WO2024088371A1 - 靶向msln的抗原结合蛋白 - Google Patents

靶向msln的抗原结合蛋白 Download PDF

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WO2024088371A1
WO2024088371A1 PCT/CN2023/127029 CN2023127029W WO2024088371A1 WO 2024088371 A1 WO2024088371 A1 WO 2024088371A1 CN 2023127029 W CN2023127029 W CN 2023127029W WO 2024088371 A1 WO2024088371 A1 WO 2024088371A1
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seq
amino acid
acid sequence
sequence shown
binding protein
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PCT/CN2023/127029
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French (fr)
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王华菁
钟阵威
成超
谢二敏
陈晓锐
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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting MSLN, a chimeric antigen receptor comprising the antigen binding protein, and applications thereof.
  • MSLN Mesothelin
  • the mesothelin gene encodes a 69kDa precursor protein, which is hydrolyzed into two chains by furin (paired alkaline amino acid protease, Furin)-like convertase.
  • furin paired alkaline amino acid protease, Furin
  • the membrane-bound protein of about 40KD at the C-terminus is mature mesothelin, and the fragment of about 30KD at the N-terminus, called megakaryocyte promoting factor (MPF), falls off and is released outside the cell. Both MPF and membrane-anchored MSLN are N-glycosylated.
  • MPF can promote the formation of megakaryocyte clones in vitro.
  • Membrane-anchored MSLN can interact with MUC16 and play an important role in the cell adhesion process. Therefore, membrane-anchored MSLN is currently selected as a target in targeted therapy. Therefore, MSLN currently refers specifically to the C-terminal 40KD fragment of MSLN, namely membrane-anchored MSLN.
  • Mesothelin is a glycoprotein present on the cell surface of mesothelial cell lines of the peritoneal, pleural and pericardial cavities. Mesothelin is dominantly expressed (overexpressed) in mesotheliomas, i.e. cancer/tumor cells, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer and endometrial cancer. In contrast, its expression is limited in normal cells such as mesothelial cells.
  • the present application provides an isolated antigen binding protein that can specifically bind to MSLN.
  • the present application also provides a chimeric antigen receptor comprising the antigen binding protein, and a cell comprising and/or expressing the chimeric antigen receptor, wherein the cell has one or more of the following characteristics: (1) strong proliferation ability; (2) ability to kill target cells expressing MSLN; (3) secreting cytokines under stimulation of target cells; and (4) inhibiting the growth of tumor cells.
  • the present application provides a separated antigen-binding protein, which comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.
  • the isolated antigen binding protein is capable of specifically binding to the mesothelin (MLSN) protein.
  • the MSLN is a human MSLN.
  • the isolated antigen-binding protein is capable of competing with a reference antibody for binding to MSLN, wherein the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are selected from any of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:27.
  • the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:10, SEQ ID NO:2, SEQ ID NO:19 and SEQ ID NO:26.
  • the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:25.
  • the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:25, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:10, SEQ ID NO:2, SEQ ID NO:19 and SEQ ID NO:26, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:27.
  • the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen-binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:50.
  • the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44.
  • the isolated antigen-binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:51.
  • the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:22 and SEQ ID NO:29.
  • the isolated antigen binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:52.
  • the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:23 and SEQ ID NO:30.
  • the isolated antigen-binding protein comprises H-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.
  • the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from any one of the following groups:
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:12
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:13
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 21
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 22
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 23
  • the H- FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:28
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:29
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:30
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:32
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:34
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:13
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:36
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:13
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:38
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:40
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:42
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:44
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.
  • the isolated antigen-binding protein comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.
  • the isolated antigen binding protein comprises an antibody or an antigen binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
  • the antigen binding fragment is a VHH.
  • the isolated antigen binding protein is a VHH, which comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.
  • the isolated antigen binding protein comprises an antibody heavy chain constant region derived from the heavy chain constant region of IgG.
  • the antibody heavy chain constant region is derived from human IgG.
  • the antibody heavy chain constant region is derived from the heavy chain constant region of human IgG1.
  • the antibody heavy chain constant region comprises the Fc region of IgG.
  • the Fc region comprises the amino acid sequence shown in SEQ ID NO:61.
  • the present application also provides a chimeric antigen receptor, which comprises a targeting portion, and the targeting portion comprises the antigen binding protein described in the present application.
  • the chimeric antigen receptor comprises a co-stimulatory domain, which comprises a co-stimulatory domain derived from one or more proteins selected from the following groups: 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, a ligand of CD83, CD40, and MyD88.
  • co-stimulatory domain which comprises a co-stimulatory domain derived from one or more proteins selected from the following groups: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2
  • the costimulatory domain is an intracellular costimulatory signal region derived from 4-1BB.
  • the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:53.
  • the chimeric antigen receptor comprises an intracellular signaling domain, which comprises an intracellular signaling domain derived from one or more proteins selected from the following group: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and a domain comprising at least one ITAM.
  • an intracellular signaling domain derived from one or more proteins selected from the following group: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian
  • 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:55.
  • the chimeric antigen receptor comprises a transmembrane region, which comprises a transmembrane domain 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.
  • a transmembrane region which comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, T
  • the transmembrane region is a transmembrane region derived from CD8.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:54.
  • the chimeric antigen receptor in the chimeric antigen receptor, it includes a hinge region between the targeting portion and the transmembrane region, and the hinge region comprises a hinge region derived from one or more proteins selected from the following group: 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 comprises a hinge region derived from one or more proteins selected from the following group: 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
  • the hinge region is a hinge region derived from CD8.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:56.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide is derived from the signal peptide of the CD8 protein.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:57.
  • the chimeric antigen receptor further comprises a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:58.
  • the present application also provides a polypeptide comprising the antigen-binding protein.
  • the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein and/or the chimeric antigen receptor.
  • the nucleic acid molecule comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is the EF1 ⁇ promoter.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the vector comprises a viral vector.
  • the vector comprises a lentiviral vector.
  • the present application also provides a cell, which comprises the antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule and/or the vector.
  • the cell is an immune effector cell.
  • the cells comprise 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
  • macrophages NK cells
  • monocytes monocytes
  • dendritic cells granulocytes
  • lymphocytes lymphocytes
  • leukocytes granulocytes
  • peripheral blood mononuclear cells embryonic stem cells
  • lymphoid progenitor cells and/or pluripotent stem cells.
  • the cell is a T cell.
  • the cell contains and/or expresses low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:58.
  • the present application also provides a method for preparing modified immune effector cells, which comprises culturing the cells under conditions such that the antigen binding protein and/or the chimeric antigen receptor are expressed.
  • the present application also provides a method for preparing modified immune effector cells, which comprises introducing the vector into immune effector cells.
  • the present application also provides a pharmaceutical composition, which comprises the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides the use of the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition in the preparation of a drug for preventing, treating and/or alleviating diseases or conditions associated with abnormal expression of MSLN.
  • the disease or condition associated with abnormal expression of MSLN comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a tumor expressing the MSLN antigen.
  • the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell carcinoma, bile duct carcinoma, triple-negative breast cancer, and/or endometrial cancer.
  • the present application also provides a method for preventing, treating and/or alleviating diseases or conditions associated with abnormal expression of MSLN, the method comprising administering the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition to a subject in need.
  • the disease or condition associated with abnormal expression of MSLN comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a tumor expressing the MSLN antigen.
  • the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell carcinoma, bile duct carcinoma, triple-negative breast cancer, and/or endometrial cancer.
  • Figure 1 shows the phage Pool ELISA results.
  • Figure 2A-D shows the screening results of positive clones.
  • FIG3 shows the flow cytometry detection of the specific binding of the antigen binding protein of the present application to MSLN.
  • Figure 4 shows the ForteBio test results.
  • Figure 5 shows the pCORE-VHH plasmid map.
  • Figure 6 shows a schematic diagram of the MSLN-VHH CAR structure.
  • FIG7A shows the electropherogram of the PCR results.
  • FIG. 7B shows the electrophoresis of vector restriction enzyme digestion
  • FIG8 shows the flow cytometry positive rate detection of SK-OV3-MSLN overexpression cell line.
  • FIG9 shows the expansion multiples of CAR-T cells after repeated stimulation by target cells.
  • FIG. 10 shows the results of cell killing by CAR-T cells in vitro.
  • FIG11A shows the results of IFN- ⁇ cytokine secretion by CAR-T cells in vitro.
  • FIG11B shows the results of IL-2 cytokine secretion by CAR-T cells in vitro.
  • FIG. 12A shows a tumor growth curve after administration of the CAR-T cells described in the present application.
  • FIG. 12B shows a graph of the animal body weight after administration of the CAR-T cells described in the present application.
  • MSLN also known as mesothelin, or CAK1 antigen or promegakaryocytic enhancer factor
  • MSLN protein is a protein present on normal mesothelial cells and is overexpressed in some tumor cells.
  • the term may include MSLN protein or its functionally active fragment.
  • the term may also include homologs, analogs or variants of MSLN protein.
  • the MSLN may include human MSLN.
  • isolated antigen-binding protein generally refers to a protein with antigen-binding ability that is separated from its naturally occurring state.
  • isolated antigen-binding protein may include a portion that binds to an antigen and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes its binding to an antigen.
  • the antigen-binding protein may include, for example, a protein framework region (FR) derived from an antibody or an alternative protein framework region or an artificial framework region with a transplanted variable region (CDR) or CDR derivative.
  • the antigen-binding protein may include an antibody or an antigen-binding fragment thereof.
  • the antigen-binding protein may bind to the MSLN protein.
  • the antigen-binding protein may compete with a reference antibody for binding to the MSLN protein.
  • the antigen-binding protein may include an antibody heavy chain variable region VH.
  • the antigen-binding protein may include at least one CDR derived from an antibody heavy chain variable region VH.
  • the VH may include HCDR3, HCDR2 and/or HCDR1.
  • the VH may include a framework region H-FR1, the C-terminus of the H-FR1 being directly or indirectly connected to the N-terminus of the HCDR1.
  • the VH may include a framework region H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2.
  • the VH may include a framework region H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3.
  • the VH may include a framework region H-FR4, wherein the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3.
  • the antigen-binding protein may be a VHH.
  • the antigen-binding protein may include an antibody heavy chain constant region, wherein the antibody heavy chain constant region may be derived from IgG.
  • the antibody heavy chain constant region may be
  • the antibody heavy chain constant region may be derived from human IgG1.
  • antibody as used includes intact antibodies and binding fragments thereof. Typically, the fragments compete with the intact antibody from which they are derived for antigen-specific binding.
  • the antibody or its binding fragment can be chemically bound to other proteins, or expressed as a fusion protein with other proteins.
  • the antibody can be a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
  • the binding protein of the antibody or its binding fragment can include MSLN.
  • the antibody or its binding fragment can be specific for MSLN.
  • antigen binding fragment refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody.
  • the antigen binding fragment may include Fab, Fab', F(ab')2, Fv fragments and single-chain Fv fragments, tandem Fv fragments, VHH, bispecific antibodies.
  • the antigen binding fragment may be a VHH.
  • the antigen binding fragment may bind to MSLN.
  • the antigen binding fragment may be specific to MSLN.
  • VHH generally refers to an antibody comprising a variable antigen binding domain of a heavy chain antibody.
  • VHH may also be referred to as a nanobody (Nb) and/or a single domain antibody.
  • Nb nanobody
  • the VHH may bind to MSLN.
  • the VHH may be specific to MSLN.
  • the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region.
  • the term "heavy chain constant region” consists of three domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region.
  • the term "light chain constant region” consists of one domain CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs).
  • CDRs complementary determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor.
  • the term "reference antibody” refers to an antibody that can competitively bind to the same epitope of MSLN as the separated antigen-binding protein.
  • the reference antibody can include a heavy chain variable region VH.
  • the reference antibody can have three CDR sequences.
  • the VH of the reference antibody can include HCDR1, HCDR2 and HCDR3.
  • the CDR sequence can be consistent with the CDR sequence of the separated antigen-binding protein.
  • IgG refers to polypeptides belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3.
  • chimeric antigen receptor generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds to an antigen or target.
  • a hinge region is included between the extracellular domain and the transmembrane region.
  • the chimeric antigen receptor may also include a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the chimeric antigen receptor may include a signal peptide.
  • the extracellular domain of CAR binds to the target antigen on the surface of the target cell, resulting in CAR clustering and transmitting the activation stimulus to the CAR-containing cell.
  • the extracellular structure may include the above-mentioned antigen binding protein.
  • the extracellular structure may specifically bind to MSLN.
  • intracellular domain refers to an intracellular domain that means any truncated portion that is sufficient to transduce an activation signal.
  • the intracellular domain may include an intracellular signal region and/or a costimulatory signal region.
  • intracellular signal region refers to an intracellular region that can generate a signal that promotes the immune effector function of a CAR-containing cell (eg, a CART cell or a NK cell expressing CAR).
  • the intracellular signal region may include an intracellular signal region of one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and at least one ITAM domain.
  • the intracellular signal region may be a signal transduction domain derived from CD3 ⁇ .
  • costimulatory signal region refers to a part of the CAR that can transduce effector signals in the intracellular signal region.
  • the costimulatory signal region may include an intracellular costimulatory signal region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligands, CD40 and MyD88.
  • the costimulatory signal region may be an intracellular costimulatory signal region derived from 4-1BB.
  • the term "membrane spanning region” refers to a domain of a peptide, polypeptide or protein that can cross a cytoplasmic membrane. These domains can be used to anchor the extracellular domain to the cell membrane.
  • the membrane spanning region can include a membrane spanning domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • the transmembrane the transmembrane
  • the term "hinge region” refers to a portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 according to the EU numbering system of Kabat.
  • the hinge region is usually a dimer molecule composed of two polypeptides having the same amino acid sequence.
  • the hinge region generally includes about 25 amino acid residues and is flexible, allowing the antigen binding region to move independently.
  • the hinge region can be subdivided into three domains: upper, middle, and lower hinge domains.
  • the hinge region can include 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 can be derived from the hinge region of CD8.
  • the term "low-density lipoprotein receptor-related protein” refers to a cell surface protein that is an endocytic receptor. It is widely distributed in the body and has great differences between tissues. Its main function is to take up cholesterol into cells for cell proliferation and the synthesis of steroid hormones and bile salts.
  • the low-density lipoprotein receptor-related protein can be from any vertebrate.
  • the low-density lipoprotein receptor-related protein or its fragment can be located at the C-terminus of the intracellular signaling region.
  • the low-density lipoprotein receptor-related protein or its fragment can include one or more selected from the following group: low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or its fragment can be low-density lipoprotein receptor-related protein 6 or its fragment.
  • signal peptide refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein, which guides the nascent protein to the endoplasmic reticulum and subsequent surface expression during or after translation.
  • the signal peptide is derived from the signal peptide of a CD8 protein.
  • polypeptide polypeptide
  • peptide protein
  • protein polymers of amino acid residues.
  • This term may be used to refer to amino acid polymers in which one or more amino acid residues are synthetic chemical mimetics of their corresponding natural amino acids, and may also be used to refer to natural amino acid polymers, those containing modified residues, and non-natural amino acid polymers.
  • the polypeptide may include the antigen-binding protein.
  • nucleic acid molecule includes DNA molecules and RNA molecules.
  • a nucleic acid molecule can be single-stranded or double-stranded, but preferably double-stranded DNA.
  • promoter generally refers to a DNA sequence that can regulate the expression of a selected DNA sequence operably connected to the promoter, thereby affecting the expression of the selected DNA sequence in the cell.
  • the nucleic acid molecule can encode an antigen binding protein and/or a chimeric antigen receptor.
  • the nucleic acid molecule can include a promoter.
  • the promoter can be a constitutive promoter.
  • the promoter can be an EF1 ⁇ promoter.
  • the term "vector” generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached.
  • the vector can be a viral vector.
  • the vector can be a lentiviral vector.
  • the term “cell” refers to a cell into which a nucleic acid can be transfected, and the term “cell” includes prokaryotic cells for plasmid propagation and eukaryotic cells for nucleic acid expression and encoded polypeptide production.
  • a cell can include the antigen binding protein, the nucleic acid molecule and/or the vector.
  • the cell can be an immune effector cell.
  • immune refers to a cell that can be transfected with a nucleic acid.
  • effector cells generally refer to immune cells that participate in immune responses and perform effector functions. For example, the performance of effector functions may include clearing foreign antigens or promoting immune effector responses, etc.
  • immune effector 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.
  • immune effector cells may be T cells.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a mammalian individual.
  • the pharmaceutical composition may include the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and/or the cell, and an optional pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be used to prevent, treat and/or alleviate a disease or condition associated with abnormal expression of MSLN.
  • the disease or condition associated with abnormal expression of MSLN may include a tumor.
  • the tumor includes a solid tumor and/or a non-solid tumor.
  • the term “specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules).
  • an antibody that specifically binds to a target may be an antibody that binds to the target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other targets.
  • an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include but does not require exclusive binding.
  • the term "subject” generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat or monkey.
  • proteins, polypeptides and/or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs having the same or similar functions as the proteins or polypeptides.
  • the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., specifically binding to MSLN).
  • the functional variant may comprise a protein or polypeptide having an amino acid change through at least 1, for example 1-30, 1-20 or 1-10, for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change.
  • the substitution may be a conservative substitution.
  • the homologue can be a homologue having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence homology.
  • sequence homology percentage ratio can be calculated in the following manner: two sequences to be compared are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical 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 in two sequences to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce sequence homology percentage ratio.
  • nucleic acid base for example, A, T, C, G, I
  • amino acid residue for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys
  • sequence homology percentage ratio For determining the comparison carried out of sequence homology percentage ratio, can realize in various ways known in the art, for example, use 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 maximum alignment over the full length of the sequences being compared or within a region of interest.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA and BLAST A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved tools for biological sequence comparisons," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R.
  • the term “comprising” generally means including, encompassing, containing or encompassing. In some cases, it also means “for”, “consisting of".
  • the term "about” generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.
  • the antigen binding protein may include an antibody or an antigen binding fragment thereof.
  • the antigen binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.
  • the CDR of an antibody is also called the complementarity determining region, which is part of the variable region.
  • the amino acid residues in this region can contact the antigen or antigen epitope.
  • Antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat/Chothia, etc. These coding systems are known in the art, and can be found, for example, at http://www.bioinf.org.uk/abs/index.html#kabatnum.
  • a person skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. There may be differences in the CDR region using different coding systems.
  • the CDR encompasses the CDR sequence obtained by any CDR division method; it also encompasses variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted and/or having one or more amino acids added.
  • the homologues may be amino acid sequences having at least about 85% (e.g., 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 homology with the amino acid sequence of the CDR.
  • the isolated antigen-binding proteins are defined by the Kabat coding system.
  • the isolated antigen-binding protein may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.
  • the isolated antigen binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:27.
  • the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:10, SEQ ID NO:2, SEQ ID NO:19 and SEQ ID NO:26.
  • the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:25.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:25; the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:10, SEQ ID NO:2, SEQ ID NO:19 and SEQ ID NO:26; and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:27.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise an amino acid sequence selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • antibody framework region FR refers to the part between the CDRs with higher divergence (i.e., hypervariable) in the antibody variable region.
  • Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and are provided for presenting the skeleton of three CDRs in three-dimensional space to form an antigen binding surface.
  • the isolated antigen binding protein may comprise H-FR1, the C-terminus of the H-FR1 being directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprising the amino acid sequence shown in SEQ ID NO:50.
  • the H-FR1 may include the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44.
  • the isolated antigen binding protein may comprise H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:51.
  • the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:22 and SEQ ID NO:29.
  • the isolated antigen binding protein may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:52.
  • the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:23 and SEQ ID NO:30.
  • the isolated antigen binding protein may comprise H-FR4, the N-terminus of which is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.
  • the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:4
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14
  • the H-FR4 comprises Contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:16
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:21
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:22
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:23
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:28
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:29
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:30
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:32
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:34
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:13
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:14
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:36
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:13
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:14
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:38
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:40
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 contains the amino acid sequence shown in SEQ ID NO:42
  • the H-FR2 contains the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 contains the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 contains the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:44
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the H-FR4 comprises Contains the amino acid sequence shown in SEQ ID NO:7.
  • the isolated antigen-binding protein may comprise a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.
  • the antigen binding fragment may be VHH, and the VHH may comprise the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.
  • the isolated antigen-binding protein may include a heavy chain constant region.
  • the heavy chain constant region refers to a region comprising at least three heavy chain constant domains CH1, CH2, and CH3.
  • Non-limiting exemplary heavy chain constant regions include ⁇ , ⁇ , and ⁇ .
  • Non-limiting exemplary heavy chain constant regions also include ⁇ and ⁇ .
  • Each heavy chain constant region corresponds to an antibody isotype.
  • an antibody comprising a ⁇ constant region is an IgG antibody
  • an antibody comprising a ⁇ constant region is an IgD antibody
  • an antibody comprising an ⁇ constant region is an IgA antibody.
  • an antibody comprising a ⁇ constant region is an IgM antibody
  • an antibody comprising an ⁇ constant region is an IgE antibody.
  • IgG antibodies include, but are not limited to, IgG1 (comprising a ⁇ 1 constant region), IgG2 (comprising a ⁇ 2 constant region), IgG3 (comprising a ⁇ 3 constant region), and IgG4 (comprising a ⁇ 4 constant region) antibodies;
  • IgA antibodies include, but are not limited to, IgA1 (comprising an ⁇ 1 constant region) and IgA2 (comprising an ⁇ 2 constant region) antibodies;
  • IgM includes, but is not limited to, IgM1 and IgM2.
  • the separated antigen-binding protein may include an antibody heavy chain constant region, which may be derived from IgG.
  • the separated antigen-binding protein may include an antibody heavy chain constant region, which may be derived from human IgG.
  • the separated antigen-binding protein may include an antibody heavy chain constant region, which may be derived from human IgG1.
  • the heavy chain constant region of the antigen-binding protein may include the Fc region of IgG.
  • the Fc region may include the amino acid sequence shown in SEQ ID NO:61.
  • the present application also provides a chimeric antigen receptor (CAR), which may include a targeting portion that binds to the MSLN protein.
  • CAR chimeric antigen receptor
  • the targeting portion that binds to the MSLN protein may be the antigen binding protein described in the present application.
  • the CAR of the present application may comprise a VHH
  • the VHH may comprise SEQ ID NO: 8, SEQ ID NO: 15, The amino acid sequence shown in any one of SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.
  • the CAR may include an extracellular targeting portion that binds to the MSLN protein and an intracellular domain.
  • the CAR may include an intracellular costimulatory signal region, which may provide a stimulation signal.
  • the costimulatory signal region may include an intracellular costimulatory signal region of 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, CD83 ligands, CD40 and MyD88.
  • the costimulatory signal region may be an intracellular costimulatory signal region derived from 4-1BB.
  • the costimulatory signal region may include an amino acid sequence shown in SEQ ID NO:53.
  • the CAR may include an intracellular signaling region, which may include a domain with at least one ITAM motif.
  • the intracellular signaling domain can transmit the activation signal to the interior of the cell.
  • the intracellular signaling region may include an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and other domains comprising at least one ITAM.
  • EBV Epstein-Barr virus
  • PBj14Nef Kaposi's sarcoma herpes virus
  • HSKV Kaposi's sarcoma herpes virus
  • the intracellular signal region can be a signal transduction domain derived from CD3 ⁇ .
  • the intracellular signal region can include the amino acid sequence shown in SEQ ID NO:55.
  • the CAR may include a transmembrane domain, which is a sequence in a cell surface protein that spans the cell membrane and may include a hydrophobic alpha helix.
  • the transmembrane domain may be derived from any type I transmembrane protein.
  • the transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix.
  • the transmembrane region can comprise a transmembrane domain 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 may be a transmembrane region derived from CD8.
  • the transmembrane region may comprise SEQ ID NO: The amino acid sequence shown in 54.
  • the CAR may include a hinge region, and the hinge region may be located between the extracellular targeting portion and the transmembrane domain.
  • the hinge region may include a hinge region of one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region may be a hinge region derived from CD8.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:56.
  • the CAR may further comprise a signal peptide at the N-terminus of the targeting portion that binds to the MSLN protein.
  • the signal peptide may be a signal peptide derived from a CD8 protein.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:57.
  • the CAR may also include a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the CAR.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may include low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may include the amino acid sequence shown in SEQ ID NO:58.
  • the nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof may include the nucleotide sequence shown in SEQ ID NO:59.
  • the sequence of the low-density lipoprotein receptor-related protein or its fragment in the CAR can be connected to the C-terminal sequence of the CAR through a self-cleaving peptide (e.g., 2A peptides such as T2A, P2A, E2A, etc.).
  • a self-cleaving peptide e.g., 2A peptides such as T2A, P2A, E2A, etc.
  • the low-density lipoprotein receptor-related protein or its fragment can be connected to the C-terminus of the intracellular signal region through T2A.
  • the cleavage peptide can include the amino acid sequence shown in SEQ ID NO:60.
  • the CAR may sequentially include a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, and for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region, and the intracellular signal region.
  • the MSLN protein e.g., the antigen binding protein, and for example, the VHH described in the present application
  • the CAR may sequentially include the VHH, a hinge region derived from CD8, a transmembrane region derived from CD8, a co-stimulatory signal region derived from 4-1BB, and an intracellular signal region derived from CD3 ⁇
  • the VHH may include the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 45.
  • the CAR may sequentially include a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal No region, the intracellular signal region and the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the MSLN protein e.g., the antigen binding protein, for example, the VHH described in the present application
  • the hinge region e.g., the antigen binding protein, for example, the VHH described in the present application
  • the transmembrane domain e.g., the transmembrane domain
  • the co-stimulatory signal No region e.g., the intracellular signal region and the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the CAR may sequentially include the VHH, a hinge region derived from CD8, a transmembrane region derived from CD8, a costimulatory signal region derived from 4-1BB, an intracellular signal region derived from CD3 ⁇ , and a low-density lipoprotein receptor-related protein or a fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 58, and the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 45.
  • the CAR may sequentially comprise a signal peptide, a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, and for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region, and the intracellular signal region.
  • a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, and for example, the VHH described in the present application)
  • the hinge region e.g., the antigen binding protein, and for example, the VHH described in the present application
  • the transmembrane domain e.g., the co-stimulatory signal region
  • the intracellular signal region e.g., the intracellular signal region.
  • the CAR may sequentially comprise a signal peptide, a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region, the intracellular signal region, and the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the MSLN protein e.g., the antigen binding protein, for example, the VHH described in the present application
  • the present application also provides one or more nucleic acid molecules, which can be isolated nucleotides, deoxynucleotides and/or ribonucleotides of any length, and can encode the isolated antigen binding protein and/or the chimeric antigen receptor.
  • the nucleic acid molecule may include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be an EF1 ⁇ promoter.
  • the present application also provides one or more nucleic acid molecules, the nucleic acid molecules comprising a sequence capable of expressing the chimeric antigen receptor and the low-density lipoprotein receptor-related protein or its fragment in a cell.
  • the nucleic acid sequence encoding the chimeric antigen protein can be connected to the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or its fragment by a cleavage peptide.
  • the present application also provides a kind of vector, and the vector can include the nucleic acid molecule.
  • the vector can transform, transduce or transfect host cells so that the genetic material elements carried by it are expressed in the host cells.
  • the vector can include a promoter, a transcriptor, an enhancer, a replicon, a selection element and a reporter gene.
  • the vector can include components that assist in entering the cell.
  • the 5' end and the 3' end of the nucleic acid molecule can also contain long terminal repeat sequences.
  • the vector can be a viral vector.
  • the vector can be a lentiviral vector.
  • the application also provides cells, which may include the isolated antigen-binding proteins, the chimeric antigen receptor, the nucleic acid molecules and/or the vector.
  • the cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical to the original parent cell (in the form of the total DNA complement or in the genome).
  • the cell may be an immune effector cell.
  • the cell may include a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphocyte progenitor cell, and/or a pluripotent stem cell.
  • NK cell natural killer cell
  • the cell may be a T cell.
  • the cell may comprise and/or express the CAR.
  • the cell may comprise and/or express the CAR and the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the present application also provides a pharmaceutical composition, which may include the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or suitable formulations of preservatives.
  • the acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical composition of the present invention may include liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.
  • the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and/or intranasal administration or direct injection into a tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application also provides a method for preparing the isolated antigen binding protein and/or the chimeric antigen receptor.
  • the method may include culturing the cell under conditions such that the antigen receptor and/or the chimeric antigen receptor is expressed.
  • the present application also provides a method for preparing modified immune effector cells, the method may include introducing into the immune cells into the vector.
  • the present application also provides the use of the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a drug, which can be used to prevent, alleviate and/or treat diseases and/or conditions.
  • the present application also provides a method for preventing, alleviating and/or treating a disease and/or condition, which may include administering the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition to a subject.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition, which are used for preventing, alleviating and/or treating diseases and/or disorders.
  • the diseases and/or disorders may include diseases and/or disorders associated with abnormal expression of MSLN.
  • the disease and/or disorder may include a tumor.
  • the tumor may include a solid tumor and/or a non-solid tumor.
  • the tumor may include hematological tumors and/or lymphomas.
  • the tumor may include a tumor expressing the MSLN antigen.
  • the tumor may include ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell carcinoma, bile duct cancer, triple-negative breast cancer and/or endometrial cancer.
  • the subject may include a human or a non-human animal.
  • the present application provides a polypeptide comprising the isolated antigen-binding protein.
  • the present application provides a kit or a drug delivery device, which comprises the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the present application also provides a method for detecting the presence and/or content of MSLN, the method comprising administering the isolated antigen binding protein, the polypeptide, the chimeric antigen receptor, and/or the cell described in the present application.
  • the method can be an in vitro method.
  • the method can be a method for non-diagnosis and treatment purposes.
  • the present application also provides a kit for detecting the presence and/or content of MSLN, which comprises the isolated antigen binding protein, the polypeptide, the chimeric antigen receptor, and/or the cell described in the present application.
  • the recombinant human MSLN was expressed in immunized alpacas and the extracellular domain (ECD) was attached to the Fc fragment to facilitate subsequent protein purification.
  • ECD extracellular domain
  • the MSLN-Fc antigen was used to immunize the alpacas, and the animal immunization was outsourced to Aikonde Biomedical Technology (Suzhou) Co., Ltd., with a single immunization dose of 1-2 mg protein.
  • 5 mL of peripheral blood was collected, serum was separated, and ELISA was used to detect the immune effect.
  • the ELISA immune titer reached more than 1:16000 (coated antigen 5ug/mL, OD value greater than 2.0).
  • RNA extraction kit provided by QIAGEN. Finally, the extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRAND SUPERMIX kit.
  • variable region (VHH) of the heavy chain antibody was amplified by nested PCR according to a protocol known in the art, using the following primers: Primers for the first round of PCR:
  • CALL001 5′-GTCCTGGCTGCTCTTCTACAAGG-3′ (SEQ ID NO:46);
  • CALL002 5′-GGTACGTGCTGTTGAACTGTTCC-3′ (SEQ ID NO: 47).
  • VHH-Back 5′-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3′(SEQ ID NO:48);
  • the target fragment was recovered from the gel and cloned into the phage display vector pMES4 (GenBank GQ907248) using restriction endonucleases (from Thermo) PstI and Eco91I. After desalting the plasmid, it was electrotransformed into electrocompetent Escherichia coli TG1 to construct the phage display nanoantibody library NanoMSLN, and the library was evaluated for diversity.
  • the library size was calculated to be 1 ⁇ 10 9 by gradient dilution plating, and then 24 single clones were picked for sequencing. A total of 21 sequences were successfully sequenced, of which 2 were useless sequences and there were 2 duplicate sequences. Therefore, a total of 85.7% (18/21) of the sequences were available, and the total diversity of NanoMSLN was 8.5 ⁇ 10 8 .
  • the plate was coated with 10 ⁇ g/ml of IgG-Fc and MSLN-His proteins and placed at 4°C overnight. The next day, it was washed three times with 1 ⁇ PBST (PBS containing 0.05% Tween20), blocked with 0.5% BSA at room temperature for 2 hours, washed three times with 1 ⁇ PBST, and 100 ⁇ l of phage library NanoMSLN was added to the IgG1-Fc well for negative screening. After 1 hour, the phages in the IgG1-Fc were transferred to the MSLN-His wells for positive screening. After 1.5 hours, it was washed 10 times with 1 ⁇ PBST to wash away the phages that did not bind to the antigen.
  • 1 ⁇ PBST PBS containing 0.05% Tween20
  • 96-well plates were coated with 2 ⁇ g/ml MSLN-His, IgG1-Fc and 0.5% BSA and left overnight. The next day, the plates were washed three times with 1 ⁇ PBST and then blocked with 0.5% BSA for 2 hours. After blocking, the plates were washed three times with 1 ⁇ PBST, 100ul of phage library of each round was added, and the plates were incubated at room temperature for 1 hour with shaking.
  • the reserved outputs from the 1st, 2nd and 3rd rounds were plated on 2YT/carb.
  • 96 single colonies were randomly picked every other day and placed in 800 ⁇ l 2YT/carb/M13KO7, and cultured overnight to produce phages.
  • 384-well plates were coated with 2 ⁇ g/ml MSLN-His and IgG1-Fc one day in advance and left overnight. The plates were washed three times with 1 ⁇ PBST the next day, and then blocked with 0.5% BSA for 2 hours, and the phage supernatant was collected by centrifugation.
  • the plates were washed three times with 1 ⁇ PBST, 30ul phage supernatant was added, and incubated at room temperature for 1 hour.
  • the plates were washed three times with 1 ⁇ PBST, and the secondary antibody Anti-M13-HRP was added, and the plates were incubated at room temperature for 30-60 minutes.
  • the plates were washed 7 times with 1 ⁇ PBST, and TMB colorimetric solution was added. After 3-5 minutes, 2M phosphoric acid was added to terminate the reaction, and the absorbance at 450nm was read.
  • the OD value of the sample well is more than 2 times greater than that of the control well (0.5% BSA or IgG1-Fc), it is judged as positive.
  • the positive phage is re-infected with TG1 and sequenced.
  • the amino acid sequence of each clone is analyzed according to the sequence alignment software BioEdit. Clones with the same CDR1, CDR2, and CDR3 sequences are considered to be the same antibody strain.
  • the amino acid sequence of CDR1 of MSLN45 is shown in SEQ ID NO:1
  • the amino acid sequence of CDR2 is shown in SEQ ID NO:2
  • the amino acid sequence of CDR3 is shown in SEQ ID NO:3
  • the amino acid sequence of VHH is shown in SEQ ID NO:8
  • the amino acid sequence of CDR1 of MSLN46 is shown in SEQ ID NO:9
  • the amino acid sequence of CDR2 is shown in SEQ ID NO:10
  • the amino acid sequence of CDR3 is shown in SEQ ID NO:11
  • the amino acid sequence of VHH is shown in SEQ ID NO:15
  • the amino acid sequence of CDR1 of MSLN47 is shown in SEQ ID NO:1
  • the amino acid sequence of CDR2 is shown in SEQ ID NO:2
  • the amino acid sequence of CDR3 is shown in SEQ ID NO:3
  • the amino acid sequence of VHH is shown in SEQ ID NO:17
  • the amino acid sequence of CDR1 of MSLN45 is
  • the amino acid sequence of CDR1 of MSLN50 is shown in SEQ ID NO:25, the amino acid sequence of CDR2 is shown in SEQ ID NO:26, the amino acid sequence of CDR3 is shown in SEQ ID NO:27, and the amino acid sequence of VHH is shown in SEQ ID NO:31;
  • the amino acid sequence of CDR1 of MSLN51 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, the amino acid sequence of CDR3 is shown in SEQ ID NO:3, and the amino acid sequence of VHH is shown in SEQ ID NO:33;
  • the amino acid sequence of CDR1 of MSLN52 is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is shown in SEQ ID NO:10, the amino acid sequence of CDR3 is shown in SEQ ID NO:11, and the amino acid sequence of VHH is shown in SEQ ID NO:35;
  • the amino acid sequence of CDR1 of MSLN53 is shown in SEQ ID NO
  • the expression plasmid was transfected into EXPI293 using the PEI transfection method, expressed in a 37°C cell culture incubator for 5 days, then the cell supernatant was collected and the antibody was purified using a ProteinA affinity chromatography column. Finally, an antibody protein with a purity of more than 90% was obtained.
  • SK-OV3 cells were revived and passaged. The cells were harvested on the day of the experiment. After counting, the cell density was adjusted to 1 ⁇ 10 6 /ml, 30ul per well (3 ⁇ 10 4 /well). Representative MSLN nanoantibodies 45-59 and positive antibody P4, negative antibody Caplacizumab, were added at 200nM as the highest concentration, 3-fold ratio, 7 gradients, PBS control, 30ul per well, mixed and incubated at 4°C for 1 hour. Wash twice with PBS containing 0.1% BSA, 500g, 5min, 4°C, and spin dry.
  • Use the AHC probe (Sartorius, Cat. No.: 18-5060), set the binding time to 180sec, the dissociation time to 360sec, the Baseline to 60sec, and regenerate three times, 5sec each time. Then use the software to fit the binding-dissociation curve and calculate the affinity of the antibody.
  • Example 3 VHH sequence CAR-T vector construction, lentiviral packaging and CAR-T cell preparation
  • the Genesis Platform plasmid (Genesis self-constructed) was double-digested with SphI and NotI, and the vector linearized fragment was recovered and mixed with the target antibody sequence VHH at a ratio of 1:3 (molar ratio). After homologous recombination, the competent cells of E. coli DH5 ⁇ were transformed, and after heat shock for 90s, they were spread on LB solid medium containing ampicillin resistance and cultured at 37°C overnight. Single clone colonies were picked, and the correctness of the plasmid was verified by sequencing. The plasmid was extracted by shaking and stored for later use. The core plasmid is shown in Figure 5, and the CAR-T structure is shown in Figure 6. 41BB (SEQ ID NO: 53) and CD3 ⁇ (SEQ ID NO: 55) were selected as costimulatory domains.
  • the vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third generation lentiviral vector system.
  • the three plasmids are the packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein; the PMD2.G plasmid encoding the envelope protein VSV-G; and the constructed core plasmid containing the target gene CAR.
  • the gene encoding CAR in the core plasmid based on the BBz platform plasmid is expressed under the control of the elongation factor-1 ⁇ (EF-1 ⁇ ) promoter.
  • EF-1 ⁇ elongation factor-1 ⁇
  • the method for producing CAR-T cells containing VHH sequences is as follows: human peripheral blood mononuclear cells are obtained by density gradient centrifugation; peripheral blood mononuclear cells are resuspended in medium containing 200U/ml interleukin 2 to a cell density of 2x106 /ml, and CD3/CD28 magnetic beads are added at a ratio of 1:3 (cell: magnetic beads) to activate T cells; the activated peripheral blood mononuclear cells are cultured in a 37°C CO2 incubator for 24 hours; the above-obtained lentiviral supernatant is added at a ratio of 3 for viral infection multiplicity (MOI), polybrene is added to a final concentration of 5ug/ml, the cell suspension is placed in a well plate and centrifuged at 1200rpm for 1 hour using a horizontal centrifuge; the well plate is returned to a 37°C CO2 incubator for 24 hours; centrifuged at 300g for 5 minutes, the supernatant is removed,
  • the full-length gene of MSLN was introduced into SK-OV3 cells by lentiviral packaging and lentiviral infection to prepare SK-OV3-MSLN overexpression cell lines.
  • pLV-C-GFPS was selected as a vector, and the full-length sequence of MSLN was inserted to construct the recombinant plasmid pLV-C-GFPS-MSLN.
  • the PCR and restriction digestion results are shown in Figure 7.
  • the PCR results are as follows (A): from left to right, Tiangen Marker IV, 1 and 2 are full-length bands of MSLN (about 1900bp); the results of double digestion of the vector by Not I and Xba I are as follows (B): Tiangen Marker IV, A and B vector digestion bands (about 8200bp); after lentiviral infection, the recombinant plasmid was introduced into SK-OV3 cells, and SK-OV3-MSLN positive cell lines were obtained by flow sorting, and expanded culture was used as target cells. The positive rate of SK-OV3-MSLN overexpressing cell lines was detected by flow cytometry. The results are shown in FIG8 . The positive rate of SK-OV3-MSLN overexpressing cells reached 97.98%.
  • CAR-T cells were prepared. When the amplification reached the 9th to 12th day, the positive rate was detected, and the CAR-T cells were resuspended in serum-free X-VIVO medium (manufacturer: LONZA) to a density of 4 ⁇ 10 5 /ml as effector cells; OV-CAR-3 cells were resuspended in serum-free X-VIVO medium to a density of 4 ⁇ 10 5 /ml as target cells; after the effector-target ratio was mixed at 1:1, they were statically cultured in a 37°C, 5% CO 2 incubator, and the color of the culture medium was observed every 2 days. When the culture medium turned from orange to yellow, 1 volume of culture medium was added.
  • SK-OV3-MSLN cells were used as target cells, cells infected with CAR-T lentivirus were used as effector cells, and uninfected T cells were used as control effector cells.
  • the specific experimental process is as follows: the infection efficiency of CAR was detected, and the infection ratio of CAR was adjusted to the same level in each group using uninfected T cells; the number of target cells in 200 ⁇ l X-VIVO medium was 1x10 4 /well, which was used as the experimental group; the wells containing only the same amount of effector cells as the experimental group were used as the effector cell self-release background group; the wells containing only the same amount of target cells as the experimental group were used as the target cell self-release background group; the obtained cells were cultured in a 37°C CO 2 incubator for 18 hours; 20 ⁇ l 10x lysate was added to some wells containing only target cells, and the reaction lasted for 45 minutes, which was used as the maximum release of target cells.
  • the obtained cell culture wells were centrifuged at 300g for 5 minutes, and 50 ⁇ l of supernatant was collected for detection of the release of lactate dehydrogenase LDH.
  • the detection method was referred to the instructions of CytoTox96 non-radioactive cytotoxicity kit (manufacturer: Promega).
  • the released LDH in the culture medium supernatant can be detected by coupled enzyme reaction.
  • the cell killing activity calculation formula is:
  • Toxicity % 100x (experimental group - effector cell self-release - target cell self-release + culture basic value) / (target cell maximum release - target cell self-release).
  • the killing effect of each antibody sequence on the target cells is analyzed, and antibodies with obvious killing effects are selected for in vivo functional evaluation.
  • the cell killing results are shown in Figure 10.
  • the results show that under the condition of an effector-target ratio of 1:1, MSLN-46 has the lowest cell killing, and there is little difference in cell killing between MSLN-52 and MLSN-53, which is equivalent to the positive control P4.
  • SK-OV-3-MSLN cells were used as target cells, cells infected with CAR-T lentivirus were used as effector cells, and uninfected T cells were used as control effector cells.
  • the detection method was referred to the instructions of the R&D DuoSet ELISA kit (man
  • the IFN- ⁇ detection results are shown in Figure 11 (A), and the IL2 detection results are shown in Figure 12 (B).
  • MSLN-46 secreted more IFN- ⁇ , but had a higher self-activation rate.
  • MSLN-53 had the highest secretion, followed by MSLN-52, both of which were better than the positive control P4.
  • mice were divided into 3 groups: T cells, P4, MSLN-VHH, 5 mice in each group, each group was administered by tail vein, and 1E7 frozen CAR-T cells were infused back.
  • the body weight and tumor size changes of each group of mice after administration were recorded, and their various biological reactions were observed. The results are shown in Figure 12A when observed until D72.

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Abstract

一种分离的抗原结合蛋白，其能够靶向MSLN，所述分离的抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。以及包含所述抗原结合蛋白的嵌合抗原受体，及其在预防和/或治疗肿瘤方面的用途。

Description

靶向MSLN的抗原结合蛋白 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向MSLN的抗原结合蛋白，包含所述抗原结合蛋白的嵌合抗原受体，及其应用。
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。
间皮素是一种在腹膜、胸膜和心包腔的间皮细胞系的细胞表面存在的糖蛋白。间皮素在间皮瘤即癌症/肿瘤细胞、卵巢癌、胰腺癌、胃癌、肺癌和子宫内膜癌中优势表达(过表达)。与此相反，其表达在正常细胞例如间皮细胞中受限。
因此，作为有开发潜力的靶点，亟需开发更多能够有效靶向MSLN的结合蛋白，对MSLN过表达引起的疾病起到更有效的治疗作用。
发明内容
本申请提供了一种分离的抗原结合蛋白，其能够特异性结合MSLN。本申请还提供了包含所述抗原结合蛋白的嵌合抗原受体，以及包含和/或表达该嵌合抗原受体的细胞，所述细胞具有以下一种或多种特点：(1)扩增能力强；(2)能够杀伤表达MSLN的靶细胞；(3)在靶细胞刺激下分泌细胞因子；(4)抑制肿瘤细胞的生长。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合间皮素(MLSN)蛋白。
在某些实施方式中，所述MSLN为人MSLN。
在某些实施方式中，所述分离的抗原结合蛋白能够与参比抗体竞争结合MSLN，所述参比抗体包含HCDR1，HCDR2以及HCDR3，所述HCDR1，HCDR2和HCDR3的氨基酸序列选自下述任一组：
1)所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列；
2)所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列；
3)所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；以及
4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，且所述HCDR3包含SEQ ID NO:11、SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:27中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO:10、SEQ ID NO:2、SEQ ID NO:19和SEQ ID NO:26中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，且所述HCDR1包含SEQ ID NO:9、SEQ ID NO:1、SEQ ID NO:18和SEQ ID NO:25中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:9、SEQ ID NO:1、SEQ ID NO:18和SEQ ID NO:25中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10、SEQ ID NO:2、SEQ ID NO:19和SEQ ID NO:26中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:11、SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:27中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3包含选自下述任一组的氨基酸序列：
1)所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列；
2)所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列；
3)所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；以及
4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述H-FR1包含SEQ ID NO:4、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:21、SEQ ID NO:28、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42和SEQ ID NO:44中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:51所示的氨基酸序列。
在某些实施方式中，所述H-FR2包含SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:22和SEQ ID NO:29中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO:23和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4包含选自下述任一组氨基酸序列：
1)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
2)所述H-FR1包含SEQ ID NO:12所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
3)所述H-FR1包含SEQ ID NO:16所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
4)所述H-FR1包含SEQ ID NO:21所示的氨基酸序列，所述H-FR2包含SEQ ID NO:22所示的氨基酸序列，所述H-FR3包含SEQ ID NO:23所示的氨基酸序列，且所述H- FR4包含SEQ ID NO:7所示的氨基酸序列；
5)所述H-FR1包含SEQ ID NO:28所示的氨基酸序列，所述H-FR2包含SEQ ID NO:29所示的氨基酸序列，所述H-FR3包含SEQ ID NO:30所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
6)所述H-FR1包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
7)所述H-FR1包含SEQ ID NO:34所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
8)所述H-FR1包含SEQ ID NO:36所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
9)所述H-FR1包含SEQ ID NO:38所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
10)所述H-FR1包含SEQ ID NO:40所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
11)所述H-FR1包含SEQ ID NO:42所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；以及
12)所述H-FR1包含SEQ ID NO:44所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，且所述VH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述抗原结合片段为VHH。
在某些实施方式中，所述分离的抗原结合蛋白为VHH，所述VHH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链恒定区，所述抗体重链恒定区源自IgG的重链恒定区。
在某些实施方式中，所述抗体重链恒定区源自人IgG。
在某些实施方式中，所述抗体重链恒定区源自人IgG1的重链恒定区。
在某些实施方式中，所述抗体重链恒定区包含IgG的Fc区。
在某些实施方式中，所述Fc区包含SEQ ID NO:61所示的氨基酸序列。
另一方面，本申请还提供了一种嵌合抗原受体，其包含靶向部分，所述靶向部分包含本申请所述的抗原结合蛋白。
在某些实施方式中，所述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:53所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号传导域：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PBj14Nef、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:55所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、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，所述跨膜区包含SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，在所述嵌合抗原受体中，其在靶向部分和跨膜区之间包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、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，所述铰链区包含SEQ ID NO:56所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含信号肽。
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:57所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段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:58所示的氨基酸序列。
另一方面，本申请还提供了一种多肽，其包含所述抗原结合蛋白。
另一方面，本申请还提供了一种或多种分离的核酸分子，其编码所述分离的抗原结合蛋白和/或所述嵌合抗原受体。
在某些实施方式中，所述核酸分子包含启动子。
在某些实施方式中，所述启动子为组成型启动子。
在某些实施方式中，所述启动子为EF1α启动子。
另一方面，本申请还提供了一种载体，其包含所述核酸分子。
在某些实施方式中，所述载体包含病毒载体。
在某些实施方式中，所述载体包含慢病毒载体。
另一方面，本申请还提供了一种细胞，其包含所述抗原结合蛋白，所述嵌合抗原受体，所述核酸分子和/或所述载体。
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，所述细胞包含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。
在某些实施方式中，所述细胞含包含和/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:58所示的氨基酸序列。
另一方面，本申请还提供了制备经修饰的免疫效应细胞的方法，所述方法包括在使得所述抗原结合蛋白和/或所述嵌合抗原受体表达的条件下，培养所述细胞。
另一方面，本申请还提供了制备经修饰的免疫效应细胞的方法，其包括像免疫效应细胞中引入所述载体。
另一方面，本申请还提供了药物组合物，其包含所述的分离的抗原结合蛋白，所述嵌合抗原受体，所述多肽，所述核酸分子，所述载体和/或所述细胞，以及任选地药学上可接受的载体。
另一方面，本申请还提供了所述的分离的抗原结合蛋白，所述的嵌合抗原受体，所述的多肽，所述的核酸分子，所述的载体，所述的细胞，和/或所述的药物组合物，在制备药物中的用途，所述药物用于预防、治疗和/或缓解与MSLN异常表达相关的疾病或病症。
在某些实施方式中，所述与MSLN异常表达相关的疾病或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括表达MSLN抗原的肿瘤。
在某些实施方式中，所述肿瘤包括卵巢癌、胰腺癌、胃癌、间皮细胞癌、胆管癌、三阴性乳腺癌和/或子宫内膜癌。
另一方面，本申请还提供了一种预防、治疗和/或缓解与MSLN异常表达相关的疾病或病症的方法，所述方法包括向有需要的受试者施用所述的分离的抗原结合蛋白，所述的嵌合抗原受体，所述的多肽，所述的核酸分子，所述的载体，所述的细胞，和/或所述的药物组合物。
在某些实施方式中，所述与MSLN异常表达相关的疾病或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括表达MSLN抗原的肿瘤。
在某些实施方式中，所述肿瘤包括卵巢癌、胰腺癌、胃癌、间皮细胞癌、胆管癌、三阴性乳腺癌和/或子宫内膜癌。
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显示的是噬菌体Pool ELISA结果。
图2A-D显示的是阳性克隆的筛选结果。
图3显示的是流式检测本申请所属的抗原结合蛋白与MSLN的特异性结合。
图4显示的是ForteBio检测结果。
图5显示的是pCORE-VHH质粒图谱。
图6显示的是MSLN-VHH CAR结构示意图。
图7A显示的是PCR结果电泳图。
图7B显示的是载体酶切电泳图
图8显示的是SK-OV3-MSLN过表达细胞株流式阳性率检测。
图9显示的是CAR-T细胞经靶细胞反复刺激后的扩增倍数。
图10显示的是CAR-T细胞体外细胞杀伤结果。
图11A显示的是CAR-T细胞体外IFN-γ细胞因子分泌结果。
图11B显示的是CAR-T细胞体外IL-2细胞因子分泌结果。
图12A显示的是施用本申请所述CAR-T细胞后，肿瘤生长曲线图。
图12B显示的是施用本申请所述CAR-T细胞后，动物体重曲线图。
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“MSLN”也称间皮素，或CAK1抗原或前幼巨核细胞增强因子，是存在于正常间皮细胞上的蛋白质，并且在一些肿瘤细胞中过表达。在本申请中，该术语可以包含MSLN蛋白或其功能活性片段。在本申请中，该术语还可以包含MSLN蛋白的同源物、类似物或变体。例如，所述MSLN可以包括人MSLN。
在本申请中，术语“分离的抗原结合蛋白”通常是指脱离了其天然存在状态的具有抗原结合能力的蛋白。所述“分离的抗原结合蛋白”可以包含结合抗原的部分和任选的，允许抗原结合部分采用促进其结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的可变区(CDR)或CDR衍生物的备选蛋白框架区或人工框架区。例如，所述抗原结合蛋白可以包括抗体或其抗原结合片段。例如，所述抗原结合蛋白可以结合MSLN蛋白。例如，所述抗原结合蛋白可以与参比抗体竞争结合MSLN蛋白。例如，所述抗原结合蛋白可以包括抗体重链可变区VH。例如，所述抗原结合蛋白可以包括源自抗体重链可变区VH中的至少一个CDR。例如，所述VH可以包含HCDR3、HCDR2和/或HCDR1。例如，所述VH可以包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连。例如，所述VH可以包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间。例如，所述VH可以包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间。例如，所述VH可以包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连。例如，所述抗原结合蛋白可以为VHH。例如，所述的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自IgG。例如，所述抗体重链恒定区可 以源自人IgG。例如，所述抗体重链恒定区可以源自人IgG1。
使用的术语“抗体”包括完整抗体和其结合片段。通常，片段与其来源的完整抗体竞争性地与抗原特异性结合。任选，抗体或其结合片段可以与其它蛋白质化学结合，或者与其它蛋白质以融合蛋白质的形式表达。例如，所述抗体可以是单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。例如，所述抗体或其结合片段的结合蛋白质可以包括MSLN。例如，所述抗体或其结合片段对MSLN可以有特异性。
术语“抗原结合片段”是指完整抗体的一部分并且是指完整抗体的抗原决定可变区。例如，所述抗原结合片段的可以包括Fab、Fab'、F(ab')2、Fv片段和单链Fv片段、串联Fv片段、VHH、双特异性抗体。例如，所述抗原结合片段可以是VHH。例如，所述抗原结合片段可以结合MSLN。例如，所述抗原结合片段对MSLN可以有特异性。
在本申请中，术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体。VHH也可称为纳米抗体(Nanobody)(Nb)和/或单域抗体。例如，所述VHH可以结合MSLN。例如，所述VHH对MSLN可以有特异性。
在本申请中，所述抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链。每条重链由重链可变区(VH)和重链恒定区组成。术语“重链恒定区”由三个结构域CH1，CH2和CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区组成。术语“轻链恒定区”由一个结构域CL组成。VH和VL区可以进一步细分为高变区，称为互补决定区(CDR)，散布有更保守的区域，称为构架区(FR)。每个VH和VL由以下列顺序从氨基末端到羧基末端排列的三个CDR和四个FR组成:FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合。
在本申请中，术语“参比抗体”是指可与所述分离的抗原结合蛋白竞争性结合于MSLN相同表位的抗体。所述参比抗体可以包括重链可变区VH。例如，所述参比抗体可以具有3个CDR序列。例如，所述参比抗体的VH可以包括HCDR1、HCDR2和HCDR3。例如，所述的CDR序列可以与所述分离的抗原结合蛋白的CDR序列一致。
在本申请中，术语“IgG”是指属于基本上由公认的免疫球蛋白γ基因编码的抗体类别的多肽。在人类中，此类别包括IgG1、IgG2、IgG3和IgG4。在小鼠中，此类别包括IgG1、IgG2a、IgG2b和IgG3。
在本申请中，术语“嵌合抗原受体”(CAR)通常是指至少包含特异性地结合抗原或靶标的胞外结构域、跨膜区以及胞内结构域的重组多肽。例如，所述胞外结构域与所述跨膜区之间包括铰链区。例如，所述嵌合抗原受体还可以包括低密度脂蛋白受体相关蛋白或其片段。 例如，所述嵌合抗原受体可以包括信号肽。CAR的胞外结构域与靶细胞表面上的靶抗原结合导致CAR聚簇并将激活刺激传送给含CAR细胞。CAR重定向免疫效应细胞的特异性，并且触发了增殖、细胞因子产生、能够以不依赖主要组织相容性(MHC)的方式介导表达靶抗原的细胞死亡的分子的吞噬作用和/或产生。例如，所述胞外结构可以包括上述的抗原结合蛋白。例如，所述胞外结构可以特异性结合MSLN。
在本申请中，术语“胞内结构域”指意思是包括足以转导活化信号的任何截短部分的细胞内结构域。所述胞内结构域可以包括细胞内信号区域和/或共刺激信号区域。术语“细胞内信号区域”指可以产生促进含CAR细胞(例如CART细胞或表达CAR的NK细胞)的免疫效应子功能的信号的胞内区域。例如，所述胞内信号区域可以包括选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。例如，所述胞内信号区域可以是源自CD3ζ的信号传导结构域。术语“共刺激信号区域”是指所述胞内信号区域中能够转导效应信号的CAR的一部分。例如，所述共刺激信号区域可以包括源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，所述共刺激信号区域可以是源自4-1BB的胞内共刺激信号区域。
在本申请中，术语“跨膜区”指能够跨越细胞质膜的肽、多肽或蛋白质的结构域。可采用这些结构域将胞外结构域锚定在细胞膜上。例如，所述跨膜区可以包含选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。例如，所述跨膜区可以源自CD8的跨膜区。
在本申请中，术语“铰链区”表示抗体重链多肽中连接CH1结构域和CH2结构域的一部分，例如，从根据Kabat的EU编号系统的约第216位至约第230位。铰链区通常是由两条具有相同氨基酸序列的多肽组成的二聚体分子。铰链区一般包括约25个氨基酸残基，是柔性的，允许抗原结合区独立的移动。铰链区可以细分为3个结构域：上、中、下铰链结构域。 例如，所述铰链区可以包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。例如，所述铰链区可以源自CD8的铰链区。
在本申请中，术语“低密度脂蛋白受体相关蛋白(Low-density lipoproteinreceptor-related protein)”指一种属于内吞性受体的细胞表面蛋白，在生物体内广泛分布并有很大的组织间差异性，主要功能是摄取胆固醇进入细胞内用以细胞增殖和固醇类激素及胆汁酸盐的合成。例如，所述低密度脂蛋白受体相关蛋白可以来自于任何脊椎动物。例如，所述低密度脂蛋白受体相关蛋白或其片段可以位于所述胞内信号区域的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白6或其片段。
在本申请中，术语“信号肽”是指处于新生CAR蛋白的氨基末端(N-末端)的前导序列，其在翻译时或在翻译后将新生蛋白引导到内质网并后续表面表达。例如，所述信号肽源自CD8蛋白的信号肽。
在本申请中，术语“多肽”、“肽”和“蛋白”在本文中可以互换，是指氨基酸残基的聚合物。这个术语可用于指一个或多个氨基酸残基是其相应的天然氨基酸的人工合成化学模拟物的氨基酸聚合物，也可用于指天然的氨基酸聚合物，那些含修饰残基的氨基酸聚合物以及非天然的氨基酸聚合物。例如，所述多肽可以包括所述的抗原结合蛋白。
在本申请中，术语“核酸分子”包括DNA分子和RNA分子。一个核酸分子可以是单链或双链的，但优选为双链DNA。术语“启动子”通常是指一个DNA序列，可以调节与启动子操作性连接的所选DNA序列的表达，从而影响细胞中所选DNA序列的表达。例如，所述核酸分子可以编码包括所属抗原结合蛋白和/或所述嵌合抗原受体。例如，所述核酸分子可以包括启动子。例如，所述启动子可以是组成型启动子。例如，所述启动子可以是EF1α启动子。
在本申请中，术语“载体”通常是指可以附着本申请的一种或多种核酸分子的分子。例如，所述载体可以是病毒载体。例如，所述载体可以是慢病毒载体。
在本申请中，术语“细胞”指可以向其转染核酸的细胞，术语“细胞”包括用于质粒繁殖的原核细胞，和用于核酸表达和编码多肽产生的真核细胞。例如，细胞可以包括所述的抗原结合蛋白，所述的核酸分子和/或所述的载体。例如，所述细胞可以是免疫效应细胞。术语“免疫 效应细胞”通常是指参与免疫应答，行使效应功能的免疫细胞。例如，所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等。例如，免疫效应细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如，免疫效应细胞可以是T细胞。
在本申请中，术语“药物组合物”通常指适合施用于哺乳动物个体的化学或生物组合物。例如，所述药物组合物可以包括所述抗原结合蛋白，所述嵌合抗原受体，所述多肽，所述核酸分子，所述载体和/或所述细胞，以及可选的药学上可接受的载体。所述药物组合物可以用于预防、治疗和/或缓解与MSLN异常表达相关的疾病或病症。例如，所述与MSLN异常表达相关的疾病或病症可以包括肿瘤。例如，所述肿瘤包括实体瘤和/或非实体瘤。
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/或以更长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，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MSLN)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MSLN)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“包含”通常是指包括、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
分离的抗原结合蛋白
在本申请中，所述抗原结合蛋白可以包括抗体或其抗原结合片段。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，所述抗体可以包括单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
CDR
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、 AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在本申请中，所述分离的抗原结合蛋白通过Kabat编码系统定义。
在本申请中，所述分离的抗原结合蛋白可以包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，所述HCDR3可包含SEQ ID NO:11、SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:27中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR2，所述HCDR2可包含SEQ ID NO:10、SEQ ID NO:2、SEQ ID NO:19和SEQ ID NO:26中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，所述HCDR1可包含SEQ ID NO:9、SEQ ID NO:1、SEQ ID NO:18和SEQ ID NO:25中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:9、SEQ ID NO:1、SEQ ID NO:18和SEQ ID NO:25中任一项所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:10、SEQ ID NO:2、SEQ ID NO:19和SEQ ID NO:26中任一项所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:11、SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:27中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3可包含选自下述任一组的氨基酸序列：
1)所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列；
2)所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO: 2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列；
3)所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；以及
4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
FR
本申请中，抗体框架区FR指抗体可变区中存在于分歧性更高的(即高变)CDR之间的部分。此类框架区典型地称为框架1至4(FR1、FR2、FR3和FR4)且提供用于在三维空间中呈现三个CDR的骨架，以形成抗原结合表面。
在本申请中，所述分离的抗原结合蛋白可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:50所示的氨基酸序列。
在本申请中，所述H-FR1可包含SEQ ID NO:4、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:21、SEQ ID NO:28、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42和SEQ ID NO:44中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:51所示的氨基酸序列。
在本申请中，所述H-FR2可包含SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:22和SEQ ID NO:29中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:52所示的氨基酸序列。
在本申请中，所述H-FR3可包含SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO:23和SEQ ID NO:30中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR1，H-FR2，H-FR3和H-FR4。
例如，所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:12所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包 含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:16所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:21所示的氨基酸序列，所述H-FR2包含SEQ ID NO:22所示的氨基酸序列，所述H-FR3包含SEQ ID NO:23所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:28所示的氨基酸序列，所述H-FR2包含SEQ ID NO:29所示的氨基酸序列，所述H-FR3包含SEQ ID NO:30所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:34所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:36所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:38所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:40所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:42所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
例如，所述H-FR1包含SEQ ID NO:44所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包 含SEQ ID NO:7所示的氨基酸序列。
VH/VHH
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH，且所述VH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在本申请中，所述抗原结合片段可以是VHH，且所述VHH可以包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
重链恒定区
在本申请中，所述的分离的抗原结合蛋白可以包括重链恒定区。所述重链恒定区是指包含至少三个重链恒定结构域CH1、CH2、和CH3的区域。非限制性示例性重链恒定区包括γ、δ、和α。非限制性示例性重链恒定区还包括ε和μ。每个重链恒定区对应于一种抗体同种型。例如，包含γ恒定区的抗体为IgG抗体，包含δ恒定区的抗体为IgD抗体，包含α恒定区的抗体为IgA抗体。此外，包含μ恒定区的抗体为IgM抗体，包含ε恒定区的抗体为IgE抗体。某些同种型可以进一步细分为亚类。例如，IgG抗体包括但不限于，IgG1(包含γ1恒定区)、IgG2(包含γ2恒定区)、IgG3(包含γ3恒定区)、和IgG4(包含γ4恒定区)抗体；IgA抗体包括但不限于，IgA1(包含α1恒定区)和IgA2(包含α2恒定区)抗体；IgM包括但不限于，IgM1和IgM2。
在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自IgG。在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自人IgG。在本申请中，所述分离的抗原结合蛋白可以包含抗体重链恒定区，所述抗体重链恒定区可以源自人IgG1。在本申请中，所述抗原结合蛋白的重链恒定区可以包含IgG的Fc区。例如，所述Fc区可以包含SEQ ID NO:61所示的氨基酸序列。
嵌合抗原受体
另一方面，本申请还提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体(CAR)可以包含结合MSLN蛋白的靶向部分，例如，结合MSLN蛋白的靶向部分可以是本申请所述的抗原结合蛋白。
例如，本申请的CAR可包含VHH，所述VHH可包含SEQ ID NO:8、SEQ ID NO:15、 SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在本申请中，所述CAR包含胞外的结合MSLN蛋白的靶向部分外，还可包含胞内结构域。
在本申请中，所述CAR可以包括胞内的共刺激信号区域，其可提供刺激信号。例如，所述共刺激信号区域可以包括选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
例如，所述共刺激信号区域可以为源自4-1BB的胞内共刺激信号区域。例如，所述共刺激信号区域可以包含SEQ ID NO:53所示的氨基酸序列。
在某些情形中，所述CAR可包含胞内信号区域，其可以包含至少有一个ITAM基序的结构域。所述胞内信号传导结构域可以将激活信号传输到细胞内部。例如，所述胞内信号区域可以包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和其他至少包含一个ITAM的结构域。
例如，所述胞内信号区域可以为源自CD3ζ的信号传导结构域。例如，所述胞内信号区域可以包含SEQ ID NO:55所示的氨基酸序列。
在某些情形中，所述CAR可包含跨膜域，所述跨膜域是细胞表面蛋白中一段跨越细胞膜的序列，其可以包含疏水性alpha螺旋。所述跨膜域可以源自任意的I型跨膜蛋白。跨膜域可以是预测为形成疏水螺旋的合成序列。例如，所述跨膜区可以包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
例如，所述跨膜区可以为源自CD8的跨膜区。例如，所述跨膜区可以包含SEQ ID NO: 54所示的氨基酸序列。
在某些情形中，所述CAR可包含铰链区，所述铰链区可以位于所述胞外的靶向部分和所述跨膜域之间。例如，所述铰链区可以包含选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
例如，所述铰链区可以为源自CD8的铰链区。例如，所述铰链区可以包含SEQ ID NO:56所示的氨基酸序列。
在本申请中，所述CAR在所述结合MSLN蛋白的靶向部分的N端还可包含信号肽。例如，所述信号肽可以为源自CD8蛋白的信号肽。例如，所述信号肽可以包含SEQ ID NO:57所示的氨基酸序列。
在本申请中，所述CAR还可包含低密度脂蛋白受体相关蛋白或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以位于所述CAR的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包括低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以是低密度脂蛋白受体相关蛋白5和/或6或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包括SEQ ID NO:58所示的氨基酸序列。例如，编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子可以包含SEQ ID NO:59所示的核苷酸序列。
在本申请中，所述CAR中的所述低密度脂蛋白受体相关蛋白或其片段的序列可通过自剪切肽(例如，T2A、P2A、E2A等2A肽)连接至CAR的C端序列。例如，所述低密度脂蛋白受体相关蛋白或其片段可以通过T2A与胞内信号区域的C端连接。例如，所述剪切肽可以包含SEQ ID NO:60所示的氨基酸序列。
在本申请中，自N端至C端，所述CAR可依次包含结合MSLN蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激信号区域和所述胞内信号区域。例如，自N端至C端，所述CAR可依次包含所述VHH，源自CD8的铰链区，源自CD8的跨膜区，源自4-1BB的共刺激信号区域，和源自CD3ζ的胞内信号区域，且所述VHH可包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在本申请中，自N端至C端，所述CAR可依次包含结合MSLN蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激信 号区域、所述胞内信号区域和所述低密度脂蛋白受体相关蛋白或其片段。例如，自N端至C端，所述CAR可依次包含所述VHH，源自CD8的铰链区，源自CD8的跨膜区，源自4-1BB的共刺激信号区域，源自CD3ζ的胞内信号区域，和包含SEQ ID NO:58示的氨基酸序列的低密度脂蛋白受体相关蛋白或其片段，且所述可包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
在本申请中，自N端至C端，所述CAR可依次包含信号肽、结合MSLN蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激信号区域和所述胞内信号区域。
在本申请中，自N端至C端，所述CAR可依次包含信号肽、结合MSLN蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激信号区域、所述胞内信号区域和所述低密度脂蛋白受体相关蛋白或其片段。
核酸分子
另一方面，本申请还提供了一种或多种核酸分子，所述核酸分子可以是任意长度的分离形式的核苷酸、脱氧核苷酸和/核糖核苷酸，可以编码所述分离的抗原结合蛋白和/或所述的嵌合抗原受体。
例如，所述核酸分子可以包括启动子。例如，所述启动子可以是组成型启动子。例如，所述启动子可以是EF1α启动子。
另一方面，本申请还提供了一种或多种核酸分子，所述核酸分子包含能够在细胞中表达所述嵌合抗原受体和所述低密度脂蛋白受体相关蛋白或其片段的序列。在本申请中，编码所述嵌合抗原蛋白的核酸序列可以与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通过剪切肽连接。
载体
另一方面，本申请还提供了一种载体，所述载体可以包括所述的核酸分子。所述载体可以转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达。例如，载体可以包括启动子、转录子、增强子、复制子、选择元件和报告基因。例如，载体可以包括协助进入细胞的成分。为了使所述核酸分子在载体中复制，所述核酸分子的5’端和3’端还可以包含长末端重复序列。
例如，所述载体可以是病毒载体。例如，所述载体可以是慢病毒载体。
细胞
另一方面，本申请还提供了细胞，所述细胞可以包括所述的分离的抗原结合蛋白、所述的嵌合抗原受体、所述的核酸分子和/或所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。
在某些实施方案中，所述的细胞可以是免疫效应细胞。在某些实施方案中，所述的细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如,
在某些实施方案中，所述的细胞可以是T细胞。
在本申请中，所述细胞可以包含和/或表达所述CAR。在本申请中，所述细胞可以包含和/或表达所述CAR和所述低密度脂蛋白受体相关蛋白或其片段。
药物组合物
另一方面，本申请还提供了一种药物组合物，可以包括所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的佐剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物可以包括液体、冷冻和冻干组合物。
在某些实施方案中，所述药学上可接受的佐剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
制备方法
另一方面，本申请还提供了制备所述分离的抗原结合蛋白和/或所述嵌合抗原受体的方法。所述方法可以包括在使得所述抗原受体和/或所述嵌合抗原受体表达的条件下，培养所述细胞。
本申请还提供了制备经修饰的免疫效应细胞的方法，所述方法可以包括向免疫细胞中引 入所述载体。
用途
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防、缓解和/或治疗疾病和/或病症的方法，所述方法可以包括向受试者施用所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物，其用于预防、缓解和/或治疗疾病和/或病症。
在本申请中，所述疾病和/或病症可以包括与MSLN表达异常相关的疾病和/或病症。
在本申请中，所述疾病和/或病症可以包括肿瘤。
在本申请中，所述肿瘤可以包括实体瘤和/或非实体瘤。
在本申请中，所述肿瘤可以包括血液瘤和/或淋巴瘤。
在本申请中，所述肿瘤可以包括表达MSLN抗原的肿瘤。
在本申请中，所述肿瘤可以包括卵巢癌、胰腺癌、胃癌、间皮细胞癌、胆管癌、三阴性乳腺癌和/或子宫内膜癌。
在本申请中，所述受试者可以包括人类或非人类动物。
另一方面，本申请提供了多肽，其包含所述分离的抗原结合蛋白。
另一方面，本申请提供了试剂盒或给药装置，其包括所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物。
另一方面，本申请还提供了一种检测MSLN的存在和/或含量的方法，所述方法包括施用本申请所述的分离的抗原结合蛋白，所述的多肽，所述嵌合抗原受体，和/或所述细胞。在某些实施方式中，所述方法可以为体外方法。在某些实施方式中，所述方法可以为非诊断和治疗为目的的方法。
另一方面，本申请还提供了一种检测MSLN的存在和/或含量的试剂盒，其包括本申请所述的分离的抗原结合蛋白，所述的多肽，所述嵌合抗原受体，和/或所述细胞。
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筛选靶向MSLN-his的纳米抗体
1.1构建噬菌体展示免疫纳米抗体库
首先表达重组人MSLN的免疫羊驼并细胞外结构域(ECD)，该结构域连着Fc片段，方便后续的蛋白纯化。根据本领域已知的方案，使用MSLN-Fc抗原对羊驼进行免疫，动物免疫外包给爱康得生物医学技术(苏州)有限公司，单次免疫剂量为1-2mg蛋白。第三次和第四次免疫结束后，分别采集5mL外周血，分离血清，采用ELISA检测免疫效果，ELISA免疫效价达到1:16000以上(包被抗原5ug/mL，OD值大于2.0)，冲击免疫后，采集150mL外周血。然后分离PBMC并使用QIAGEN公司提供的RNA提取试剂盒提取总RNA。最后使用Super-Script III FIRST STRANDSUPERMIX试剂盒将提取的RNA反转录成cDNA。
在本领域已知的方案下，通过巢式PCR扩增重链抗体的可变区(VHH)，所用引物如下：第一轮PCR的引物：
CALL001：5′-GTCCTGGCTGCTCTTCTACAAGG-3′(SEQ ID NO:46)；
CALL002：5′-GGTACGTGCTGTTGAACTGTTCC-3′(SEQ ID NO:47)。
第二轮PCR的引物：
VHH-Back：5′-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3′(SEQ ID NO:48)；
VHH-For：5′-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3′(SEQ ID NO:49)。
胶回收目标片段，并使用限制性内切酶(来自Thermo)PstⅠ和Eco91Ⅰ将其克隆至噬菌体展示的载体pMES4(GenBank GQ907248)中。将质粒脱盐后，电转至电转感受态大肠杆菌TG1中，构建噬菌体展示纳米抗体库NanoMSLN，并对文库进行多样性评价。通过梯度稀释点板，计算库容大小为1×109，之后随即挑取24个单克隆进行测序，一共测序成功21条，其中2条为无用序列，另外有2条重复序列。所以一共有85.7％(18/21)的序列可用的，而NanoMSLN总的多样性为8.5×108。
1.2靶向MSLN进行淘选
用IgG-Fc和MSLN-His蛋白10μg/ml包被平板，4℃放置过夜。隔天用1×PBST(PBS中含有0.05％Tween20)清洗3次后，0.5％BSA室温封闭2小时，1×PBST清洗3次，在IgG1-Fc孔加入100μl噬菌体库NanoMSLN进行负筛，1小时后将IgG1-Fc中的噬菌体转移至MSLN-His的孔中进行正筛，1.5小时后用1×PBST清洗10次，以洗掉不结合抗原的噬菌体，最后用pH＝2.2的Glycine-HCl进行洗脱，100μl/well，然后用pH＝8.0的Tris-HCl中和，取一半洗脱出来的噬菌体感染处于对数生长期的TG1，半小时后超感染M13KO7，过夜培养。隔天沉淀 噬菌体用于下一轮的筛选。相似的筛选过程重复4轮，第二轮正筛后，用1×PBST清洗20次，第三轮清洗30次，第四轮清洗40次。
1.3 Pool ELISA
提前一天用2μg/ml的MSLN-His，IgG1-Fc和0.5％BSA包被96孔板，静置过夜。隔天用1×PBST清洗三次，然后用0.5％BSA封闭2小时。封闭结束后用1×PBST清洗三次，加入100ul每一轮的噬菌体库，室温震荡孵育1小时，用1×PBST清洗三次后加入二抗Anti-M13-HRP(Sino Biological，货号：11973-MM05T-H)和Anti-Flag-HRP(abcam，货号：ab1162)，室温孵育30-60分钟，用1×PBST清洗7次后加入TMB显色液，3-5分钟后加入2M磷酸终止反应，读取450nm波长的吸收值。
结果如图1所示，结果显示用噬菌体展示免疫纳米抗体库NanoMSLN筛选3轮后，出现明显的特异性富集。
1.4噬菌体酶联免疫方法(ELISA)鉴定特异性阳性单克隆
3轮淘选后，将第1，2，3轮预留的output涂板2YT/carb，隔天各随机挑取96个单菌落于800μl的2YT/carb/M13KO7中，过夜震荡培养，产生噬菌体。提前一天用2μg/ml的MSLN-His和IgG1-Fc包被384孔板，静置过夜。隔天用1×PBST清洗三次，然后用0.5％BSA封闭2小时，同时离心收集噬菌体上清。封闭结束后用1×PBST清洗三次，加入30ul噬菌体上清，室温震荡孵育1小时，用1×PBST清洗三次后加入二抗Anti-M13-HRP，室温孵育30-60分钟，用1×PBST清洗7次后加入TMB显色液，3-5分钟后加入2M磷酸终止反应，读取450nm波长的吸收值。当样品孔的OD值大于对照孔(0.5％BSA或者IgG1-Fc)2倍以上时，判定为阳性。最后将阳性噬菌体重新感染TG1，并进行测序。根据序列比对软件BioEdit分析各个克隆的氨基酸序列。把CDR1、CDR2、CDR3序列相同的克隆视为同一抗体株。
经过单克隆ELISA和阳性克隆测序后共获得15条特异性序列。其中，MSLN45的CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示，VHH的氨基酸序列如SEQ ID NO:8所示；MSLN46的CDR1的氨基酸序列如SEQ ID NO:9所示，CDR2的氨基酸序列如SEQ ID NO:10所示，CDR3的氨基酸序列如SEQ ID NO:11所示，VHH的氨基酸序列如SEQ ID NO:15所示；MSLN47的CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如CDR2所示，CDR3的氨基酸序列如SEQ ID NO:3所示，VHH的氨基酸序列如SEQ ID NO:17所示；MSLN48的CDR1的氨基酸序列如SEQ ID NO:18所示，CDR2的氨基酸序列如SEQ ID NO:19所示，CDR3的氨基酸序列如SEQ ID NO:20所示，VHH的氨基酸序列如SEQ ID NO:24所示； MSLN50的CDR1的氨基酸序列如SEQ ID NO:25所示，CDR2的氨基酸序列如SEQ ID NO:26所示，CDR3的氨基酸序列如SEQ ID NO:27所示，VHH的氨基酸序列如SEQ ID NO:31所示；MSLN51的CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示，VHH的的氨基酸序列如SEQ ID NO:33所示；MSLN52的CDR1的氨基酸序列如SEQ ID NO:9所示，CDR2的氨基酸序列如SEQ ID NO:10所示，CDR3的氨基酸序列如SEQ ID NO:11所示，VHH的氨基酸序列如SEQ ID NO:35所示；MSLN53的CDR1的氨基酸序列如SEQ ID NO:9所示，CDR2的氨基酸序列如SEQ ID NO:10所示，CDR3的氨基酸序列如SEQ ID NO:11所示，VHH的氨基酸序列如SEQ ID NO:37所示；MSLN55的CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示，VHH的氨基酸序列如SEQ ID NO:39所示；MSLN57的CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示，VHH的氨基酸序列如SEQ ID NO:41所示；MSLN58的CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示，VHH的氨基酸序列如SEQ ID NO:43所示；MSLN59的CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示，VHH的氨基酸序列如SEQ ID NO:45所示。
实施例2鉴定靶向MSLN的纳米抗体的结合活性
2.1 VHH-Fc融合蛋白在真核细胞中的表达和纯化
从测序结果中挑选特异性的抗体序列，将其克隆至载体pcDNA3.4中，将重组质粒转化到大肠杆菌DH5α(天根生化科技有限公司，货号：CB101-02)中，然后将菌液均匀地涂在含有100μg/ml氨苄青霉素的2YT固体培养基上，37℃静置过夜。隔天挑取单克隆，37℃摇床培养6个小时左右，取一半的菌液送测序，另一半保存在4℃。将测序正确的克隆放大培养，质粒中抽。采用PEI转染的方法将表达质粒转染至EXPI293中，在37℃细胞培养箱中表达5天，之后收集细胞上清，用ProteinA亲和层析柱纯化抗体。最终获得纯度达90％以上的抗体蛋白。
2.2酶联免疫吸附实验检测VHH-Fc对人MSLN蛋白的特异性结合
用2μg/ml的MSLN-His和5％脱脂牛奶包被高吸附384孔板，4℃静置过夜，1×PBST清洗3次，5％脱脂牛奶室温封闭2小时。封闭期间，稀释抗体MSLN-VHH，起始浓度为200nM，2.5倍稀释，共11个梯度。封闭结束后，1×PT清洗3次，加入对应的稀释的一抗，30ul/well， 室温震荡孵育1小时，用1×PBST清洗三次后，加入二抗Anti-human IgG Fc Antibody HRP(abcam，货号：ab99759)，室温孵育30-60分钟，用1×PBST清洗7次后加入TMB显色液，3-5分钟后加入2M磷酸终止反应，读取450nm波长的吸收值。
结果如图2A-D所示，选择其中单克隆ELISA信号高的13条序列做ELISA检测，除了MSLN49，其他序列均可结合MSLN-his蛋白。
2.3通过流式细胞术鉴定二价VHH-Fc与SK-VO3的结合活性
复苏SK-OV3细胞并传代，实验当天收获细胞，计数后调整细胞密度为1×106/ml，每孔30ul(3×104/well)。将具有代表性的MSLN纳米抗体45-59和阳性抗体P4，阴性抗体Caplacizumab，以200nM为最高浓度，3倍比，7个梯度，设PBS对照，每孔30ul，混匀后4℃孵育1小时。用含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干。1:200稀释荧光二抗Goat anti-Human IgG(H+L)Secondary Antibody[DyLight 650]，每孔30ul，混匀后4℃孵育30分钟；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干，30ul/well重悬，上高通量流式检测仪(IQue)读值，将收集到的数据用Graphpad做三参数拟合曲线。
结果如图3所示，结果显示，MSLN46，52和53可以特异性结合细胞表面的MSLN。
2.4 ForteBio检测具有阻断活性抗体的亲和力
稀释具有流式结合活性的MSLN纳米抗体至100nM，将抗体加入384孔板中。稀释人MSLN-His抗原至100nM，2倍稀释，共7个梯度，加入384孔板。使用AHC探针(Sartorius，货号：18-5060)，设置结合时间为180sec，解离时间360sec，Baseline为60sec，再生循环三次，每次5sec。之后用软件拟合结合-解离曲线，计算抗体的亲和力。
结果如图4所示，ForteBio检测结果显示，MSLN46，52和53的亲和力均在nM级别，显著高于阳性对照抗体P4，KD值分别是2.15E-09M，2.58E-09M，1.41E-09M。
实施例3 VHH序列CAR-T载体构建、慢病毒包装及CAR-T细胞制备
3.1 VHH序列CAR-T载体构建
使用SphI和NotI对原能平台质粒(原能自构)进行双酶切，回收载体线性化片段，与目的抗体序列VHH分别按1:3的比例混合(摩尔比例)，同源重组以后，转化大肠杆菌DH5α感受态细胞，热激90s后涂布在含氨苄抗性的LB固体培养基上，37℃过夜培养。挑单克隆菌落，测序验证质粒正确性，摇菌抽质粒保存备用，核心质粒如图5所示，CAR-T结构如图6所示，选用41BB(SEQ ID NO:53)和CD3ζ(SEQ ID NO:55)作为共刺激域。
3.2慢病毒包装及滴度测定
用于构建本发明的慢病毒质粒载体的载体系统属于第三代慢病毒载体系统，该系统共有 三个质粒即编码Gag-Pol蛋白和Rev蛋白的包装质粒psPAX2；编码包膜蛋白VSV-G的PMD2.G质粒；构建的含有编码目的基因CAR的核心质粒。基于BBz平台质粒的核心质粒中编码CAR的基因由延长因子-1α(EF-1α)启动子调控表达。慢病毒的包装过程如下：1x106的293T细胞悬于2ml 10％FBS的DMEM培养基中铺于6孔板的单个孔中，过夜培养；吸去1ml培养基，混入1ml含有2.88ug包装质粒(psPAX2：PMD2.G：核心质粒＝3:2:4)和8.64ul FuGENEHD转染试剂，加144ul的Opti-MEM培养基(质粒质量的50倍)，轻轻混匀后在CO2培养箱中37℃培养12小时；去除含质粒的培养基，PBS洗涤一次后更换为2ml含5％FBS的DMEM培养基，培养48小时；收集2.5ml病毒上清，该上清3000rpm离心5分钟，分装冻存-80℃待用，并检测其滴度。
3.3 VHH序列CAR-T细胞制备
含有VHH序列的CAR-T细胞的产生方法如下：通过密度梯度离心法获得人外周血单核细胞；用含有200U/ml白介素2培养基重悬外周血单核细胞至细胞密度为2x106/ml，按1:3的比例(细胞:磁珠)加入CD3/CD28磁珠对T细胞进行激活；激活的外周血单核细胞静置在37℃CO2培养箱中培养24小时；按病毒感染复数(MOI)为3的比例加入以上所得慢病毒上清，加polybrene至终浓度为5ug/ml，将细胞悬液置于孔板中用水平离心机1200rpm离心1小时；将孔板放回37℃CO2培养箱中培养24小时；300g离心5分钟，去上清，并用新鲜的含500U/ml白介素2的X-VIVO培养基重悬细胞至细胞密度为0.6x106/ml，置于37℃CO2培养箱中培养；每2天对细胞进行计数，并补加新鲜的含500U/ml白介素2的X-VIVO培养基将细胞密度调回0.6x106/ml；培养9-14天的CAR-T细胞检测细胞阳性率：用于感染细胞的慢病毒携带GFP，慢病毒感染细胞后通过流式细胞仪检测GFP阳性率从而得到CAR表达阳性率，阳性率>20％的细胞可以用于进行肿瘤杀伤实验。
实施例4过表达细胞株SK-OV3-MSLN的构建及检测
采用慢病毒包装、慢病毒感染的方式，将MSLN全长基因导入SK-OV3细胞中，制备SK-OV3-MSLN过表达细胞株。选用pLV-C-GFPS为载体，插入MSLN全长序列，构建重组质粒pLV-C-GFPS-MSLN，PCR及酶切结果如图7所示，PCR结果如下(A)：从左往右，依次为天根Marker IV，1和2为MSLN全长条带(约1900bp)；载体经Not I和Xba I双酶切结果如下(B)：依次为天根Marker IV，A和B载体酶切后条带(约8200bp)；通过慢病毒感染后，将重组质粒导入SK-OV3细胞，通过流式分选获得SK-OV3-MSLN阳性细胞株，扩大培养作为靶细胞。流式检测SK-OV3-MSLN过表达细胞株的阳性率，结果如图8，SK-OV3-MSLN过表达细胞阳性率达到97.98％。
实施例5 VHH序列CAR-T的细胞杀伤及因子检测
5.1 CAR-T体外反复刺激实验
根据实施例3制备CAR-T细胞，在扩增到第9-12天时，检测阳性率，取CAR-T细胞用无血清X-VIVO培养基(厂家:LONZA)重悬至4×105/ml的密度，作为效应细胞；将OV-CAR-3细胞用无血清X-VIVO培养基重悬至4×105/ml的密度，作为靶细胞；效靶比1:1混合后，在37℃，5％CO2培养箱中静置培养，每2天观察一次培养基颜色，培养基由橙色转黄色时补1倍体积的培养基。第4至5天时进行细胞计数，计数扩增倍数。计数后取5×105CAR-T细胞，再次进行第二、三轮扩增，方法同前描述的，将第一轮扩增倍数乘以第二轮再乘以第三轮的，计算总的扩增倍数。
反复刺激结果如图9，结果显示，CAR-T细胞(MSLN-52)经过三轮反复刺激后，累积扩增倍数达到543.75倍，与对照抗体P4(重链可变区的氨基酸序列如SEQ ID NO:63所示，轻链可变区的氨基酸序列如SEQ ID NO:64所示)相当。
5.2 CAR-T体外细胞杀伤实验
将SK-OV3-MSLN细胞作为靶细胞，感染了CAR-T慢病毒的细胞作为效应细胞，未感染的T细胞可作为对照效应细胞。具体实验过程如下，检测CAR的感染效率，用未感染的T细胞调整感染CAR比例至每组一致；按效应细胞：靶细胞(效靶比)＝3:1、1:1、0.3:1、0.1:1200μl X-VIVO培养基中，靶细胞数量为1x104/孔，作为实验组；只含有与实验组等量的效应细胞的孔作为效应细胞自释放背景组；只含有与实验组等量的靶细胞的孔作为靶细胞自释放背景组；将得到的细胞在37℃CO2培养箱中培养18小时；在部分仅含靶细胞的孔中加入20μl10x的裂解液，反应45分钟，作为靶细胞最大释放。将所得细胞培养孔板分别300g离心5分钟，收集50μl上清用于检测乳酸脱氢酶LDH的释放量，检测方法参照CytoTox96非放射性细胞毒性试剂盒的说明书(厂家：Promega)。释放的LDH在培养基上清中，可通过偶联的酶反应来检测。细胞杀伤活性计算公式为：
杀伤毒性％＝100x(实验组-效应细胞自释放-靶细胞自释放+培养基本底值)/(靶细胞最大释放-靶细胞自释放)。
根据细胞杀伤公式，分析各抗体序列对靶细胞的杀伤作用，选择具有明显杀伤作用的抗体进入体内功能学评价。
细胞杀伤结果如图10，结果显示，在效靶比1：1的条件下，MSLN-46的细胞杀伤最低，MSLN-52和MLSN-53细胞杀伤区别不大，与阳性对照P4相当。
5.3 CAR-T体外细胞因子分泌实验
SK-OV-3-MSLN细胞作为靶细胞，感染了CAR-T慢病毒的细胞作为效应细胞，未感染的T细胞可作为对照效应细胞。具体实验过程如下，检测CAR的感染效率，用未感染的T细胞调整感染CAR比例至每组一致；按效应细胞：靶细胞(效靶比)＝1:1 200μl X-VIVO培养基中，靶细胞数量为2x104/孔，作为实验组；只含有与实验组等量的效应细胞的孔作为背景组；将得到的细胞在37℃CO2培养箱中培养18小时；将所得细胞培养孔板分别300g离心5分钟，收集50μl上清用于检测IL2、IFN-γ表达水平，检测方法参考R&D DuoSet ELISA试剂盒(厂家：R&D SYSTEM)说明书。
IFN-γ检测结果如图11(A)，IL2检测结果如图12(B)，MSLN-46的IFN-γ分泌较多，但自激活较高，IL-2的结果中MSLN-53分泌最高，MSLN-52其次，均优于阳性对照P4。
实施例6 VHH序列CAR-T体内药效验证
SK-OV-3-MSLN 3E6/只小鼠，右上肢背中皮下荷瘤，待肿瘤至78mm3时，将NDG小鼠各分为3组：T细胞、P4、MSLN-VHH每组5只，每组给药途径为尾静脉，回输1E7冻存CAR-T细胞。记录给药后各组小鼠的体重，肿瘤大小变化，观察其各种生物学反应情况。观察至D72，结果如图12A所示，小鼠体重无明显变化，状态良好，未出现不明原因的死亡；如图12B所示，MSLN-VHH小鼠出现明显抑瘤作用，D40 4只小鼠达到完全消瘤，安全性和抗肿瘤作用得到验证。
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 (80)

  1. 分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其能够特异性结合间皮素(MLSN)蛋白。
  3. 根据权利要求2所述的分离的抗原结合蛋白，其中所述MSLN为人MSLN。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其能够与参比抗体竞争结合MSLN，所述参比抗体包含HCDR1，HCDR2以及HCDR3，所述HCDR1，HCDR2和HCDR3的氨基酸序列选自下述任一组：
    1)所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列；
    2)所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列；
    3)所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；以及
    4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含HCDR3，且所述HCDR3包含SEQ ID NO:11、SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:27中任一项所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其包含HCDR2，且所述HCDR2包含SEQ ID NO:10、SEQ ID NO:2、SEQ ID NO:19和SEQ ID NO:26中任一项所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其包含HCDR1，且所述HCDR1包含SEQ ID NO:9、SEQ ID NO:1、SEQ ID NO:18和SEQ ID NO:25中任一项所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:9、SEQ ID NO:1、SEQ ID NO:18和SEQ ID  NO:25中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10、SEQ ID NO:2、SEQ ID NO:19和SEQ ID NO:26中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:11、SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:27中任一项所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3的包含选自下述任一组的氨基酸序列：
    1)所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列；
    2)所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列；
    3)所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；以及
    4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接项链，且所述H-FR1包含SEQ ID NO:50所示的氨基酸序列。
  11. 根据权利要求10所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:4、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:21、SEQ ID NO:28、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42和SEQ ID NO:44中任一项所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:51所示的氨基酸序列。
  13. 根据权利要求12所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:22和SEQ ID NO:29中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:52所示的氨基酸序列。
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO:23和SEQ ID NO:30中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1，H-FR2，H-FR3和H-FR4包含选自下述任一组氨基酸序列：
    1)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    2)所述H-FR1包含SEQ ID NO:12所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    3)所述H-FR1包含SEQ ID NO:16所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    4)所述H-FR1包含SEQ ID NO:21所示的氨基酸序列，所述H-FR2包含SEQ ID NO:22所示的氨基酸序列，所述H-FR3包含SEQ ID NO:23所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    5)所述H-FR1包含SEQ ID NO:28所示的氨基酸序列，所述H-FR2包含SEQ ID NO:29所示的氨基酸序列，所述H-FR3包含SEQ ID NO:30所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    6)所述H-FR1包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    7)所述H-FR1包含SEQ ID NO:34所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    8)所述H-FR1包含SEQ ID NO:36所示的氨基酸序列，所述H-FR2包含SEQ ID NO:13所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述 H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    9)所述H-FR1包含SEQ ID NO:38所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    10)所述H-FR1包含SEQ ID NO:40所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；
    11)所述H-FR1包含SEQ ID NO:42所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；以及
    12)所述H-FR1包含SEQ ID NO:44所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，且所述VH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  20. 根据权利要求19所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  21. 根据权利要求19-20中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  22. 根据权利要求19-21中任一项所述的分离的抗原结合蛋白，其中所述抗原结合片段为VHH。
  23. 根据权利要求22所述的分离的抗原结合蛋白，其中所述VHH包含SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:24、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43和SEQ ID NO:45中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包含抗体重链恒定区，所述抗体重链恒定区源自IgG的重链恒定区。
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人IgG。
  26. 根据权利要求24-25中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人IgG1的重链恒定区。
  27. 根据权利要求24-26中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区包含IgG的Fc区。
  28. 根据权利要求27所述的分离的抗原结合蛋白，其中所述Fc区包含SEQ ID NO:61所示的氨基酸序列。
  29. 嵌合抗原受体，其包含靶向部分，所述靶向部分包含权利要求1-28中任一项所述的抗原结合蛋白。
  30. 根据权利要求29所述的嵌合抗原受体，其包含共刺激域，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。
  31. 根据权利要求29-30中任一项所述的嵌合抗原受体，其中所述共刺激域为源自4-1BB的胞内共刺激信号区域。
  32. 根据权利要求30-31中任一项所述的嵌合抗原受体，其中所述共刺激域包含SEQ ID NO:53所示的氨基酸序列。
  33. 根据权利要求29-32中任一项所述的嵌合抗原受体，其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。
  34. 根据权利要求33所述的嵌合抗原受体，其中所述胞内信号传导域为源自CD3ζ的信号传导域。
  35. 根据权利要求33-34中任一项所述的嵌合抗原受体，其中所述胞内信号传导域包含SEQ ID NO:55所示的氨基酸序列。
  36. 根据权利要求29-35中任一项所述的嵌合抗原受体，其包含跨膜区，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域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、 NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  37. 根据权利要求36所述的嵌合抗原受体，其中所述跨膜区为源自CD8的跨膜区。
  38. 根据权利要求36-37所述的嵌合抗原受体，其中所述跨膜区包含SEQ ID NO:54所示的氨基酸序列。
  39. 根据权利要求29-38中任一项所述的嵌合抗原受体，其在靶向部分和跨膜区之间包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  40. 根据权利要求39所述的嵌合抗原受体，其中所述铰链区为源自CD8的铰链区。
  41. 根据权利要求39-40中任一项所述的嵌合抗原受体，其中所述铰链区包含SEQ ID NO:56所示的氨基酸序列。
  42. 根据权利要求29-41中任一项所述的嵌合抗原受体，其还包含信号肽。
  43. 根据权利要求42所述的嵌合抗原受体，其中所述信号肽源自CD8蛋白的信号肽。
  44. 根据权利要求42-43中任一项所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:57所示的氨基酸序列。
  45. 根据权利要求29-44中任一项所述的嵌合抗原受体，还包含低密度脂蛋白受体相关蛋白或其片段。
  46. 根据权利要求45所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  47. 根据权利要求45-46中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
  48. 根据权利要求45-47中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:58所示的氨基酸序列。
  49. 多肽，其包含权利要求1-28中任一项所述的抗原结合蛋白。
  50. 一种或多种分离的核酸分子，其编码权利要求1-28中任一项所述的抗原结合蛋白和/或权利要求29-48中任一项所述的嵌合抗原受体。
  51. 根据权利要求50所述的核酸分子，其还包含启动子。
  52. 根据权利要求51所述的核酸分子，其中所述启动子为组成型启动子。
  53. 根据权利要求51-52中任一项所述的核酸分子，其中所述启动子为EF1α启动子。
  54. 载体，其包含权利要求50-53中任一项所述的核酸分子。
  55. 根据权利要求54所述的载体，其包含病毒载体。
  56. 根据权利要求54-55中任一项所述的载体，其包含慢病毒载体。
  57. 细胞，其包含权利要求1-28中任一项所述的抗原结合蛋白，权利要求29-48中任一项所述的嵌合抗原受体，权利要求49所述的多肽，权利要求50-53中任一项所述的核酸分子，和/或权利要求54-56中任一项所述的载体。
  58. 根据权利要求57所述的细胞，其为免疫效应细胞。
  59. 根据权利要求57-58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。
  60. 根据权利要求57-59中任一项所述的细胞，其为T细胞。
  61. 根据权利要求57-60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。
  62. 根据权利要求61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。
  63. 根据权利要求61-62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。
  64. 根据权利要求61-63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:58所示的氨基酸序列。
  65. 根据权利要求61-64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外源低密度脂蛋白受体相关蛋白或其片段。
  66. 制备权利要求1-28中任一项所述的抗原结合蛋白和/或权利要求29-48中任一项所述的嵌合抗原受体的方法，所述方法包括在使得权利要求1-28中任一项所述的抗原结合蛋白和/或权利要求29-48中任一项所述的嵌合抗原受体表达的条件下，培养权利要求57-65中任一项所述的细胞。
  67. 制备经修饰的免疫效应细胞的方法，其包括向免疫效应细胞中引入权利要求54-56中任一项所述的载体。
  68. 药物组合物，其包含权利要求1-28中任一项所述的分离的抗原结合蛋白，权利要求29-48中任一项所述的嵌合抗原受体，权利要求49所述的多肽，权利要求50-53中任一项所述的核酸分子，权利要求54-56中任一项所述的载体，和/或权利要求57-65中任一项 所述的细胞，以及任选地药学上可接受的载体。
  69. 权利要求1-28中任一项所述的分离的抗原结合蛋白，权利要求29-48中任一项所述的嵌合抗原受体，权利要求49所述的多肽，权利要求50-53中任一项所述的核酸分子，权利要求54-56中任一项所述的载体，和/或权利要求57-65中任一项所述的细胞，和/或权利要求68所述的药物组合物，在制备药物中的用途，所述药物用于预防、治疗和/或缓解与MSLN异常表达相关的疾病或病症。
  70. 根据权利要求69所述的用途，其中所述与MSLN异常表达相关的疾病或病症包括肿瘤。
  71. 根据权利要求70所述的用途，其中所述肿瘤包括实体瘤。
  72. 根据权利要求70所述的用途，其中所述肿瘤包括非实体瘤。
  73. 根据权利要求70-72中任一项所述的用途，其中所述肿瘤包括表达MSLN抗原的肿瘤。
  74. 根据权利要求70-73中任一项所述的用途，其中所述肿瘤包括卵巢癌、胰腺癌、胃癌、间皮细胞癌、胆管癌、三阴性乳腺癌和/或子宫内膜癌。
  75. 一种预防、治疗和/或缓解与MSLN异常表达相关的疾病或病症的方法，所述方法包括向有需要的受试者施用权利要求1-28中任一项所述的分离的抗原结合蛋白，权利要求29-48中任一项所述的嵌合抗原受体，权利要求49所述的多肽，权利要求50-53中任一项所述的核酸分子，权利要求54-56中任一项所述的载体，和/或权利要求57-65中任一项所述的细胞，和/或权利要求68所述的药物组合物。
  76. 根据权利要求75所述的方法，其中所述与MSLN异常表达相关的疾病或病症包括肿瘤。
  77. 根据权利要求76所述的方法，其中所述肿瘤包括实体瘤。
  78. 根据权利要求76所述的方法，其中所述肿瘤包括非实体瘤。
  79. 根据权利要求76-78中任一项所述的方法，其中所述肿瘤包括表达MSLN抗原的肿瘤。
  80. 根据权利要求76-79中任一项所述的方法，其中所述肿瘤包括卵巢癌、胰腺癌、胃癌、间皮细胞癌、胆管癌、三阴性乳腺癌和/或子宫内膜癌。
PCT/CN2023/127029 2022-10-28 2023-10-27 靶向msln的抗原结合蛋白 WO2024088371A1 (zh)

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