项所述的表达载体，其中所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列通过编码剪切肽的序列连接。
  143. 根据权利要求142所述的表达载体，其中所述剪切肽选自下组：P2A、T2A、F2A和E2A。
  144. 根据权利要求142-143中任一项所述的表达载体，其中所述剪切肽包含SEQ ID NO:10所示的氨基酸序列。
  145. 根据权利要求142-144中任一项所述的表达载体，其中编码所述剪切肽的核酸序列包含SEQ ID NO:9所示的核酸序列。
  146. 根据权利要求83-145中任一项所述的表达载体，其包含至少一条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列。
  147. 根据权利要求83-146中任一项所述的表达载体，其包含两条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列。
  148. 根据权利要求147所述的表达载体，其中所述两条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列通过编码剪切肽的核酸序列连接。
  149. 根据权利要求148所述的表达载体，其中所述剪切肽选自下组：P2A、T2A、F2A和E2A。
  150. 根据权利要求148-149中任一项所述的表达载体，其中所述编码剪切肽的核酸序列包含SEQ ID NO:9所示的核酸序列。
  151. 根据权利要求83-150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。
  152. 根据权利要求151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。
  153. 根据权利要求151-152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。
  154. 根据权利要求151-153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包含SEQ ID NO:17所示的核酸序列。
  155. 根据权利要求151-154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与所述编码靶向MSLN的嵌合抗原受体的胞内信号传导域的核酸序列直接相连。
  156. 根据权利要求151-154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与所述编码靶向MSLN的嵌合抗原受体的核酸序列通过剪切肽相连。
  157. 根据权利要求83-156中任一项所述的表达载体，其中所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列和编码靶向MSLN的嵌合抗原受体的核酸序列位于同一条核酸分子中。
  158. 根据权利要求151-157中任一项所述的表达载体，其中所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列位于同一条核酸分子中。
  159. 根据权利要求151-158中任一项所述的表达载体，其中第一条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、第二条所述编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列位于同一条核酸分子中。
  160. 根据权利要求151-159中任一项所述的表达载体，其从5’端至3’端分别依次包含：编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  161. 根据权利要求151-160中任一项所述的表达载体，其从5’端至3’端分别依次包含：编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向CLDN18.2的嵌合抗原受体的核酸序列、编码剪切肽的核酸序列、编码靶向MSLN的嵌合抗原受体的核酸序列、编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  162. 根据权利要求151-161中任一项所述的表达载体，其从5’端至3’端分别依次包含：编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码MSLN VHH的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码胞内信号传导域的核酸序列以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  163. 根据权利要求151-162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CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码MSLN VHH的核酸序列、编码CD28铰链区的核酸序列、编码CD28跨膜区的核酸序列、编码CD3ζ胞内信号传导域的核酸序列以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  164. 根据权利要求151-163中任一项所述的表达载体，其从5’端至3’端分别依次包含：编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码共刺激域的核酸序列、编码剪切肽的核酸序列、编码信号肽的核酸序列、编码MSLN VHH的核酸序列、编码铰链区的核酸序列、编码跨膜区的核酸序列、编码胞内信号传导域的核酸序列以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  165. 根据权利要求151-164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CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码CLDN18.2 scFv的核酸序列、编码CD8铰链区的核酸序列、编码CD8跨膜区的核酸序列、编码4-1BB共刺激域的核酸序列、编码剪切肽的核酸序列、编码CD8信号肽的核酸序列、编码MSLN VHH的核酸序列、编码CD28铰链区的核酸序列、编码CD28跨膜区的核酸序列、编码CD3ζ胞内信号传导域的核酸序列以及编码所述低密度脂蛋白受体相关蛋白或其片段的核酸序列。
  166. 细胞，其包含权利要求83-165中任一项所述的表达载体。
  167. 根据权利要求166所述的细胞，其为免疫效应细胞。
  168. 根据权利要求166-167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。
  169. 根据权利要求166-168中任一项所述的细胞，其为T细胞。
  170. 权利要求1-82和166-168中任一项所述的细胞、和/或权利要求83-165中任一项所述的表达载体在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  171. 权利要求1-169中任一项所述的靶向CLDN18.2的嵌合抗原受体和权利要求1-169中任 一项所述的靶向MSLN的嵌合抗原受体在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  172. 根据权利要求170-171中任一项所述的用途，其中所述疾病和/或病症包括肿瘤。
  173. 根据权利要求170-172中任一项所述的用途，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中任一项所述的用途，其中所述肿瘤包括同时表达CLDN18.2和MSLN两种抗原的肿瘤。
  175. 根据权利要求172-174中任一项所述的用途，其中所述肿瘤包括胃癌、胰腺癌和/或胃食管交界处癌。
  176. 一种预防和/或治疗疾病和/或病症的方法，其包括向有需要的受试者施用权利要求1-82和166-168中任一项所述的细胞。
  177. 根据权利要求176所述的方法，其中所述疾病和/或病症包括肿瘤。
  178. 根据权利要求177中任一项所述的方法，其中所述肿瘤包括实体瘤和/或非实体瘤。
  179. 根据权利要求177-178中任一项所述的方法，其中所述肿瘤包括同时表达CLDN18.2和MSLN两种抗原的肿瘤。
  180. 根据权利要求177-179中任一项所述的方法，其中所述肿瘤包括胃癌、胰腺癌和/或胃食管交界处癌。
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