WO2022078286A1 - 特异性结合msln的嵌合抗原受体及其应用 - Google Patents

特异性结合msln的嵌合抗原受体及其应用 Download PDF

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WO2022078286A1
WO2022078286A1 PCT/CN2021/123047 CN2021123047W WO2022078286A1 WO 2022078286 A1 WO2022078286 A1 WO 2022078286A1 CN 2021123047 W CN2021123047 W CN 2021123047W WO 2022078286 A1 WO2022078286 A1 WO 2022078286A1
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sequence
seq
cdr
variant
nucleic acid
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PCT/CN2021/123047
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English (en)
French (fr)
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常建辉
朱雁林
吴子明
蔡珍珍
李虹
卢洲
马平凡
田海军
陈扬德
薛彤彤
肖亮
葛均友
王晶翼
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四川科伦博泰生物医药股份有限公司
科纳思药业有限公司
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Priority to CN202180063982.7A priority Critical patent/CN116234911A/zh
Publication of WO2022078286A1 publication Critical patent/WO2022078286A1/zh

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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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/46444—Interleukins [IL]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • 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
    • 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
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    • 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
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    • 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
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    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes

Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to MSLN, and a chimeric antigen receptor (CAR) comprising the antibody or its antigen-binding fragment.
  • CAR chimeric antigen receptor
  • the invention also relates to engineered immune cells expressing the CAR, or co-expressing the CAR and another biologically active molecule (eg, PD-1 antibody and/or mIL-15), and preparation of the engineered immune cells Methods.
  • the present invention also relates to the use of these antibodies, CARs and immune cells for the prevention and/or treatment of diseases related to the expression of mesothelin, such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer and other cancers and prevention And/or a method for treating MSLN-positive tumors such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, and ovarian cancer.
  • diseases related to the expression of mesothelin such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer and other cancers and prevention And/or a method for treating MSLN-positive tumors such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, and ovarian cancer.
  • MSLN Mesothelin
  • MSLN is a glycosylphosphatidylinositol-linked glycoprotein
  • the precursor protein of MSLN is hydrolyzed by protease into 31kDa megakaryocyte-potentiating factor (MPF) and 40kDa mesothelial white.
  • MPF megakaryocyte-potentiating factor
  • CA125/MUC16 is a ligand of mesothelin, which binds to mesothelin through the repeat fragment of the N-terminal extracellular domain and participates in cell adhesion together.
  • MSLN has highly specific expression, low expression on mesothelial cells of peritoneal cavity, pleural cavity and pericardial cavity in normal tissues, and high expression in solid tumors such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, and ovarian cancer. Expression, especially in malignant mesothelioma (85%-90%), pancreatic cancer (80%-85%), epithelial ovarian cancer (60%-65%) and lung cancer (60%-65%) , related to cell proliferation, cell adhesion function and anti-apoptotic process. These biological characteristics suggest that MSLN may serve as an ideal tumor therapy target with multiple indications.
  • Chimeric antigen receptor (CAR)-T cell therapy is regarded as one of the most promising cancer treatments and has become a new hope for centuries to fight cancer. It cultured immune cells collected from patients in vitro, transduced specific exogenous genes in vitro, amplified them in vitro and then infused them back into patients, so as to achieve the purpose of treating tumors in a non-MHC-restricted manner.
  • CAR-T cell therapy has achieved remarkable efficacy in the treatment of hematological malignancies, with a complete remission rate of over 90% for relapsed and refractory B-cell leukemia.
  • Solid tumors account for about 90% of all malignant tumors, and their therapeutic drugs are in great demand.
  • the current therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly due to the complex tumor microenvironment and high tumor heterogeneity of solid tumors.
  • CAR-T cell therapy targeting MSLN is expected to become one of the ways to overcome MSLN-positive tumors. Therefore, it is urgent and necessary to develop CAR-T therapy targeting MSLN with high specificity and good efficacy.
  • the inventors first developed a fully human antibody with low immunogenicity capable of specifically recognizing/binding to MSLN.
  • the present invention designs and constructs a chimeric antigen receptor (CAR) comprising the MSLN antibody or its antigen-binding fragment, and further designs and constructs a targeting MSLN that co-expresses PD-1 antibody and/or mIL-15 CAR.
  • CARs of the present invention are capable of targeting immune effector cells specifically and reactively to MSLN-expressing cells (eg, malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer cells) in a non-MHC-restricted manner for their elimination .
  • the CAR targeting MSLN of the present invention has the potential for preventing and/or treating diseases related to the expression of mesothelin, such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer and other MSLN-positive tumors , with significant clinical value.
  • diseases related to the expression of mesothelin such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer and other MSLN-positive tumors , with significant clinical value.
  • a first aspect of the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to MSLN (eg, human MSLN).
  • MSLN eg, human MSLN
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • variant described in any one of (1a), (1b), (1c) has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); preferably, said substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDRs of the following six heavy and light chains defined according to the Kabat numbering system: CDR-H1 of sequence SEQ ID NO: 3; CDR-H2 of sequence SEQ ID NO: 4; sequence of SEQ ID NO: 4 : CDR-H3 of 5; CDR-L1 of SEQ ID NO: 6; CDR-L2 of SEQ ID NO: 7; CDR-L3 of SEQ ID NO: 8; or
  • the antibody or antigen-binding fragment thereof comprises human-derived immunoglobulin framework regions (FRs).
  • the antibody or antigen-binding fragment thereof comprises:
  • a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; wherein the variant and the sequence from which it is derived at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% compared to , or 100% sequence identity, or have one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletion or addition); preferably, the substitution is a conservative substitution.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • variant described in any of (2a), (2b), (2c) has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions); preferably, said substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDRs of the following six heavy and light chains as defined by the Kabat numbering system CDR-H1 of sequence SEQ ID NO: 18; CDR-H2 of sequence SEQ ID NO: 19; sequence of SEQ ID NO: 19 : CDR-H3 of 20; CDR-L1 of SEQ ID NO: 21; CDR-L2 of SEQ ID NO: 22; CDR-L3 of SEQ ID NO: 23; or
  • CDR-H1 of sequence SEQ ID NO: 29 the following six CDRs of heavy and light chains as defined by the Chothia numbering system: CDR-H1 of sequence SEQ ID NO: 29; CDR-H2 of sequence SEQ ID NO: 30; sequence of SEQ ID NO: : CDR-H3 of 20; CDR-L1 of sequence is SEQ ID NO:21; CDR-L2 of sequence is SEQ ID NO:22; CDR-L3 of sequence is SEQ ID NO:23.
  • the antibody or antigen-binding fragment thereof comprises human-derived immunoglobulin framework regions (FRs).
  • the antibody or antigen-binding fragment thereof comprises:
  • a VH comprising the sequence shown in SEQ ID NO: 16 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO: 17 or a variant thereof; wherein the variant and the sequence from which it is derived at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% compared to , or 100% sequence identity, or have one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletion or addition); preferably, the substitution is a conservative substitution.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • variant described in any one of (3a), (3b), (3c) has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions); preferably, said substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDRs of the following six heavy and light chains defined according to the Kabat numbering system: CDR-H1 of sequence SEQ ID NO: 33; CDR-H2 of sequence SEQ ID NO: 34; sequence of SEQ ID NO: 34 : CDR-H3 of 35; CDR-L1 of sequence SEQ ID NO: 36; CDR-L2 of sequence SEQ ID NO: 37; CDR-L3 of sequence SEQ ID NO: 38; or
  • CDR-H1 of sequence SEQ ID NO: 44 the following six CDRs of heavy and light chains as defined by the Chothia numbering system: CDR-H1 of sequence SEQ ID NO: 44; CDR-H2 of sequence SEQ ID NO: 45; sequence of SEQ ID NO: 45 : CDR-H3 of 35; CDR-L1 of sequence is SEQ ID NO:36; CDR-L2 of sequence is SEQ ID NO:37; CDR-L3 of sequence is SEQ ID NO:38.
  • the antibody or antigen-binding fragment thereof comprises human-derived immunoglobulin framework regions (FRs).
  • the antibody or antigen-binding fragment thereof comprises:
  • a VH comprising the sequence shown in SEQ ID NO: 31 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO: 32 or a variant thereof; wherein the variant and the sequence from which it is derived at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% compared to , or 100% sequence identity, or have one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletion or addition); preferably, the substitution is a conservative substitution.
  • the antibody or antigen-binding fragment thereof of the invention may be selected from camelid Ig, IgNAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab)' 3 fragment, single chain antibody (e.g. scFv, di-scFv or (scFv) 2 ), minibodies, diabodies, tribodies, tetrabodies, disulfide stabilized Fv proteins (dsFv) and single domain antibodies (sdAbs, Nanobodies) .
  • camelid Ig, IgNAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab)' 3 fragment single chain antibody (e.g. scFv, di-scFv or (scFv) 2 ), minibodies, diabodies, tribodies, tetrabodies, disulfide stabilized Fv proteins (dsFv) and single domain antibodies (sdAbs, Nanobodies) .
  • an antibody of the invention may be an antigen-binding fragment (eg, scFv) that includes one or more linkers that connect two antibody domains or regions (eg, a heavy chain may variable (VH) region and light chain variable (VL) region).
  • the antibodies of the invention may include single chain antibody fragments, such as scFvs and diabodies, particularly human single chain antibody fragments, typically comprising one or more linkages linking two antibody domains or regions (eg VH and VL regions) son.
  • Linkers are typically peptide linkers, eg, flexible and/or soluble peptide linkers, eg, glycine- and serine-rich peptide linkers. Linkers include those rich in glycine and serine and/or in some cases threonine. In some embodiments, the linker also includes charged residues (eg, lysine and/or glutamic acid), which can improve solubility. In some embodiments, the linker further includes one or more prolines.
  • linkers rich in glycine and serine (and/or threonine) comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98% or 99% of such amino acids. In some embodiments, they comprise at least at or about 50%, 55%, 60%, 70% or 75% glycine, serine and/or threonine. In some embodiments, the linker consists essentially entirely of glycine, serine and/or threonine. For example, linkers include linkers having single or repeats (eg, 1, 2, 3, 4, and 5 repeats) of the sequence GGGGS or GGGS.
  • the VH and VL of an antibody or antigen-binding fragment thereof of the invention are linked by one or more linkers; preferably, the linkers comprise one or several (eg 1, 2 or 3 a) a sequence shown as (G m S) n , wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, the linker has the sequence of SEQ ID NO:52.
  • the linkers comprise one or several (eg 1, 2 or 3 a) a sequence shown as (G m S) n , wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, the linker has the sequence of SEQ ID NO:52.
  • the antibody or antigen-binding fragment thereof is a single chain antibody, eg, a scFv, di-scFv, or (scFv) 2 .
  • the single chain antibody comprises, in order from its N-terminus to its C-terminus:
  • VH-linker comprising the sequence shown in SEQ ID NO: 1 or a variant thereof - a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO: 16 or a variant thereof - a VL comprising the sequence shown in SEQ ID NO: 17 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO: 31 or a variant thereof - a VL comprising the sequence shown in SEQ ID NO: 32 or a variant thereof;
  • VL-linker comprising the sequence shown in SEQ ID NO: 2 or a variant thereof - a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof;
  • VL-linker comprising the sequence shown in SEQ ID NO: 17 or a variant thereof - a VH comprising the sequence shown in SEQ ID NO: 16 or a variant thereof;
  • VL-linker comprising the sequence shown in SEQ ID NO: 32 or a variant thereof - a VH comprising the sequence shown in SEQ ID NO: 31 or a variant thereof;
  • the variant is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% compared to the sequence from which it is derived , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or substitution, deletion or addition of one or several amino acids (eg 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, said substitutions are conservative substitutions.
  • the single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) the amino acid sequence shown in any one of SEQ ID NOs: 54, 56, 58; (2) the same as SEQ ID NOs: 54 , 56, 58 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence; or (3) an amino acid set forth in any one of SEQ ID NOs: 54, 56, 58 sequence comparison with one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) , deletion or addition); preferably, the substitution is a conservative substitution.
  • the antibodies or antigen-binding fragments thereof of the invention further comprise constant regions derived from human immunoglobulins.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgGl, IgG2, IgG3, or IgG4), the antibody or antigen-binding fragment thereof has a heavy chain constant region derived from a human immunoglobulin (eg, IgGl, IgG2, IgG3, or IgG4).
  • the light chain comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, the variant being compared to the wild-type sequence from which it was derived with one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4 1 or 5 amino acid substitutions, deletions or additions); and/or,
  • CH heavy chain constant region
  • the light chain of the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acids from the wild-type sequence from which it is derived. Substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions of 1, 2, 3, 4, or 5 amino acids , missing or added).
  • CL light chain constant region
  • the heavy chain constant region is an IgG, IgM, IgE, IgD or IgA heavy chain constant region.
  • the heavy chain constant region is an IgG heavy chain constant region, eg, an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa or lambda light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present invention.
  • Antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly by recombinant host cells (Reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.
  • a second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and/or a light chain variable region thereof.
  • the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (1) the nucleotide sequence set forth in any one of SEQ ID NOs: 55, 57 and 59; (2) and The nucleotide sequences shown in any one of SEQ ID NOs: 55, 57 and 59 have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity the sequence of.
  • a third aspect of the present invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.
  • the vector of the invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral, adenoviral or retroviral vector.
  • a fourth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, eg, mouse cells, human cells, etc.).
  • the present invention also relates to a method of preparing an antibody or antigen-binding fragment thereof of the present invention, comprising, culturing a host cell as described above under conditions that permit expression of the antibody or antigen-binding fragment thereof, and removing the The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
  • the present invention relates to a CAR targeting MSLN with features including non-MHC-restricted MSLN recognition capability that confers on immune cells (eg, T cells, NK cells, monocytes, macrophages, or dendritic cells) expressing the CAR The ability to recognize MSLN-expressing cells (eg, tumor cells) independent of antigen processing and presentation.
  • immune cells eg, T cells, NK cells, monocytes, macrophages, or dendritic cells
  • the fifth aspect of the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain (anti-MSLN binding domain), a spacer domain, a transmembrane domain and an intracellular signaling structure area.
  • CAR chimeric antigen receptor
  • the antigen binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize MSLN.
  • the antigen-binding domain comprises an anti-MSLN binding domain comprising an antibody or antigen-binding fragment thereof capable of specifically binding MSLN (eg, human MSLN).
  • the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof of the first aspect.
  • the anti-MSLN binding domain comprises VH and VL, wherein,
  • the VH comprises the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 of sequence SEQ ID NO: 3 or a variant thereof; CDR-H2 of sequence SEQ ID NO: 4 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 5 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the Kabat numbering system: the sequence is CDR-L1 of SEQ ID NO: 6 or a variant thereof; CDR-L2 having the sequence of SEQ ID NO: 7 or a variant thereof; CDR-L3 having the sequence of SEQ ID NO: 8 or a variant thereof; or,
  • the VH comprises the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 of sequence SEQ ID NO: 9 or a variant thereof; CDR-H2 of sequence SEQ ID NO: 10 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 11 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the IMGT numbering system: the sequence is CDR-L1 of SEQ ID NO: 12 or a variant thereof; CDR-L2 having the sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 having the sequence of SEQ ID NO: 8 or a variant thereof; or,
  • the VH comprises the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 having the sequence of SEQ ID NO: 14 or a variant thereof; CDR-H2 having the sequence of SEQ ID NO: 15 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 5 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 of sequence SEQ ID NO: 6 or a variant thereof; The sequence is CDR-L2 of SEQ ID NO: 7 or its variant; the sequence is CDR-L3 of SEQ ID NO: 8 or its variant;
  • the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the The substitutions are conservative substitutions.
  • the VH comprises the sequence set forth in SEQ ID NO: 1 or a variant thereof
  • the VL comprises the sequence set forth in SEQ ID NO: 2 or a variant thereof; wherein the variant
  • the body is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 96%, at least 97% compared to the sequence from which it was derived.
  • substitutions are conservative substitutions.
  • the anti-MSLN binding domain comprises VH and VL, wherein,
  • the VH comprises the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 having the sequence of SEQ ID NO: 18 or a variant thereof; CDR-H2 having the sequence of SEQ ID NO: 19 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 20 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the Kabat numbering system: the sequence is CDR-L1 of SEQ ID NO: 21 or a variant thereof; CDR-L2 having the sequence of SEQ ID NO: 22 or a variant thereof; CDR-L3 having the sequence of SEQ ID NO: 23 or a variant thereof; or,
  • the VH comprises the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 having a sequence of SEQ ID NO: 24 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 25 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 26 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the IMGT numbering system: the sequence is CDR-L1 of SEQ ID NO: 27 or a variant thereof; CDR-L2 having the sequence of SEQ ID NO: 28 or a variant thereof; CDR-L3 having the sequence of SEQ ID NO: 23 or a variant thereof; or,
  • the VH comprises the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 having the sequence of SEQ ID NO: 29 or a variant thereof; CDR-H2 having the sequence of SEQ ID NO: 30 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 20 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the Chothia numbering system: the sequence is CDR-L1 of SEQ ID NO: 21 or a variant thereof; The sequence is CDR-L2 of SEQ ID NO: 22 or a variant thereof; the sequence is CDR-L3 of SEQ ID NO: 23 or a variant thereof;
  • the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the The substitutions are conservative substitutions.
  • the VH comprises the sequence set forth in SEQ ID NO: 16, or a variant thereof
  • the VL comprises the sequence set forth in SEQ ID NO: 17, or a variant thereof; wherein the variant
  • the body is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 96%, at least 97% compared to the sequence from which it was derived.
  • substitutions are conservative substitutions.
  • the anti-MSLN binding domain comprises VH and VL, wherein,
  • the VH comprises the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 of sequence SEQ ID NO: 33 or a variant thereof; CDR-H2 of sequence SEQ ID NO: 34 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 35 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the Kabat numbering system: the sequence is CDR-L1 of SEQ ID NO: 36 or a variant thereof; CDR-L2 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-L3 whose sequence is SEQ ID NO: 38 or a variant thereof; or,
  • the VH comprises the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 having a sequence of SEQ ID NO: 39 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 40 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 41 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the IMGT numbering system: the sequence is CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 having the sequence of SEQ ID NO: 43 or a variant thereof; CDR-L3 having the sequence of SEQ ID NO: 38 or a variant thereof; or,
  • the VH comprises the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 having the sequence of SEQ ID NO: 44 or a variant thereof; CDR-H2 having the sequence of SEQ ID NO: 45 or a variant thereof; The sequence is CDR-H3 of SEQ ID NO: 35 or a variant thereof; the VL comprises the following three light chain CDRs defined according to the Chothia numbering system: the sequence is CDR-L1 of SEQ ID NO: 36 or a variant thereof; The sequence is CDR-L2 of SEQ ID NO: 37 or its variant; the sequence is CDR-L3 of SEQ ID NO: 38 or its variant;
  • the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the The substitutions are conservative substitutions.
  • the VH comprises the sequence set forth in SEQ ID NO: 31 or a variant thereof
  • the VL comprises the sequence set forth in SEQ ID NO: 32 or a variant thereof; wherein the variant
  • the body is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 96%, at least 97% compared to the sequence from which it was derived.
  • substitutions are conservative substitutions.
  • the anti-MSLN binding domain of any of the above embodiments is a single chain antibody, eg, a scFv, di-scFv, or (scFv) 2 .
  • the VH and VL contained in the anti-MSLN binding domain described in any of the above embodiments, or the VH and VL of the antibody or antigen-binding fragment thereof contained in the anti-MSLN binding domain are passed through Linker connection.
  • the linker comprises one or several (eg, 1, 2, or 3) sequences as shown in (G m S) n , wherein m is selected from an integer of 1-6, and n An integer selected from 1-6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 1 or 2. In certain embodiments, the linker has the sequence of SEQ ID NO:52.
  • the anti-MSLN binding domain comprises a single chain antibody comprising an amino acid sequence selected from the group consisting of:
  • substitutions are conservative substitutions.
  • the anti-MSLN binding domain comprises a single chain antibody comprising an amino acid sequence selected from the group consisting of:
  • substitutions are conservative substitutions.
  • the anti-MSLN binding domain comprises a single chain antibody comprising an amino acid sequence selected from the group consisting of:
  • substitutions are conservative substitutions.
  • the antigen binding domain comprises the anti-MSLN binding domain as a first binding domain, and further comprises a second binding domain that does not bind MSLN.
  • the antigen bound by the second binding domain that does not bind MSLN is selected from the group consisting of: CD19, GPC3, PSMA, MUCl, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
  • the transmembrane domain contained in the chimeric antigen receptor of the present invention may be any protein structure known in the art as long as it can be thermodynamically stable in cell membranes (especially eukaryotic cell membranes).
  • the transmembrane domains of CARs suitable for use in the present invention may be derived from natural sources.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain may be a synthetic non-naturally occurring protein segment, eg, a protein segment comprising predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is a transmembrane region of a protein selected from the group consisting of alpha, beta or zeta chains of T cell receptors, CD28, CD45, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1, and any combination thereof.
  • the transmembrane domain is a transmembrane region of a protein selected from the group consisting of CD8 ⁇ , CD4, PD-1, CD152 and CD154.
  • the transmembrane domain comprises the transmembrane region of CD8 ⁇ .
  • the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:64.
  • the chimeric antigen receptor of the present invention comprises a spacer domain located between the extracellular antigen binding domain and the transmembrane domain.
  • the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (eg, IgGl or IgG4).
  • an immunoglobulin eg, IgGl or IgG4
  • CH2 and CH3 extend the antigen-binding domain of the CAR from the cell membrane of the CAR-expressing cell and may more accurately mimic the size and domain of native TCRs structure.
  • the spacer domain comprises a hinge domain.
  • a hinge domain can be a stretch of amino acids commonly found between two domains of a protein, which can allow flexibility in the protein and allow movement of one or both domains relative to each other.
  • the hinge domain can be any amino acid sequence that provides this flexibility of the extracellular antigen binding domain and this mobility relative to the transmembrane domain.
  • the hinge domain is the hinge region or portion thereof of a naturally occurring protein.
  • the hinge domain comprises the hinge region of CD8 ⁇ , or a portion thereof, eg, a fragment comprising at least 15 (eg, 20, 25, 30, 35, or 40) contiguous amino acids of the hinge region of CD8 ⁇ .
  • the spacer domain comprises the amino acid sequence set forth in SEQ ID NO:62.
  • the CAR of the present invention may further comprise a signal peptide at its N-terminus.
  • a signal peptide is a polypeptide sequence that targets the sequence to which it is linked to a desired site.
  • the signal peptide can target the CAR to which it is attached to the secretory pathway of the cell and allow the CAR to be further integrated and anchored into the lipid bilayer.
  • Useful signal peptides for CARs are known to those skilled in the art.
  • the signal peptide comprises a heavy chain signal peptide (eg, heavy chain signal peptide of IgG1), a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or CD8 ⁇ signal peptide.
  • the signal peptide is selected from CD8 ⁇ signal peptides.
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:60.
  • the CARs of the invention are co-expressed with additional biologically active molecules.
  • the additional biologically active molecule may have its own signal peptide, which is named signal peptide-2 in order to distinguish it from the signal peptide in the previous paragraph.
  • Signal peptide-2 directs the transport of additional bioactive molecules to specific sites within the cell or outside the cell membrane.
  • the signal peptide-2 may be the same as or different from the signal peptide described in the previous paragraph.
  • the signal peptide-2 may be different from the signal peptide described in the previous paragraph.
  • the intracellular signaling domain included in the CAR of the present invention is involved in transducing the signal generated by the combination of the CAR of the present invention with MSLN into the immune effector cells, activating at least one normal effector function of the immune effector cells expressing the CAR , or enhance the secretion of at least one cytokine (eg, IL-2, IFN- ⁇ ) by CAR-expressing immune effector cells.
  • cytokine eg, IL-2, IFN- ⁇
  • the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain.
  • the primary signaling domain may be any intracellular signaling domain comprising an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of a protein selected from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary signaling domain comprises the intracellular signaling domain of CD3 ⁇ .
  • the costimulatory signaling domain may be an intracellular signaling domain from a costimulatory molecule.
  • the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4- 1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10.
  • the costimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137(4-1BB), or a combination of fragments of both.
  • the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains. In such embodiments, the two or more costimulatory signaling domains may be the same or different.
  • the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain.
  • the primary signaling domain and at least one costimulatory signaling domain can be tandemly attached to the carboxy-terminus of the transmembrane domain in any order.
  • the intracellular signaling domain may comprise the intracellular signaling domain of CD3 ⁇ and the intracellular signaling domain of CD137.
  • the intracellular signaling domain of CD3 ⁇ comprises the amino acid sequence set forth in SEQ ID NO:68.
  • the intracellular signaling domain of CD137 comprises the amino acid sequence set forth in SEQ ID NO:66.
  • the intracellular signaling domain of the chimeric antigen receptor has the sequence set forth in SEQ ID NO:70.
  • the present invention provides a chimeric antigen receptor capable of specifically binding to MSLN, the chimeric antigen receptor comprises an anti-MSLN binding domain, a spacer domain, a transmembrane domain, an intracellular domain from its N-terminus to its C-terminus in order signaling domain.
  • the intracellular signaling domain is a costimulatory signaling domain and a primary signaling domain from the N-terminus to the C-terminus.
  • the spacer domain comprises the hinge region of CD8 (eg, CD8 ⁇ ) having the sequence set forth in SEQ ID NO:62.
  • the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8 ⁇ ) having the sequence set forth in SEQ ID NO:64.
  • the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3 ⁇ having SEQ ID NO : the sequence shown in 68.
  • the costimulatory signaling domain comprises the intracellular signaling domain of CD137, which has the sequence set forth in SEQ ID NO:66.
  • the intracellular signaling domain of the chimeric antigen receptor has the sequence set forth in SEQ ID NO:70.
  • the chimeric antigen receptor comprises the signal peptide, an anti-MSLN binding domain, a spacer domain, a transmembrane domain, an intracellular signaling structure in order from its N-terminus to its C-terminus domain (from N-terminal to C-terminal costimulatory signaling domain and primary signaling domain).
  • the signal peptide comprises the heavy chain signal peptide of IgGl or the CD8 ⁇ signal peptide. In certain exemplary embodiments, the signal peptide comprises a CD8 ⁇ signal peptide having the sequence set forth in SEQ ID NO:60.
  • the CAR of the invention comprises an amino acid sequence selected from the group consisting of:
  • sequence identity substantially retains at least one of the amino acid sequences from which it is derived a biological activity (eg, the ability to direct the specificity and reactivity of immune effector cells to cells expressing MSLN in a non-MHC-restricted manner); or,
  • substitutions are conservative substitutions.
  • the CAR of the invention comprises an amino acid sequence selected from the group consisting of:
  • sequence identity substantially retains at least one of the amino acid sequences from which it is derived a biological activity (eg, the ability to direct the specificity and reactivity of immune effector cells to cells expressing MSLN in a non-MHC-restricted manner); or,
  • substitutions are conservative substitutions.
  • the CAR of the invention comprises an amino acid sequence selected from the group consisting of:
  • sequence identity substantially retains at least one of the amino acid sequences from which it is derived a biological activity (eg, the ability to direct the specificity and reactivity of immune effector cells to cells expressing MSLN in a non-MHC-restricted manner); or,
  • substitutions are conservative substitutions.
  • the CAR of the fifth aspect of the invention may also be co-expressed with additional biologically active molecules.
  • Self-cleaving peptides prevent amino acids from forming covalent bonds during translation and keep translation going, so that the translation product is "self-cleaving", which specifically binds to MSLN's chimeric antigen receptor and additional biological activity Molecular separation. Therefore, when the CAR described in the fifth aspect of the present invention can also be co-expressed with another biologically active molecule, the chimeric antigen receptor that can specifically bind to MSLN becomes an independent CAR with extracellular antigen binding domain and spacer domain.
  • transmembrane domains and intracellular signaling domains of CARs, while other biologically active molecules can be secreted out of cells or expressed as membrane chimeric polypeptides or proteins.
  • additional bioactive molecules are enriched in the tumor microenvironment to synergize with the anti-MSLN-CAR to exert anti-tumor effects.
  • the additional biologically active molecule is selected from one or more of the following components: an antibody or antigen-binding fragment thereof that specifically binds an immune checkpoint (eg, anti-PD-1, PD -L1, CTLA-4, or LAG-3 antibodies or antigen-binding fragments thereof), cytokines (eg, IL-15, IL-7, IL-12, IL-18, IL-21), or membrane chimeric Polypeptides (eg, mIL-15, mIL-7, mIL-12, mIL-18, mIL-21).
  • an immune checkpoint eg, anti-PD-1, PD -L1, CTLA-4, or LAG-3 antibodies or antigen-binding fragments thereof
  • cytokines eg, IL-15, IL-7, IL-12, IL-18, IL-21
  • membrane chimeric Polypeptides eg, mIL-15, mIL-7, mIL-12, mIL-18, mIL-21.
  • the nucleic acid sequence encoding the anti-MSLN-CAR is linked to the nucleic acid sequence of the additional biologically active molecule by the nucleic acid sequence of the self-cleaving peptide.
  • the anti-MSLN-CAR can be at the N-terminus or C-terminus of the additional biologically active molecule. In certain exemplary embodiments, the anti-MSLN-CAR is 5' to the additional biologically active molecule. Any self-cleaving peptide capable of causing cleavage of the fusion protein into two separate proteins can be used in the present invention.
  • the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 72, the nucleotide sequence of which can be optimized according to the needs of gene recombination.
  • the fusion protein comprising the CAR and the additional biologically active molecule has the following structure:
  • N'-signal peptide--extracellular antigen-binding domain that specifically binds to human MSLN--spacer domain-transmembrane domain-intracellular signaling domain-self-cleaving peptide-signal peptide-2--additional organisms Active Molecules - C'. wherein the signal peptide-2 is the same as or different from the signal peptide of N'.
  • the additional bioactive molecule has a different signal peptide immediately upstream from the N-terminus of the MSLN-CAR.
  • the signal peptide-2 is a human IL2 signal peptide, having the sequence set forth in SEQ ID NO:74.
  • the plurality of nucleic acid sequences encoding the more than one additional biologically active molecules are separated by encoding from Sequence ligation of cleavage peptides (eg P2A).
  • the additional biologically active molecule is an anti-PD-1 linear antibody, preferably, the linear antibody has the sequence set forth in SEQ ID NO:77.
  • the additional biologically active molecule is membrane chimeric IL-15, preferably, the membrane chimeric IL-15 has the sequence set forth in SEQ ID NO:81.
  • Methods of generating chimeric antigen receptors and immune effector cells comprising the chimeric antigen receptors are known in the art and can include transfecting the cells with at least one polynucleotide encoding a CAR, and Polynucleotides are expressed in cells.
  • a nucleic acid molecule encoding a CAR of the invention can be included in an expression vector (eg, a lentiviral vector) capable of being expressed in a host cell, such as a T cell, to manufacture the CAR.
  • the sixth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of the fifth aspect.
  • nucleotide sequence encoding a chimeric antigen receptor of the present invention can have a variety of different sequences.
  • a "nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are a degenerate form of each other and that encode the same amino acid sequence.
  • the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) the sequence shown in SEQ ID NO: 84 or a degenerate variant thereof; (2) A sequence that is substantially identical to the sequence described in (1), eg, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, compared to (1), at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% A sequence of %, or 100% sequence identity, or, a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and the sequence substantially retains the nucleoside from which it was derived At least one biological activity of the acid sequence (eg, capable of encoding the ability to direct the specificity and reactivity of immune effector cells to cells expressing M
  • the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) the sequence shown in SEQ ID NO: 89 or a degenerate variant thereof; (2) A sequence that is substantially identical to the sequence described in (1), eg, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, compared to (1), at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% A sequence of %, or 100% sequence identity, or, a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and the sequence substantially retains the nucleoside from which it was derived At least one biological activity of the acid sequence (eg, capable of encoding the ability to direct the specificity and reactivity of immune effector cells to cells expressing M
  • the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) the sequence shown in SEQ ID NO: 91 or a degenerate variant thereof; (2) A sequence that is substantially identical to the sequence described in (1), eg, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, compared to (1), at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% A sequence of %, or 100% sequence identity, or, a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and the sequence substantially retains the nucleoside from which it was derived At least one biological activity of the acid sequence (eg, capable of encoding the ability to direct the specificity and reactivity of immune effector cells to cells expressing M
  • the CARs of the present invention can also be co-expressed with additional bioactive molecules to synergistically exert anti-tumor effects.
  • the seventh aspect of the present invention also provides a nucleic acid construct comprising a first nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect, and further comprising a second nucleic acid sequence encoding an additional biologically active molecule .
  • the additional biologically active molecule encoded by the second nucleic acid sequence has anti-tumor activity.
  • the additional biologically active molecule encoded by the second nucleic acid sequence is selected from one or more of the following components: immune checkpoint inhibitors (eg, anti-PD-1, PD-L1 , CTLA-4, or LAG-3 antibodies or antigen-binding fragments thereof), cytokines (eg, IL-15, IL-7, IL-12, IL-18, or IL-21), or membrane chimeric polypeptides (eg, mIL-15, mIL-7, mIL-12, mIL-18, or mIL-21).
  • immune checkpoint inhibitors eg, anti-PD-1, PD-L1 , CTLA-4, or LAG-3 antibodies or antigen-binding fragments thereof
  • cytokines eg, IL-15, IL-7, IL-12, IL-18, or IL-21
  • membrane chimeric polypeptides eg, mIL-15, mIL-7, mIL-12, mIL-18, or mIL-21.
  • the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide-2 at its N-terminus.
  • the signal peptide-2 is different from the signal peptide contained in the chimeric antigen receptor encoded by the first nucleic acid sequence.
  • the signal peptide-2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide
  • IL2 signal peptide refers to the signal peptide sequence contained in the IL2 native gene sequence, preferably, the IL2 native base Since the human IL2 native gene, the IL2 signal peptide is the human IL2 signal peptide.
  • the IL2 signal peptide comprises the sequence set forth in SEQ ID NO:74.
  • the first nucleotide sequence is upstream of the second nucleotide sequence.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (eg, P2A, E2A, F2A, T2A, or any combination thereof).
  • a self-cleaving peptide eg, P2A, E2A, F2A, T2A, or any combination thereof.
  • the self-cleaving peptide is P2A (eg, as set forth in SEQ ID NO:72).
  • the sequence encoding the self-cleaving peptide is linked to the 3' end of the first nucleotide sequence, and is linked to the 5' end of the second nucleotide sequence.
  • the additional biologically active molecule is selected from immune checkpoint inhibitors that are anti-PD-1 or PD-L1 antibodies or antigen-binding fragments thereof (eg, scFvs).
  • the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region of any one of the following groups: (1) Nivolumab or a variant thereof The heavy chain variable region and/or the light chain variable region of the body, (2) the heavy chain variable region and/or the light chain variable region of Pembrolizumab or its variants, (3) the heavy chain of Atezolizumab or its variants variable region and/or light chain variable region, (4) heavy chain variable region and/or light chain variable region of Durvalumab or a variant thereof, (5) heavy chain variable region of Avelumab or a variant thereof and /or light chain variable region, (6) VH having the sequence shown in SEQ ID NO: 79 or a variant thereof and/or VL having the sequence shown in SEQ ID NO: 80 or a variant thereof.
  • the variant is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 94% compared to the sequence from which it is derived 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or substitution, deletion or addition of one or several amino acids (e.g. 1, 2) compared to the sequence from which it is derived 1, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
  • the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof is a single chain antibody (eg, scFv).
  • the additional biologically active molecule comprises an anti-PD-1 single chain antibody having the sequence VH set forth in SEQ ID NO:79 and/or having SEQ ID NO: VL of the sequence shown in 80.
  • the VH and VL are linked by a linker.
  • the linker comprises a sequence as represented by (G m S) n , wherein m is selected from an integer of 1-6. In certain embodiments, m is 3, 4, or 5; n is selected from an integer of 1-10. In certain embodiments, n is 2, 3, 4, 5, or 6.
  • the anti-PD-1 single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) the amino acid sequence shown in SEQ ID NO:77; (2) the same as the amino acid sequence shown in SEQ ID NO:77 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least A sequence of 97%, at least 98%, at least 99%, or 100% identity; (3) compared with the sequence shown in SEQ ID NO: 77, has one or several amino acid substitutions, deletions or additions (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
  • the nucleic acid construct of the seventh aspect comprises, in order from its 5' end to its 3' end: the first nucleic acid sequence, a nucleotide sequence encoding a self-cleaving peptide, a signal peptide encoding The nucleotide sequence of -2, the nucleotide sequence encoding an immune checkpoint inhibitor.
  • the nucleic acid construct of the seventh aspect comprises a nucleotide sequence selected from the group consisting of: (1) the nucleotide sequence shown in SEQ ID NO: 85 or a degenerate variant thereof; (2) A sequence substantially identical to the sequence described in (1), eg, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , sequences of at least 99%, or 100% sequence identity.
  • the additional biologically active molecule is selected from a membrane chimeric polypeptide that is mIL-15.
  • the membrane chimeric polypeptide mIL-15 comprises an amino acid sequence selected from the group consisting of: (1) the sequence shown in SEQ ID NO: 81; (2) the same as the sequence shown in SEQ ID NO: 81 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least A sequence of 97%, at least 98%, at least 99%, or 100% identity; (3) compared with the sequence shown in SEQ ID NO: 81, there is a substitution, deletion or addition of one or several amino acids (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
  • the nucleic acid construct of the seventh aspect comprises, in order from its 5' end to its 3' end: the first nucleic acid sequence, a nucleotide sequence encoding a self-cleaving peptide, a membrane embedding The nucleotide sequence of the synthetic polypeptide.
  • the nucleic acid construct of the seventh aspect comprises a nucleotide sequence selected from the group consisting of: (1) the nucleotide sequence shown in SEQ ID NO: 86 or a degenerate variant thereof; (2) A sequence substantially identical to the sequence described in (1), for example having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% %, or sequences with 100% sequence identity.
  • the additional biologically active molecule encoded by the second nucleic acid sequence comprises at least two components selected from the group consisting of immune checkpoint inhibitors (eg, anti-PD-1, PD-L1, CTLA -4, or LAG-3 antibody or antigen-binding fragment thereof), cytokine (eg, IL-15, IL-7, IL-12, IL-18, or IL-21), or membrane chimeric polypeptide (eg , mIL-15, mIL-7, mIL-12, mIL-18, or mIL-21).
  • immune checkpoint inhibitors eg, anti-PD-1, PD-L1, CTLA -4, or LAG-3 antibody or antigen-binding fragment thereof
  • cytokine eg, IL-15, IL-7, IL-12, IL-18, or IL-21
  • membrane chimeric polypeptide eg , mIL-15, mIL-7, mIL-12, mIL-18, or mIL-21.
  • the nucleotide sequences encoding the at least two components contained in the second nucleic acid sequence are mutually accessible by encoding self-cleaving peptides (eg, P2A, E2A, F2A, T2A, or any thereof). combination), in certain embodiments, the self-cleaving peptide is P2A (eg, as set forth in SEQ ID NO: 72).
  • the additional biologically active molecule encoded by the second nucleic acid sequence comprises: (i) an anti-PD-1 antibody or antigen-binding fragment thereof (eg, scFv) and (ii) mIL-15.
  • the nucleic acid construct of the seventh aspect comprises the first nucleic acid sequence, a nucleotide sequence encoding a self-cleaving peptide, and a signal peptide-2 encoding in order from its 5' end to its 3' end.
  • the nucleic acid construct of the seventh aspect comprises a nucleotide sequence selected from the group consisting of: (1) the nucleotide sequence set forth in SEQ ID NO: 87 or a degenerate variant thereof; ( 2) A sequence substantially identical to the sequence described in (1), eg, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • the eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule of the sixth aspect, or the nucleic acid construct of the seventh aspect.
  • the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, viral vectors.
  • the vector is an expression vector.
  • the vector is an episomal vector.
  • the vector is a viral vector.
  • the viral vector is a lentiviral, adenoviral, or retroviral vector.
  • the vector is an episomal or non-integrating viral vector, such as an integration-deficient retrovirus or lentivirus.
  • a ninth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule of the sixth aspect, the nucleic acid construct of the seventh aspect or the vector of the eighth aspect.
  • the vectors described above can be introduced into host cells by various suitable means, such as calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, TALEN methods, ZFN methods, non-viral vectors mediated transfection (e.g. liposomes) or viral vector-mediated transfection (e.g. lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological methods for transfer into host cells means, such as transposon technology, CRISPR-Cas9 and other technologies.
  • the host cell comprises the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule, the host cell expressing the chimeric antigen receptor of the present invention.
  • the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, the host cell expressing the chimeric antigen receptor of the invention and additional biologically active molecules .
  • the host cells are selected from mammalian (eg, human) immune cells.
  • the immune cells are derived from a patient or healthy donor.
  • the immune cells are selected from T lymphocytes, natural killer (NK) cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are derived from T lymphocytes or NK cells.
  • the tenth aspect of the present invention provides a method for preparing a cell expressing the chimeric antigen receptor of the present invention, comprising: (1) providing a host cell; (2) adding the isolated nucleic acid molecule or containing the nucleic acid molecule according to the sixth aspect The vector for the nucleic acid molecule is introduced into the host cell to obtain a host cell capable of expressing the chimeric antigen receptor.
  • Also provided is a method for cells co-expressing the chimeric antigen receptor of the present invention and another biologically active molecule comprising: (1) providing a host cell; (2) combining the nucleic acid construct or comprising the nucleic acid construct of the seventh aspect The vector of the nucleic acid construct is introduced into the host cell to obtain a host cell capable of co-expressing the chimeric antigen receptor and the additional biologically active molecule.
  • the host cells are selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages, and any combination thereof.
  • the immune cells are selected from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells, and any combination of these cells.
  • the immune cells are provided from a patient or a healthy donor and undergo pretreatment; the pretreatment includes sorting, activation and/or proliferation of immune cells; in a certain In some embodiments, the pretreatment comprises contacting the immune cells with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.
  • the nucleic acid molecule or vector in step (2), is introduced into immune cells by viral infection. In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into immune cells by means of non-viral vector transfection, such as vector system by transposon, CRISPR/Cas9 vector, TALEN method, ZFN method, Methods such as electroporation, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • non-viral vector transfection such as vector system by transposon, CRISPR/Cas9 vector, TALEN method, ZFN method, Methods such as electroporation, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • the method further comprises: expanding the immune cells obtained in step (2).
  • Immune cells derived from patients or healthy donors can be transformed into immune cells expressing a CAR that specifically binds to MSLN and optionally other biologically active molecules by the above-mentioned preparation method provided by the present invention.
  • the eleventh aspect of the present invention also provides an engineered immune cell expressing the CAR of the present invention that specifically binds to MSLN.
  • the engineered immune cells comprise the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule.
  • the engineered immune cells also express additional biologically active molecules.
  • the engineered immune cells comprise the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct.
  • the engineered immune cells of the present invention can also co-express another biologically active molecule, which can specifically bind to the chimeric antigen receptor of MSLN to become Independent CARs with extracellular antigen-binding domains, spacer domains, transmembrane domains, and intracellular signaling domains, while additional biologically active molecules can be secreted extracellularly or expressed as membrane chimeric polypeptides or protein.
  • the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells of a patient or healthy donor, and any combination thereof. These immune cells are prepared into engineered immune cells by introducing the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect or the vector described in the eighth aspect by the method provided in the tenth aspect.
  • the engineered immune cells may have binding specificities other than MSLN targets, eg: the engineered immune cells also express a CAR that is not specific for MSLN; preferably, the non-specific
  • the CAR for MSLN has specificity for a target selected from CD19, GPC3, PSMA, MUCl, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
  • genes involved in the immune exclusion of engineered immune cells eg, TRAC, TRBC, B2M, HLA-A, HLA-B, or HLA-C
  • genes of immune co-suppressive pathways or signaling molecules eg, the transcription or expression of one or both target genes of PD-1, CTLA-4 or LAG-3
  • the method by which the transcription or expression of the target gene is inhibited is selected from gene knockout (eg, CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
  • the present invention also provides immune cell compositions comprising the aforementioned engineered immune cells, and optionally unengineered and/or unsuccessfully engineered immune cells, which are unengineered and /or unsuccessfully engineered immune cells that do not express a CAR specific for MSLN.
  • immune cell compositions comprising the aforementioned engineered immune cells, and optionally unengineered and/or unsuccessfully engineered immune cells, which are unengineered and /or unsuccessfully engineered immune cells that do not express a CAR specific for MSLN.
  • the immune cell composition can contain immune cells that express and do not express CAR specific for MSLN, and the immune cell composition can still meet the needs of clinical applications.
  • the engineered immune cells expressing a CAR specific for MSLN comprise about 10%-100%, preferably 40%-80%, of the total cell number of the immune cell composition.
  • the immune cell composition is cultured into an immune cell line, thus, in another aspect, the invention also provides an immune cell line comprising the immune cell composition.
  • the present invention provides the preparation of a chimeric antigen receptor that specifically binds to MSLN, or for the preparation of cells expressing the chimeric antigen receptor or co-expressing the chimeric antigen receptor and additional biological activity Molecular immune cell kit.
  • the kit comprises the isolated nucleic acid molecule of the sixth aspect, the nucleic acid construct of the seventh aspect or the vector of the eighth aspect, or the host of the ninth aspect cells, and necessary solvents, such as sterile water, physiological saline, or cell culture medium, such as LB medium, such as EliteCell primary T lymphocyte culture system (product number: PriMed-EliteCell-024), and optionally, Also includes instructions for use.
  • the present invention provides the aforementioned kit for preparing a chimeric antigen receptor capable of specifically binding to MSLN, or a cell expressing the chimeric antigen receptor, or co-expressing the chimeric antigen receptor and Application of additional biologically active molecules to immune cells.
  • the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, and the chimeric antigen receptor (including bispecific chimeric antigen receptor) described in the fifth aspect.
  • a CAR construct co-expressed with an antigen receptor or with another biologically active molecule the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the third aspect or the eighth aspect
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity (eg, anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, pemetrexed Troxetate, cisplatin, paclitaxel, gemcitabine, capecitabine, or FOLFIRINOX).
  • a drug with anti-tumor activity eg, anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, pemetrexed Troxetate, cisplatin, paclitaxel, gemcitabine, capecitabine, or FOLFIRINOX.
  • the antibody or antigen-binding fragment thereof of the first aspect of the present invention may be administered simultaneously, separately or sequentially.
  • the pharmaceutical composition of the present invention comprises: the isolated nucleic acid molecule of the sixth aspect, the nucleic acid construct of the seventh aspect or the vector of the eighth aspect, or the ninth aspect of host cells.
  • the pharmaceutical compositions of the present invention comprise: engineered immune cells or immune cell compositions of the present invention.
  • the drug can be formulated into any dosage form known in the medical art, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection, sterile powder for injection and concentrated solution for injection), inhalation, spray, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage.
  • a preferred dosage form is an injection.
  • injectable preparations can be sterile injectable solutions.
  • sterile injectable solutions can be prepared as sterile lyophilized powders (eg, by vacuum drying or freeze-drying) for ease of storage and use.
  • Such sterile lyophilized powders can be dispersed in a suitable vehicle, eg, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg, 0.9% (w/v) NaCl), Dextrose solutions (eg, 5% dextrose), surfactant-containing solutions (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered solutions), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution eg, 0.9% (w/v) NaCl
  • Dextrose solutions eg, 5% dextrose
  • surfactant-containing solutions eg, 0.01% polysorbate 20
  • pH buffered solutions eg, phosphate buffered solutions
  • Ringer's solution eg, Ringer's solution, and any combination thereof.
  • the drug can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical , topical (eg, powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral administration (eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • parenteral administration eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection.
  • route and/or mode of administration will vary depending on the intended purpose.
  • the antibody or antigen-binding fragment thereof of the first aspect of the present invention is administered by intravenous injection or bolus injection.
  • the pharmaceutical composition of the present invention may comprise a "therapeutically effective amount” or “prophylactically effective amount” of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect, and the second aspect of the present invention.
  • the isolated nucleic acid molecule described in the sixth aspect the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the eleventh aspect
  • the engineered immune cells of the aspect or the immune cell composition of the twelfth aspect refers to an amount sufficient to prevent, prevent, or delay the onset of a disease.
  • a “therapeutically effective amount” refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.
  • the therapeutically effective amount of the drug may vary depending on factors such as the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general conditions such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered treatment, etc.
  • the present invention provides a method for preventing and/or treating a disease associated with expression of mesothelin in a subject (eg, a human), the method comprising administering to a subject in need thereof or administering an effective amount of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the second or sixth aspect, the seventh aspect
  • the disease associated with the expression of mesothelin is selected from a proliferative disease, such as a tumor. In certain embodiments, the disease associated with the expression of mesothelin is a non-tumor-related condition associated with the expression of mesothelin.
  • the tumor is an MSLN-positive tumor.
  • the tumor is selected from solid tumors (eg, malignant pleural mesothelioma, pancreatic cancer, lung cancer (eg, squamous cell carcinoma of the lung), breast cancer, ovarian cancer (eg, epithelial ovarian cancer)).
  • solid tumors eg, malignant pleural mesothelioma, pancreatic cancer, lung cancer (eg, squamous cell carcinoma of the lung), breast cancer, ovarian cancer (eg, epithelial ovarian cancer)).
  • the method comprises administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of the first aspect.
  • the method comprises administering to the subject an effective amount of the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the seventh aspect
  • the host cells are immune cells (eg, human immune cells).
  • the method comprises the steps of: (1) providing immune cells (eg, T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells); (2) introducing the isolated nucleic acid molecule described in the sixth aspect of the present invention, the nucleic acid construct described in the seventh aspect or the vector described in the eighth aspect into the step (1) described to obtain cells expressing the chimeric antigen receptor and optionally additional biologically active molecules; (3) administering the immune cells obtained in step (2) to the subject for treatment.
  • immune cells eg, T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • immune cells eg, T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • the method administers to the subject an immune cell expressing a CAR of the invention by administering a portion of the dose in divided doses, such as one, two, three or more divided doses, e.g., during treatment
  • the first percentage of the total dose is administered on the first day of treatment
  • the second hundredth of the total dose is administered on a subsequent (eg, second, third, fourth, fifth, sixth or seventh day or later) treatment day fraction, such as a third percentage of the total dose (e.g., administered on a subsequent (e.g. third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or later) treatment day) , the remaining percentage).
  • 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.
  • 50% of the total dose of cells is administered on the first day of treatment and on subsequent (eg, second, third, fourth, fifth, sixth or seventh or later) treatment days Administer 50% of the total dose of cells.
  • 1/3 of the total dose of cells is administered on the first day of treatment and on subsequent (eg, second, third, fourth, fifth, sixth or seventh days or later) Administer 1/3 of the total dose of cells on treatment days, with 1 of the total dose administered on subsequent days (eg, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or later) /3 cells.
  • the total cell dose comprises 1 ⁇ 10 7 to 10 ⁇ 10 8 CAR-positive immune cells, eg, comprises (1-5) ⁇ 10 7 to (5-10) ⁇ 10 8 CAR-positive immune cells .
  • the physician may adjust the dosage or treatment regimen based on the patient's state, the size and stage of the tumor, or clinical circumstances such as the drugs being used in combination therapy.
  • the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the engineered immune cell of the eleventh aspect or the tenth aspect is administered in combination with another agent.
  • the additional agent comprises (i) increasing a cell comprising a CAR nucleic acid or a CAR polypeptide (eg, an immune cell expressing a CAR of the invention, an engineered immune cell or an immune cell composition of the invention) (ii) ameliorating one or Agents with various side effects; (iii) additional pharmaceutically active agents with antitumor activity.
  • a CAR nucleic acid or a CAR polypeptide eg, an immune cell expressing a CAR of the invention, an engineered immune cell or an immune cell composition of the invention
  • reagents can be used in the administration of the antibody or antigen-binding fragment thereof of the first aspect of the invention, the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the second or sixth aspect, the seventh aspect
  • the immune cell composition, or the pharmaceutical composition is administered before, at the same time or after.
  • the methods described above further comprise administering to the subject a second therapy, which may be any therapy known for use in tumors, such as surgery, chemotherapy, radiation therapy, immunotherapy, Gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.
  • a second therapy which may be any therapy known for use in tumors, such as surgery, chemotherapy, radiation therapy, immunotherapy, Gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.
  • the second therapy may be administered separately or in combination with the methods described above; alternatively, the second therapy may be administered concurrently or sequentially with the methods described above.
  • the subject may be a mammal, such as a human.
  • the present invention provides the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the second or sixth aspect, the The nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the engineered immune cell of the eleventh aspect or the twelfth aspect
  • the dosage, dosage form, administration route, indication, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the medicament.
  • FR antibody framework region amino acid residues other than CDR residues in the variable region of an antibody
  • Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991 ).
  • IMGT is based on The international ImMunoGeneTics information system initiated by Lefranc et al. (IMGT)), see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • Chothia An immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. Man (1989) Nature 342:878-883).
  • antibody refers to an immunization capable of specifically binding a target (eg, carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule globulin molecules.
  • a target eg, carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term includes not only whole polyclonal or monoclonal antibodies, but also fragments thereof (eg Fab, Fab', F(ab')2, Fv), single chain (eg scFv, di-scFv, (scFv) ) 2 ) and domain antibodies (including, for example, shark and camel antibodies), as well as fusion proteins including antibodies, and immunoglobulin molecules in any other modified configuration including antigen recognition sites.
  • the antibodies of the present invention are not limited by any particular method of producing antibodies.
  • Antibodies include any type of antibody, such as IgG, IgA, or IgM (or a subclass thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant regions corresponding to the different types of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains.
  • the heavy chain constant region consists of 4 domains (CH1, hinge region, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins with host tissues or factors, including various cells of the immune system (eg, effector cells) and classical complement Binding of the first component (C1q) of the system.
  • VH and VL regions of antibodies can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901- 917; definition by Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained by an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art.
  • the CDRs contained by an antibody or antigen-binding fragment thereof of the invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or Or compete with a full-length antibody for specific binding to an antigen, which is also referred to as an "antigen-binding portion.”
  • an antigen-binding portion See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is hereby incorporated by reference in its entirety for all purposes.
  • antigen-binding fragments of antibodies are produced by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include camelid Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab )' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , minibodies, diabodies, tribodies, tetrabodies, disulfide stabilized Fv proteins ("dsFv”) and single structures Domain antibodies (sdAbs, Nanobodies) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136 middle.
  • the term "camelid Ig” or “camel VHH” refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte et al., FASEB J., 21:3490-3498 (2007)).
  • "Heavy chain antibody” or “camel antibody” refers to an antibody containing two VH domains and no light chain (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999) ); WO94/04678; WO94/25591; US Pat. No. 6,005,079).
  • IgNAR immunoglobulin neoantigen receptor
  • VNAR variable neoantigen receptor
  • CNAR constant neoantigen receptor
  • Fd means an antibody fragment consisting of VH and CH1 domains
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989))
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • F(ab') 2 fragment means an antibody fragment comprising two fragments linked by a disulfide bridge on the hinge region.
  • Fab'fragment means a fragment obtained by reducing the disulfide bond linking two heavy chain fragments in an F(ab') 2 fragment, consisting of an intact light chain and heavy chain Fd Fragments (consisting of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the one-armed VL and VH domains of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (eg, an Fd fragment, which contains only three CDRs specific for the antigen) is able to recognize and bind the antigen, albeit with possibly lower affinity than the intact binding site.
  • a single variable region eg, an Fd fragment, which contains only three CDRs specific for the antigen
  • Fc means that the second and third constant regions of the first heavy chain of an antibody are joined by disulfide bonds to the second and third constant regions of the second heavy chain.
  • Antibody Fragments The Fc fragment of an antibody has many different functions, but is not involved in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, eg, Bird et al., Science 242:423 -426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • GGGGS linker with the amino acid sequence
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • a disulfide bond may also exist between the VH and VL of the scFv.
  • the VH and VL domains can be positioned relative to each other in any suitable arrangement.
  • scFvs comprising NH2 -VH-VH-COOH, NH2 - VL-VL-COOH.
  • the scFv can form any engineering possible structure, single chain antibody (scFv), tandem antibody (tandem di-scFvs), diabody, tribody, tetrabody, disulfide stabilized Fv protein, camelid Ig , IgNAR, etc.
  • the scFv can form a di-scFv, which refers to the tandem of two or more individual scFvs to form an antibody.
  • scFvs may form (scFv) 2 , which refers to two or more individual scFvs in parallel to form an antibody.
  • the term "diabody” refers to an antibody fragment having two antigen-binding sites, the fragment comprising in the same polypeptide chain (VH-VL) linked to a light chain variable domain (VL) ) of the heavy chain variable domain (VH).
  • VH-VL polypeptide chain
  • VL light chain variable domain
  • VH heavy chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and two antigen binding sites are created.
  • Bifunctional antibodies can be bivalent or bispecific. Bifunctional antibodies are more fully described, for example, in EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:6444-6448 ( 1993). Tri- and tetra-antibodies are also described in Hudson et al., Nature Med 9: 129-134 (2003).
  • single-domain antibody has the meaning commonly understood by those of skill in the art, which refers to a combination of a single monomeric variable antibody domain (eg, a single heavy chain variable region), which retain the ability to specifically bind to the same antigen bound by the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003 ).
  • Single domain antibodies are also known as nanobodies.
  • Each of the aforementioned antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and/or compete with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (eg, an antibody provided herein) using conventional techniques known to those of skill in the art (eg, recombinant DNA techniques or enzymatic or chemical fragmentation methods). ), and the antibody is screened for specificity for antigen-binding fragments in the same manner as is used for intact antibodies.
  • antibody includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • the expression “specifically binds” or “specifically targets” refers to a non-random binding reaction between two molecules, such as between an antibody and the antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant ( KD ) for that interaction.
  • K D the equilibrium dissociation constant
  • the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods well known in the art.
  • One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • association rate constants ka or kon
  • dissociation rate constants kdis or koff
  • KD, kon and kdis values can be measured by any effective method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by an adenine, or both A position in each of the polypeptides is occupied by a lysine)
  • the molecules are identical at that position.
  • the "percent identity” between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6).
  • comparisons are made when two sequences are aligned for maximum identity.
  • Such alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.).
  • Align program DNAstar, Inc.
  • Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0) can also be used, using the PAM120 weight residue table , a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences.
  • the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences .
  • conservative substitutions means amino acid substitutions that do not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues that have similar side chains, e.g., that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.) Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, eg, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector may include sequences that replicate directly autonomously in the cell, or may include sequences sufficient to allow integration into the DNA of the host cell.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and viral vectors.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Non-limiting examples of viral vectors include, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses vesicle virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses vesicle virus
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication site.
  • episomal in the term “episomal vector” means that the vector is capable of replication without integration into the chromosomal DNA of the host and is not progressively lost by dividing host cells, and also means that the vector is extrachromosomal or episomal copy.
  • viral vector is used broadly to refer to a nucleic acid molecule (eg, a transfer plasmid) that includes a virus-derived nucleic acid element that typically facilitates transfer or integration of the nucleic acid molecule into the genome of a cell, or mediates nucleic acid transfer virus particles.
  • viral particles will typically include various viral and sometimes host cell components.
  • viral vector can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself.
  • Viral vectors and transfer plasmids contain structural and/or functional genetic elements derived primarily from viruses.
  • retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof derived primarily from retroviruses.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof (including LTRs) derived primarily from lentiviruses.
  • the terms "lentiviral vector”, “lentiviral expression vector” may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. Where elements (eg, cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.) are referred to herein, it is to be understood that the sequences of these elements are present in the lentiviral particles of the invention in RNA form and in the present invention in DNA form in the DNA plasmid of the invention.
  • an "integration-deficient" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that is unable to integrate the viral genome into the genome of a host cell.
  • the integrase protein is mutated to specifically reduce its integrase activity.
  • Integration-deficient lentiviral vectors can be obtained by modifying the pol gene encoding an integrase protein to generate a mutant pol gene encoding an integration-deficient integrase.
  • integration-deficient viral vectors have been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes) cells, NK cells, monocytes, macrophages or dendritic cells, etc.).
  • a host cell can include a single cell or a population of cells.
  • chimeric antigen receptor refers to a domain comprising at least one extracellular antigen binding domain, a spacer domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as a cytoplasmic signaling domain).
  • cytoplasmic signaling domain also referred to herein as a cytoplasmic signaling domain.
  • Intracellular signaling domain recombinant polypeptide constructs that combine antibody-based specificity for an antigen of interest (eg, MSLN) with an immune effector cell activating intracellular domain to exhibit specificity for expressing the antigen of interest (eg, MSLN) ) specific immune activity of cells.
  • CAR-expressing immune effector cells refers to immune effector cells that express CAR and have antigen specificity determined by the targeting domain of the CAR.
  • Methods of making CARs are known in the art, see, eg, Park et al, Trends Biotechnol., 29:550-557, 2011; Grupp et al, N Engl J Med., 368 : 1509-1518, 2013; Han et al., J. Hematol. Oncol., 6:47, 2013; PCT Patent Publications WO2012/079000, WO2013/059593; and U.S. Patent Publication 2012/0213783, all of which are incorporated by reference Incorporated herein in its entirety.
  • extracellular antigen binding domain refers to a polypeptide capable of specifically binding an antigen or receptor of interest. This domain will be able to interact with cell surface molecules. For example, extracellular antigen binding domains can be selected to recognize antigens that are cell surface markers of target cells associated with a particular disease state.
  • intracellular signaling domain refers to the portion of a protein that transmits effector signal function signals and directs cells to perform specialized functions.
  • the intracellular signaling domain has the ability to activate at least one normal effector function of CAR-expressing immune effector cells.
  • the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.
  • primary signaling domain refers to the portion of a protein that is capable of modulating primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner.
  • Primary signaling domains that act in a stimulatory manner typically contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • costimulatory signaling domain refers to the intracellular signaling domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, upon binding to an antigen, provide a secondary signal required for efficient activation and function of T lymphocytes.
  • Non-limiting examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), DAP10.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to: sterile water, physiological saline, pH adjusters, surfactants , adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate salts and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, glucan, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate) and the like.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (eg, aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg, 0.9% (w/v) NaCl), dextrose Solutions (eg, 5% dextrose), surfactant-containing solutions (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution eg, 0.9% (w/v) NaCl
  • dextrose Solutions eg, 5% dextrose
  • surfactant-containing solutions eg, 0.01% polysorbate 20
  • pH buffered solutions eg, phosphate buffered solution
  • Ringer's solution e.g, Ringer's solution, and any combination thereof.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, tumor) in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • a beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (ie, not worsening) of the disease state, delaying or slowing the progression of the disease, amelioration or alleviation of the disease status, and relief of symptoms (whether in part or in full), whether detectable or undetectable.
  • treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • the term “subject” refers to a mammal, such as a primate, such as a human.
  • the term “subject” is meant to include a living organism in which an immune response can be elicited.
  • the subject eg, a human
  • has a tumor eg, a tumor associated with MSLN
  • the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a disease-prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (eg, a tumor);
  • a disease-treatment effective amount refers to an amount sufficient to cure or at least partially prevent an existing disease
  • an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other concurrently administered treatments and many more.
  • immune cell refers to a cell involved in an immune response, eg, involved in promoting immune effector function.
  • immune cells include T cells (eg, alpha/beta T cells and gamma/delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
  • the immune cells of the present invention may be autologous/autologous ("self") or non-autologous ("non-self", eg, allogeneic, syngeneic or allogeneic).
  • autologous refers to cells from the same subject;
  • allogeneic refers to cells of the same species that are genetically different from the comparison cell;
  • seyngeneic refers to the comparison cell genetically Identical cells from different subjects;
  • allogeneic refers to cells from a different species than the cells being compared.
  • the cells of the present invention are allogeneic.
  • T lymphocytes and/or NK cells.
  • T cell or “T lymphocyte” is well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.
  • the T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • the T cells can be helper T cells (HTL; CD4 T cells) CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, CD4CD8 T cells or any other subset of T cells.
  • T cells can include naive T cells and memory T cells.
  • immune cells can also be used as immune cells with a CAR as described herein.
  • immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils, and macrophages.
  • Immune cells also include progenitor cells of immune cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into immune cells.
  • immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained within a population of CD34+ cells derived from cord blood, bone marrow, or flowing peripheral blood, which are administered in a subject After differentiation into mature immune cells, it can be induced to differentiate into mature immune cells in vitro.
  • HSCs hematopoietic stem cells
  • the term "engineered immune cell” refers to an immune cell that expresses any one of the CARs described herein, or has introduced any one of the isolated nucleic acids or vectors described herein.
  • the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods of introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell.
  • transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell.
  • virus-mediated methods can be used.
  • Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (eg, retroviruses, adenoviruses), liposomes, and the like.
  • Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or particle bombardment.
  • the polynucleotide can be included in a vector, such as a plasmid vector or a viral vector.
  • immune effector function refers to the function or response of an immune effector cell that enhances or facilitates an immune attack on a target cell (eg, kills the target cell, or inhibits its growth or proliferation).
  • the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.
  • Chimeric antigen receptor (CAR)-T cell therapy is considered to be one of the most promising cancer treatments compared to traditional tumor treatments such as surgery, radiotherapy, and chemotherapy.
  • CAR Chimeric antigen receptor
  • the present invention provides a chimeric antigen receptor comprising an antibody of the present invention or an antigen-binding fragment thereof that targets MSLN, and immune effector cells expressing the chimeric antigen receptor of the present invention are compared to CAR-Ts known to target MSLN Has enhanced effector functions (eg, tumor-killing activity and cytokine-releasing activity).
  • the present invention also blocks the combination of PD-1 and PD-L1 by co-expressing the CAR and PD-1 antibody of the present invention, thereby restoring the activity of T cells, thereby enhancing the immune response; and by co-expressing mIL-15, promoting The proliferation and activation of T and NK cells enhance the tumor-killing effect of CAR-T cells.
  • Figure 1 shows the structure of the chimeric antigen receptor constructed in Example 2.
  • the anti-MSLN binding domain is scFv, selected from G5, G9 and G16 respectively.
  • Figure 2 shows the assay results of the killing activity of G5-CAR-T, G9-CAR-T, and G16-CAR-T on NCI-H226 target cells.
  • Figure 3 shows the detection results of IL2, TNF- ⁇ , and IFN- ⁇ secretion levels after G16-CAR-T cells were activated by NCI-H226 target cells.
  • Figure 4 shows the assay results of the killing activity of G5-CAR-T, G9-CAR-T, and G16-CAR-T on SKOV-3 target cells.
  • Figure 5 shows the detection results of IL2, TNF- ⁇ , and IFN- ⁇ secretion levels after G5-CAR-T, G9-CAR-T, and G16-CAR-T cells were activated by SKOV-3 target cells.
  • Figure 6 shows the assay results of the killing activity of G16-CAR-T, G16-PD1-CAR-T, G16-mIL15-CAR-T, G16-PD1-mIL15-CAR-T on SKOV-3 target cells.
  • Figure 7 shows the assay results of the killing activity of G16-CAR-T on negative A431 cells.
  • Figure 8 shows the detection results of IL2, TNF- ⁇ , and IFN- ⁇ secretion levels after G16-CAR-T was treated with A431-negative cells.
  • Figure 9 shows the killing ability of G16-CAR-T on PANC1 target cells in mice.
  • Figure 10 shows the killing ability of G16-CAR-T, G16-PD1-CAR-T, and G16-PD1-mIL15-CAR-T on SKOV-3 target cells in mice.
  • sequence information involved in the present invention is provided as follows:
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method described in F.M. Ausubel et al., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995 was performed.
  • Those skilled in the art appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.
  • Phage antibody libraries were constructed using cDNA and primers amplifying the variable regions of the heavy and light chains of anti-MSLN antibodies.
  • the amplified phage was transferred to a 50ml centrifuge tube, centrifuged at 9000g for 15min at 4°C. Transfer the supernatant to a new centrifuge tube, add 20% PEG6000/2.5M NaCl, mix well and place on ice for 2h. Centrifuge at 12000g for 30min, discard the supernatant, resuspend the phage pellet with 0.5ml PBST, centrifuge at 10000g for 8min, transfer the supernatant to a new test tube, take a part for titer determination, and carry out the second and third rounds of screening.
  • Monoclonal phage production in microtiter plates Inoculate a single colony into a 96-deep well plate, each well containing 300 ul of 2YT medium, 10ug/ml Tet, and incubate at 37°C for 5-6 hours at 250rpm. Take 150ul of culture from each well, add an equal volume of sterile 50% glycerol, and store at -80°C. 25ul of helper phage ( 2.5x109 ) was added to each well and incubated at 37°C for 30 minutes.
  • Biotin-labeled MSLN (MSN-H826x, ACROBiosystems) was diluted to 100ug/ml-8ug/ml with PBS, coated in streptavidin 96-well plate, overnight at 4°C. Wells were washed once with 300ul PBS. 200ul mPBST (2% milk) was blocked at 37°C for 1 hour. Remove the blocking solution, add 80ul of phage supernatant and 80ul of mPBST to each well, and let stand at room temperature for 1 hour.
  • the plate was washed 5 times with PBST, 100ul of anti-M13-HRP (diluted to 0.4ug/ml in mPBST) was added, and the plate was allowed to stand at room temperature for 1 hour.
  • the plate was washed 5 times with PBST, 100 ul of TMB was added, the reaction was performed at room temperature for 3 min, and then 100 ul of stop solution (0.2M H 2 SO 4 ) was added.
  • Absorbance at 450 nm was detected using an enzyme-linked immunosorbent assay.
  • MSLN scFv sequencing 158 single clones were selected for sequencing according to the ELISA test results, and the scfv sequence was obtained.
  • the forward and reverse primers used for sequencing were: PKLT1F (SEQ ID NO:92); PKLT1R (SEQ ID NO:93) .
  • the sequence results were analyzed using Seqcher software, and three candidate scfvs were finally obtained, named: G16, G5 and G9.
  • the 3 candidate scFv sequences were linked with Fc (human IgG1) sequence and constructed in TGEX-KAL vector, and then transfected into expi293 cells to express and purify scFv-Fc protein.
  • the Fc (human IgG1) sequence is shown in SEQ ID NO: 94 .
  • the experimental results of SEC analysis showed that the area of the monomer peaks (main peaks) of the three candidate sequences accounted for more than 85%.
  • ND means not detected.
  • MSLN scFv-Fc protein was serially diluted and stained for three selected cell lines expressing MSLN, and the cell binding ability was detected by flow cytometry. The results are shown in the table below.
  • the EC50 of anti-MSLN antibodies G9 and G16 to three kinds of MSLN-positive cells are 2-23 nM, so they have good binding affinity to MSLN.
  • Table 3 Affinity determination results of candidate scFv-Fc on 3 kinds of MSLN-positive cells
  • Example 2 Construction of lentiviral plasmid and viral packaging
  • a CAR was further constructed. Using the intracellular domain of CD137 and the ITAM region of CD3Zeta as the activation signal, it was fused with the above scFv, and the signal peptide, CD8 hinge region, and CD8 transmembrane region were added to construct a chimeric antigen receptor expression vector.
  • the constructed chimeric Antigen receptor structures are shown in the table below.
  • the sequences encoding one or more other biologically active molecules are connected by the sequences encoding P2A self-cleaving peptides.
  • P2A self-cleaving peptide nucleotide sequence ligation to obtain co-expressed CAR.
  • the biologically active molecules linked to the P2A sequence are secreted outside the CAR-T cells or chimerically expressed on the CAR-T cell membrane to synergistically play an anti-tumor effect.
  • G16-PD1-CAR-T can relieve or eliminate immunosuppression by secreting anti-PD-1 antibody after activation, and increase the anti-tumor effect;
  • G16-mIL15-CAR-T activation Afterwards, CAR-T cells express membrane chimeric IL-15, which increases the anti-tumor effect by stimulating the secretion of some cytokines and makes the anti-tumor effect more durable.
  • the structure of the coding nucleic acid sequence of the co-expressed CAR is shown in the table below.
  • the CAR lentiviral plasmid constructed above and the transfection reagent mixture were added dropwise to the 293T (ATCC) cells, and the culture dish was gently shaken to mix well. Place the petri dish in a 37°C, 5% CO 2 incubator; after culturing for 6-8 hours, remove the medium containing the transfection reagent and replace it with fresh complete medium.
  • the virus-containing medium supernatant after 48 hours and 72 hours of continuous culture was collected, and PEG was added to precipitate overnight at 4°C, and centrifuged at 4000 ⁇ g for 1 hour at 4°C. After centrifugation, in a biological safety cabinet, carefully aspirate the liquid in the centrifuge tube, add 300 ⁇ L of virus freezing solution to resuspend the pellet, and store the virus at -80°C.
  • Human PBMC cells were isolated with lymphocyte separation medium (GE), cultured in an incubator at 37°C and 5% CO 2 , and 100 ⁇ l/mL of CD3 antibody and CD28 antibody were added. Incubate for 15 minutes.
  • GE lymphocyte separation medium
  • the tube remains in the magnetic pole and is gently inverted to pour out the cells in the tube.
  • the cells were resuspended in X-vivo 15 medium and supplemented with 10% FBS, 300U/mL IL-2, 5ng/mL IL-15 and 10ng/mL IL-7.
  • cytokines and antibody complexes IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng/mL Antibody at a final concentration of 300U/mL) -CD3 (OKT3), 2 ⁇ g/mL Anti-CD28 configuration
  • the nucleic acid sequence encoding the CAR was expressed under the drive of the promoter, and the lentivirus-transfected T cells were labeled with biotin-labeled MSLN antigen, and then detected with fluorescently labeled streptavidin, and determined by flow cytometry, It reflects the expression level of CAR on the surface of T cells.
  • the CAR positive rate of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS test results are shown in the following table. The results showed that the CAR positive rate of all CAR-T cells was greater than 20%, indicating that after lentivirus transfected effector cells, CAR was successfully expressed, and MSLN-CAR chimeric antigen receptor T cells were successfully constructed.
  • the Luciferase gene was integrated into the genome of NCI-H226 cells by lentiviral transduction to obtain NCI-H226 human lung squamous cell carcinoma cells (NCI-H226-luc) that can stably express Luciferase.
  • NCI-H226-luc cells were digested with 0.25% trypsin, and the digestion was terminated in 1640 medium containing 10% FBS. After centrifugation, the cells were resuspended, the cell density was adjusted to 1 ⁇ 10 5 cells/mL, and the cells were seeded at 100 ⁇ L/well.
  • the target cells NCI-H226-luc were placed in a 96-well plate in a 5% CO 2 37°C incubator for 30 min.
  • G5-CAR-T, G9-CAR-T, and G16-CAR-T were collected separately, collected by centrifugation and resuspended in 1640 medium with 10% FBS, and then treated with G5-CAR-T, G9-CAR-T, G16-CAR-T and blank T cells that were not transfected with CAR were used as effector cells, and then added to the cells containing NCI-H226- In a 96-well plate of luc, 100 ⁇ L/well, the final volume was supplemented to 200 ⁇ L/well, and cultured in a 5% CO 2 37°C incubator for 18-24 h. After the incubation, take the plate out of the incubator, add 20ul of fluorescence detection reagent, and use a microplate reader to detect the fluorescence reading.
  • the detection results of the killing activity of CAR-T are shown in Figure 2, all of which can exert the biological activity of lysis on tumor cells.
  • G16-CAR-T can lyse tumor cells up to 98% when the ratio of effector cells/target cells is 1.
  • G9-CAR-T and G5-CAR-T can lyse about 80% of tumor cells when the ratio of effector cells/target cells is 1.
  • NCI-H226 cells adjust the cell density to 1 ⁇ 10 5 cells/mL with medium, seed target cells in 96-well plates at an amount of 100 ⁇ L/well, and resuspend G16-CAR-T cells with medium, then use G16 -CAR-T and blank T cells not transfected with CAR were used as effector cells, and then added to a 96-well plate containing target cells at a 1:1 ratio of E/T (effector cells/target cells), 100 ⁇ L/well, final The volume was made up to 200 ⁇ L/well and cultured overnight in a 5% CO 2 37°C incubator. After the culture, the well plate was taken out of the incubator, centrifuged, and the supernatant was taken, and ELISA kits (IL2, TNF- ⁇ , IFN- ⁇ ) were used to detect the cytokine release of CAR-T cells.
  • IL2 TNF- ⁇ , IFN- ⁇ ELISA kits
  • G16-CAR-T can significantly enhance the secretion or release of IL2, TNF- ⁇ , and IFN- ⁇ , and has good immune-enhancing activity.
  • Example 6 Evaluation of the killing activity of CAR-T on SKOV-3 target cells
  • the Luciferase gene was integrated into the genome of SKOV-3 cells by lentiviral transduction to obtain human ovarian cancer SKOV-3 cells (SKOV-3-luc) that can stably express Luciferase.
  • SKOV-3-luc cells were digested with 0.25% trypsin, and the McCoy's5A medium containing 10% FBS was used to terminate digestion. After centrifugation, resuspend the cells and adjust the cell density to 1 ⁇ 10 5 cells/mL.
  • the target cells SKOV-3-luc were seeded in a 96-well plate in a 96-well plate, and the cells were placed in a 5% CO 2 37° C. incubator for 30 min.
  • G5-CAR-T, G9-CAR-T, G16-CAR-T were collected respectively, collected by centrifugation and resuspended CAR-T cells in McCoy's5A medium with 10% FBS.
  • T, G16-CAR-T and blank T cells without CAR transfection were used as effector cells, and then added to the cells containing SKOV- In a 96-well plate of 3-luc, 100 ⁇ L/well, the final volume was supplemented to 200 ⁇ L/well, and cultured in a 5% CO 2 37°C incubator for 18-24 h. After the incubation, take the plate out of the incubator, add 20ul of fluorescence detection reagent, and use a microplate reader to detect the fluorescence reading.
  • the detection results of the killing activity of CAR-T are shown in Figure 4, all of which can exert the biological activity of lysis on tumor cells.
  • G16-CAR-T can lyse tumor cells up to 95% when the ratio of effector cells/target cells is 1.
  • G9-CAR-T and G5-CAR-T can lyse about 50% of tumor cells when the ratio of effector cells/target cells is 1.
  • Collect SKOV-3-luc cells adjust the cell density to 1 ⁇ 10 5 cells/mL with medium, seed target cells in 96-well plates at 100 ⁇ L/well, and resuspend G5-CAR-T and G9 in medium -CAR-T, G16-CAR-T cells, G5-CAR-T, G9-CAR-T, G16-CAR-T and blank T cells that were not transfected with CAR were used as effector cells, and then E The ratio of /T (effector cells/target cells) was added to a 96-well plate containing target cells, 100 ⁇ L/well, the final volume was supplemented to 200 ⁇ L/well, and the cells were cultured overnight in a 5% CO 2 37°C incubator. After the culture, the well plate was taken out of the incubator, centrifuged, and the supernatant was taken, and ELISA kits (IL2, TNF- ⁇ , IFN- ⁇ ) were used to detect the cytokine release of CAR-T cells
  • G5-CAR-T, G9-CAR-T, and G16-CAR-T can promote the secretion or release of cytokines such as TNF- ⁇ and IFN- ⁇ under the stimulation of tumor cells, especially G16-CAR-T can significantly enhance the secretion or release of IL2, TNF- ⁇ , and IFN- ⁇ , and has good immune-enhancing activity.
  • SKOV-3-luc cells were digested with 0.25% trypsin, and the McCoy's5A medium containing 10% FBS was used to terminate digestion. After centrifugation, resuspend the cells and adjust the cell density to 1 ⁇ 10 5 cells/mL.
  • the target cells SKOV-3-luc were seeded in a 96-well plate in a 96-well plate, and the cells were placed in a 5% CO 2 37° C. incubator for 30 min.
  • G16-CAR-T, G16-PD1-CAR-T, G16-mIL15-CAR-T, G16-PD1-mIL15-CAR-T were collected, collected by centrifugation and resuspended CAR-T in McCoy's 5A medium with 10% FBS Cells, G16-CAR-T, G16-PD1-CAR-T, G16-mIL15-CAR-T, G16-PD1-mIL15-CAR-T, and blank T cells without CAR were used as effector cells, followed by 1 :1, 0.5:1, 0.25:1 E/T (effector cells/target cells) ratios were added to 96-well plates containing SKOV-3-luc, 100 ⁇ L/well, and the final volume was supplemented to 200 ⁇ L/well, 5% Incubate in a CO 2 37°C incubator for 18-24h. After the incubation, take the plate out of the incubator, add 20ul of fluorescence detection reagent,
  • the detection results of the killing activity of CAR-T are shown in Figure 6, all of which can exert the biological activity of lysis on tumor cells.
  • G16-CAR-T, G16-PD1-CAR-T, G16-PD1-mIL15-CAR-T have comparable biological activities on tumor cell lysis, and when the ratio of effector cells/target cells is 1, the lysis rate of tumor cells is as high as 98%. When the ratio of effector cells/target cells is 1, the lysis rate of G16-mIL15-CAR-T on tumor cells reaches about 73%.
  • Example 7 Evaluation of the killing activity of CAR-T on MSLN-negative A431 cells
  • the Luciferase gene was integrated into the genome of MSLN-negative A431 cells by lentiviral transduction to obtain A431 human skin squamous cell carcinoma cells (A431-luc) that can stably express Luciferase.
  • A431-luc cells were digested with 0.25% trypsin, and the digestion was terminated in 1640 medium containing 10% FBS. After centrifugation, the cells were resuspended, the cell density was adjusted to 1 ⁇ 10 5 cells/mL, and the target cells were seeded at 100 ⁇ L/well.
  • A431-luc was placed in a 96-well plate in a 5% CO 2 37°C incubator for 30 min.
  • G16-CAR-T was collected, collected by centrifugation, and CAR-T cells were resuspended in 1640 medium with 10% FBS.
  • G16-CAR-T and blank T cells without CAR were used as effector cells.
  • :1, 0.125:1 E/T (effector cells/target cells) ratio was added to the 96-well plate containing A431-luc, 100 ⁇ L/well, the final volume was supplemented to 200 ⁇ L/well, 5% CO 2 37 °C incubator Incubate for 18-24h. After the incubation, take the plate out of the incubator, add 20ul of fluorescence detection reagent, and use a microplate reader to detect the fluorescence reading.
  • the cytotoxic activity test results of CAR-T are shown in Figure 7.
  • the results show that G16-CAR-T has no cytotoxic activity on MSLN-negative A431 cells, indicating that G16-CAR-T has no off-target killing on MSLN-negative cells.
  • Collect A431-luc cells adjust the cell density to 1 ⁇ 10 5 cells/mL with medium, seed target cells in 96-well plates at 100 ⁇ L/well, and resuspend G16-CAR-T and untransfected cells in medium
  • the blank T cells of the CAR were used as effector cells, and then added to a 96-well plate containing target cells at a 1:1 ratio of E/T (effector cells/target cells), 100 ⁇ L/well, and the final volume was supplemented to 200 ⁇ L/well, 5 Incubate overnight in a 37°C incubator with % CO 2 . After the culture, the well plate was taken out of the incubator, centrifuged, and the supernatant was taken, and ELISA kits (IL2, TNF- ⁇ , IFN- ⁇ ) were used to detect the cytokine release of CAR-T cells.
  • test results are shown in Figure 8.
  • the results show that for MSLN-negative A431-luc cells, G16-CAR-T does not secrete IL2, TNF- ⁇ , and IFN- ⁇ , indicating that MSLN-negative cells have no effect on G16-CAR-T. Activate function.
  • Example 8 In vivo model to assess the killing ability of CAR-T cells to target cells
  • SKOV-3 cells were cultured in monolayer in vitro, and the culture conditions were McCoy's 5A medium plus 10% fetal bovine serum, and cultured at 37°C in an incubator containing 5% CO 2 air. Digestion and passage with trypsin-EDTA were performed 2-3 times a week. Cells in logarithmic growth phase were harvested, counted, and seeded.

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Abstract

本发明涉及生物医药领域，具体而言，本发明涉及特异性结合MSLN的抗体或其抗原结合片段以及包含所述抗体或其抗原结合片段的嵌合抗原受体（CAR）。本发明还涉及表达所述CAR、或者共表达所述CAR和另外的生物活性分子（例如PD-1抗体和/或mIL-15）的经改造的免疫细胞，以及制备所述经改造的免疫细胞的方法。本发明还涉及这些抗体，CAR和免疫细胞用于预防和/或治疗患有与间皮素的表达相关的疾病，例如恶性胸膜间皮瘤、胰腺癌、肺癌、乳腺癌、卵巢癌等癌症的用途以及预防和/或治疗患有与间皮素的表达相关的疾病，例如恶性胸膜间皮瘤、胰腺癌、肺癌、乳腺癌、卵巢癌等MSLN阳性肿瘤的方法。

Description

特异性结合MSLN的嵌合抗原受体及其应用 技术领域
本发明涉及生物医药领域，具体而言，本发明涉及特异性结合MSLN的抗体或其抗原结合片段以及包含所述抗体或其抗原结合片段的嵌合抗原受体(CAR)。本发明还涉及表达所述CAR、或者共表达所述CAR和另外的生物活性分子(例如PD-1抗体和/或mIL-15)的经改造的免疫细胞，以及制备所述经改造的免疫细胞的方法。本发明还涉及这些抗体，CAR和免疫细胞用于预防和/或治疗间皮素的表达相关的疾病，例如恶性胸膜间皮瘤、胰腺癌、肺癌、乳腺癌、卵巢癌等癌症的用途以及预防和/或治疗恶性胸膜间皮瘤、胰腺癌、肺癌、乳腺癌、卵巢癌等MSLN阳性肿瘤的方法。
背景技术
间皮素(Mesothelin，MSLN)是一种糖基磷脂酰肌醇连接的糖蛋白，MSLN的前体蛋白被蛋白酶水解为31kDa的巨核细胞强化因子(megakaryocyte-potentiating factor，MPF)和40kDa的间皮素。前期研究显示，CA125/MUC16是间皮素的配体，通过N-末端的细胞外功能区的重复片段与间皮素结合在一起，共同参与细胞黏附。MSLN具有高度特异性表达，在正常组织中腹膜腔、胸膜腔和心包腔的间皮细胞上都低表达，在恶性胸膜间皮瘤、胰腺癌、肺癌、乳腺癌、卵巢癌等实体瘤中高度表达，特别是在恶性间皮瘤(85％～90％)、胰腺癌(80％～85％)、卵巢上皮癌(60％～65％)和肺癌(60％～65％)中表达较高，与细胞增殖、细胞的黏附功能及抗凋亡过程相关。这些生物学特征表明MSLN可以作为一个理想的和具有多种适应症的肿瘤治疗靶点。
随着癌症发病率的逐年攀升，传统的手术、放疗和化疗等治疗方法在肿瘤治疗中的效果不佳，临床迫切需要行之有效的肿瘤治疗方法。嵌合抗原受体(CAR)-T细胞疗法被认为是最有前途的癌症治疗方法之一，成为了人类对抗癌症的新希望。它通过从病人体内采集的免疫细胞进行体外培养，在体外转导特定的外源基因，体外扩增后回输到病人体内，以非MHC限制性的方式达到治疗肿瘤的目的。CAR-T细胞疗法在血液恶行肿瘤治疗中取得了显著的疗效，对复发难治性B细胞白血病的完全缓解率超过90％。实体瘤约占所有恶行肿瘤的90％，其治疗药物需求量大。但是，CAR-T细胞治疗目前在实体瘤中的治疗效果仍不足，其主要原因是实体瘤有复杂的肿瘤微环境和肿瘤异质性高。
基于MSLN的表达特异性，针对MSLN的CAR-T细胞治疗有希望成为攻克MSLN 阳性肿瘤的方式之一。因此，发展具有高特异性和良好疗效的靶向MSLN的CAR-T疗法是迫切而必要的。
发明内容
在本申请中，发明人首先开发了免疫原性低的能够特异性识别/结合MSLN的全人源抗体。在此基础上，本发明设计构建了包含所述MSLN抗体或其抗原结合片段的嵌合抗原受体(CAR)，进一步设计构建了共表达PD-1抗体和/或mIL-15的靶向MSLN的CAR。本发明的CAR能够以非MHC限制的方式将免疫效应细胞特异性和反应性指向表达MSLN的细胞(例如恶性胸膜间皮瘤、胰腺癌、肺癌、乳腺癌、卵巢癌细胞)从而使其被清除。因此，本发明的靶向MSLN的CAR具有用于预防和/或治疗间皮素的表达相关的疾病，例如恶性胸膜间皮瘤、胰腺癌、肺癌、乳腺癌、卵巢癌等MSLN阳性肿瘤的潜力，具有重大的临床价值。
本发明的抗体
本发明第一方面提供了一种能够特异性结合MSLN(例如人MSLN)的抗体或其抗原结合片段。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(1a)如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：3或其变体的CDR-H1；序列为SEQ ID NO：4或其变体的CDR-H2；序列为SEQ ID NO：5或其变体的CDR-H3；和/或，如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：6或其变体的CDR-L1；序列为SEQ ID NO：7或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；
或
(1b)如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：9或其变体的CDR-H1；序列为SEQ ID NO：10或其变体的CDR-H2；序列为SEQ ID NO：11或其变体的CDR-H3；和/或，如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；
或
(1c)如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：14或其变体的CDR-H1；序列为SEQ ID NO：15或其变体的CDR-H2；序列为SEQ ID  NO：5或其变体的CDR-H3；和/或，如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：6或其变体的CDR-L1；序列为SEQ ID NO：7或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；
其中，(1a)、(1b)、(1c)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)如下六个根据Kabat编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：3的CDR-H1；序列为SEQ ID NO：4的CDR-H2；序列为SEQ ID NO：5的CDR-H3；序列为SEQ ID NO：6的CDR-L1；序列为SEQ ID NO：7的CDR-L2；序列为SEQ ID NO：8的CDR-L3；或
(b)如下六个根据IMGT编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：9的CDR-H1；序列为SEQ ID NO：10的CDR-H2；序列为SEQ ID NO：11的CDR-H3；序列为SEQ ID NO：12的CDR-L1；序列为SEQ ID NO：13的CDR-L2；序列为SEQ ID NO：8的CDR-L3；或
(c)如下六个根据Chothia编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：14的CDR-H1；序列为SEQ ID NO：15的CDR-H2；序列为SEQ ID NO：5的CDR-H3；序列为SEQ ID NO：6的CDR-L1；序列为SEQ ID NO：7的CDR-L2；序列为SEQ ID NO：8的CDR-L3。
在某些实施方案中，所述抗体或其抗原结合片段包括来自人的免疫球蛋白的构架区(FRs)。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含如SEQ ID NO:1所示的序列或其变体的VH和/或包含如SEQ ID NO:2所示的序列或其变体的VL；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(2a)如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：18或其变体的CDR-H1；序列为SEQ ID NO：19或其变体的CDR-H2；序列为SEQ ID NO：20或其变体的CDR-H3；和/或，如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：21或其变体的CDR-L1；序列为SEQ ID NO：22或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；
或
(2b)如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：24或其变体的CDR-H1；序列为SEQ ID NO：25或其变体的CDR-H2；序列为SEQ ID NO：26或其变体的CDR-H3；和/或，如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；
或
(2c)如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：29或其变体的CDR-H1；序列为SEQ ID NO：30或其变体的CDR-H2；序列为SEQ ID NO：20或其变体的CDR-H3；和/或，如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：21或其变体的CDR-L1；序列为SEQ ID NO：22或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；
其中，(2a)、(2b)、(2c)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)如下六个根据Kabat编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：18的CDR-H1；序列为SEQ ID NO：19的CDR-H2；序列为SEQ ID NO：20的CDR-H3；序列为SEQ ID NO：21的CDR-L1；序列为SEQ ID NO：22的CDR-L2；序列为SEQ ID NO：23的CDR-L3；或
(b)如下六个根据IMGT编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：24的CDR-H1；序列为SEQ ID NO：25的CDR-H2；序列为SEQ ID NO：26的CDR-H3；序列为SEQ ID NO：27的CDR-L1；序列为SEQ ID NO：28的CDR-L2；序列为SEQ ID NO：23的CDR-L3；或
(c)如下六个根据Chothia编号系统所定义的重链和轻链的CDRs：序列为SEQ ID  NO：29的CDR-H1；序列为SEQ ID NO：30的CDR-H2；序列为SEQ ID NO：20的CDR-H3；序列为SEQ ID NO：21的CDR-L1；序列为SEQ ID NO：22的CDR-L2；序列为SEQ ID NO：23的CDR-L3。
在某些实施方案中，所述抗体或其抗原结合片段包括来自人的免疫球蛋白的构架区(FRs)。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含如SEQ ID NO:16所示的序列或其变体的VH和/或包含如SEQ ID NO:17所示的序列或其变体的VL；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(3a)如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：33或其变体的CDR-H1；序列为SEQ ID NO：34或其变体的CDR-H2；序列为SEQ ID NO：35或其变体的CDR-H3；和/或，如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：36或其变体的CDR-L1；序列为SEQ ID NO：37或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；
或
(3b)如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：39或其变体的CDR-H1；序列为SEQ ID NO：40或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；
或
(3c)如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：44或其变体的CDR-H1；序列为SEQ ID NO：45或其变体的CDR-H2；序列为SEQ ID NO：35或其变体的CDR-H3；和/或，如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：36或其变体的CDR-L1；序列为SEQ ID NO：37或其变体 的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；
其中，(3a)、(3b)、(3c)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)如下六个根据Kabat编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：33的CDR-H1；序列为SEQ ID NO：34的CDR-H2；序列为SEQ ID NO：35的CDR-H3；序列为SEQ ID NO：36的CDR-L1；序列为SEQ ID NO：37的CDR-L2；序列为SEQ ID NO：38的CDR-L3；或
(b)如下六个根据IMGT编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：39的CDR-H1；序列为SEQ ID NO：40的CDR-H2；序列为SEQ ID NO：41的CDR-H3；序列为SEQ ID NO：42的CDR-L1；序列为SEQ ID NO：43的CDR-L2；序列为SEQ ID NO：38的CDR-L3；或
(c)如下六个根据Chothia编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：44的CDR-H1；序列为SEQ ID NO：45的CDR-H2；序列为SEQ ID NO：35的CDR-H3；序列为SEQ ID NO：36的CDR-L1；序列为SEQ ID NO：37的CDR-L2；序列为SEQ ID NO：38的CDR-L3。
在某些实施方案中，所述抗体或其抗原结合片段包括来自人的免疫球蛋白的构架区(FRs)。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含如SEQ ID NO:31所示的序列或其变体的VH和/或包含如SEQ ID NO:32所示的序列或其变体的VL；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段可以选自骆驼Ig、IgNAR、Fab片段、Fab'片段、F(ab)' 2片段、F(ab)' 3片段、单链抗体(例如scFv、di-scFv或(scFv) 2)、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(dsFv)和单结构域抗体(sdAb，纳米抗体)。
在某些实施方案中，本发明的抗体可以是包括一个或多个连接子的抗原结合片段(如scFv)，所述一个或多个连接子连接两个抗体结构域或区域(如重链可变(VH)区和轻链可变(VL)区)。因此，本发明的抗体可以包括单链抗体片段，如scFv和双抗体，特别是人单链抗体片段，通常包含连接两个抗体结构域或区域(如VH和VL区)的一个或多个连接子。
连接子通常是肽接头，例如柔性和/或可溶性肽接头，例如富含甘氨酸和丝氨酸的肽接头。连接子包括富含甘氨酸和丝氨酸和/或在一些情况下苏氨酸的那些连接子。在一些实施方案中，连接子还包括带电荷的残基(如赖氨酸和/或谷氨酸)，其可以改善溶解性。在一些实施方案中，连接子还包括一个或多个脯氨酸。
在某些实施方案中，富含甘氨酸和丝氨酸(和/或苏氨酸)的连接子包括至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的此类氨基酸。在一些实施方案中，它们包括至少为或约50％、55％、60％、70％或75％的甘氨酸、丝氨酸和/或苏氨酸。在一些实施方案中，连接子基本上完全由甘氨酸、丝氨酸和/或苏氨酸组成。例如，连接子包括具有单个或重复(例如1个、2个、3个、4个和5个重复)的序列GGGGS或GGGS的连接子。
在某些实施方案中，本发明的抗体或其抗原结合片段的VH和VL通过一个或多个连接子连接；优选地，所述连接子包含一个或几个(例如1个、2个或3个)如(G mS) n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2；更优选地，所述连接子具有SEQ ID NO：52的序列。
在某些实施方案中，所述抗体或其抗原结合片段是单链抗体，例如scFv、di-scFv或(scFv) 2。
在某些实施方案中，所述单链抗体从其N端至C端依次包括：
(1)包含如SEQ ID NO:1所示的序列或其变体的VH-连接子-包含如SEQ ID NO:2所示的序列或其变体的VL；
(2)包含如SEQ ID NO:16所示的序列或其变体的VH-连接子-包含如SEQ ID NO:17所示的序列或其变体的VL；
(3)包含如SEQ ID NO:31所示的序列或其变体的VH-连接子-包含如SEQ ID NO:32所示的序列或其变体的VL；
(4)包含如SEQ ID NO:2所示的序列或其变体的VL-连接子-包含如SEQ ID NO:1所示的序列或其变体的VH；
(5)包含如SEQ ID NO:17所示的序列或其变体的VL-连接子-包含如SEQ ID NO:16 所示的序列或其变体的VH；或
(6)包含如SEQ ID NO:32所示的序列或其变体的VL-连接子-包含如SEQ ID NO:31所示的序列或其变体的VH；
其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述单链抗体包含选自下列的氨基酸序列：(1)SEQ ID NOs:54、56、58任一项所示的氨基酸序列；(2)与SEQ ID NOs:54、56、58任一项所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列；或(3)与SEQ ID NOs:54、56、58任一项所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区。在某些实施方案中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，
所述抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。
在某些实施方案中，所述重链恒定区是IgG、IgM、IgE、IgD或IgA重链恒定区。 在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。
在某些实施方案中，所述轻链恒定区是κ或λ轻链恒定区。在某些优选的实施方案中，所述轻链恒定区是人κ轻链恒定区。
抗体的制备
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(Reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’) 2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此，本发明第二方面提供了一种分离的核酸分子，其包含编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的核苷酸序列：(1)SEQ ID NO：55、57和59任一项所示的核苷酸序列；(2)与SEQ ID NO：55、57和59任一项所示的核苷酸序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
本发明第三方面提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。在某些实施方案中，本发明的载体是例如DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体或病毒载体；优选的，所述载体是表达载体；优选地，所述载体是游离型载体；优选地，所述载体是病毒载体；更优选地，所述病毒载体是慢 病毒载体、腺病毒载体或逆转录病毒载体。
本发明第四方面提供了一种宿主细胞，其包含如上所述的分离的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
在另一个方面，本发明还涉及制备本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
嵌合抗原受体(CAR)
本发明涉及靶向MSLN的CAR，其特征包括非MHC限制的MSLN识别能力，其赋予表达该CAR的免疫细胞(例如，T细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞)不依赖于抗原加工及提呈而识别表达MSLN的细胞(例如肿瘤细胞)的能力。
因此，本发明第五方面提供了一种嵌合抗原受体(CAR)，其包含胞外抗原结合结构域(抗MSLN结合结构域)、间隔结构域、跨膜结构域以及胞内信号传导结构域。
I.胞外抗原结合结构域
本发明的嵌合抗原受体中所包含的抗原结合结构域赋予所述CAR识别MSLN的能力。
在某些实施方案中，所述抗原结合结构域包含抗MSLN结合结构域，所述抗MSLN结合结构域包含能够特异性结合MSLN(例如人MSLN)的抗体或其抗原结合片段。在某些实施方案中，所述抗体或其抗原结合片段选自第一方面所述的抗体或其抗原结合片段。
在某些实施方案中，所述抗MSLN结合结构域包含VH和VL，其中，
所述VH包含如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：3或其变体的CDR-H1；序列为SEQ ID NO：4或其变体的CDR-H2；序列为SEQ ID NO：5或其变体的CDR-H3；所述VL包含如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：6或其变体的CDR-L1；序列为SEQ ID NO：7或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；或者，
所述VH包含如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：9或其变体的CDR-H1；序列为SEQ ID NO：10或其变体的CDR-H2；序列为SEQ ID NO：11或其变体的CDR-H3；所述VL包含如下三个根据IMGT编号系统所定 义的轻链CDRs：序列为SEQ ID NO：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；或者，
所述VH包含如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：14或其变体的CDR-H1；序列为SEQ ID NO：15或其变体的CDR-H2；序列为SEQ ID NO：5或其变体的CDR-H3；所述VL包含如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：6或其变体的CDR-L1；序列为SEQ ID NO：7或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述VH包含如SEQ ID NO:1所示的序列或其变体，所述VL包含如SEQ ID NO:2所示的序列或其变体；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗MSLN结合结构域包含VH和VL，其中，
所述VH包含如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：18或其变体的CDR-H1；序列为SEQ ID NO：19或其变体的CDR-H2；序列为SEQ ID NO：20或其变体的CDR-H3；所述VL包含如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：21或其变体的CDR-L1；序列为SEQ ID NO：22或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；或者，
所述VH包含如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：24或其变体的CDR-H1；序列为SEQ ID NO：25或其变体的CDR-H2；序列为SEQ ID NO：26或其变体的CDR-H3；所述VL包含如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；或者，
所述VH包含如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：29或其变体的CDR-H1；序列为SEQ ID NO：30或其变体的CDR-H2；序列为SEQ ID NO：20或其变体的CDR-H3；所述VL包含如下三个根据Chothia编号系统所 定义的轻链CDRs：序列为SEQ ID NO：21或其变体的CDR-L1；序列为SEQ ID NO：22或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述VH包含如SEQ ID NO:16所示的序列或其变体，所述VL包含如SEQ ID NO:17所示的序列或其变体；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗MSLN结合结构域包含VH和VL，其中，
所述VH包含如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：33或其变体的CDR-H1；序列为SEQ ID NO：34或其变体的CDR-H2；序列为SEQ ID NO：35或其变体的CDR-H3；所述VL包含如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：36或其变体的CDR-L1；序列为SEQ ID NO：37或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；或者，
所述VH包含如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：39或其变体的CDR-H1；序列为SEQ ID NO：40或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；所述VL包含如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；或者，
所述VH包含如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：44或其变体的CDR-H1；序列为SEQ ID NO：45或其变体的CDR-H2；序列为SEQ ID NO：35或其变体的CDR-H3；所述VL包含如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：36或其变体的CDR-L1；序列为SEQ ID NO：37或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述VH包含如SEQ ID NO:31所示的序列或其变体，所述VL 包含如SEQ ID NO:32所示的序列或其变体；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，以上任一实施方案中所述的抗MSLN结合结构域为单链抗体，例如scFv、di-scFv或(scFv) 2。
在某些实施方案中，以上任一实施方案中所述的抗MSLN结合结构域所包含的VH和VL、或所述抗MSLN结合结构域所包含的抗体或其抗原结合片段的VH和VL通过连接子连接。在某些实施方案中，所述连接子包含一个或几个(例如1个、2个或3个)如(G mS) n所示的序列，其中m选自1-6的整数，n选自1-6的整数。在某些实施方案中，m为3、4、或5。在某些实施方案中，n为1或2。在某些实施方案中，所述连接子具有SEQ ID NO：52的序列。
在某些实施方案中，所述抗MSLN结合结构域包含单链抗体，所述单链抗体包含选自下列的氨基酸序列：
(1)SEQ ID NO:54所示的氨基酸序列或其变体，
(2)与SEQ ID NO:54所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；或，
(3)与SEQ ID NO:54所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗MSLN结合结构域包含单链抗体，所述单链抗体包含选自下列的氨基酸序列：
(1)SEQ ID NO:56所示的氨基酸序列或其变体，
(2)与SEQ ID NO:56所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；或，
(3)与SEQ ID NO:56所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗MSLN结合结构域包含单链抗体，所述单链抗体包含选自下列的氨基酸序列：
(1)SEQ ID NO:58所示的氨基酸序列或其变体，
(2)与SEQ ID NO:58所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；或，
(3)与SEQ ID NO:58所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗原结合结构域包含所述抗MSLN结合结构域作为第一结合结构域，并且进一步还包含不结合MSLN的第二结合结构域。在某些优选实施方案中，所述不结合MSLN的第二结合结构域结合的抗原选自：CD19、GPC3、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2。
II.跨膜结构域
本发明的嵌合抗原受体所包含的跨膜结构域可以是本领域已知的任何蛋白结构，只要其能够在细胞膜(特别是真核细胞膜)中热力学稳定。适用于本发明的CAR的跨膜结构域可衍生自天然来源。在此类实施方案中，所述跨膜结构域可衍生自任何膜结合的或跨膜的蛋白质。或者，所述跨膜结构域可为合成的非天然存在的蛋白质区段，例如主要包含疏水残基例如亮氨酸和缬氨酸的蛋白质区段。
在某些实施方案中，所述跨膜结构域是选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD28、CD45、CD3ε、CD3ζ、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD152、CD154和PD-1及其任意组合。在某些优选的实施方案中，所述跨膜结构域是选自下列蛋白的跨膜区：CD8α、CD4、PD-1、CD152和CD154。在某些优选的实施方案中，所述跨膜结构域包含CD8α的跨膜区。在某些示例性实施方案中，所述跨膜结构域包含如SEQ ID NO:64所示的氨基酸序列。
III.间隔结构域
本发明的嵌合抗原受体所包含间隔结构域位于胞外抗原结合结构域与跨膜结构域之间。
在某些实施方案中，所述间隔结构域包含免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区。在此类实施方案中，不受特定理论的约束，认为CH2和CH3使所述CAR的抗原结合结构域从表达CAR的细胞的细胞膜延伸出去，并且可更精确地模拟天然TCR的大小和结构域结构。
在某些实施方案中，所述间隔结构域包含铰链结构域。铰链结构域可以是通常在蛋白质的两个结构域之间发现的氨基酸区段，其可以允许蛋白质具有柔性并且允许一个或两个结构域相对于彼此的运动。因此，所述铰链结构域可以是任何氨基酸序列，只要其能够提供胞外抗原结合结构域的这种柔性以及其相对于跨膜结构域的这种运动性。
在某些实施方案中，所述铰链结构域是天然存在的蛋白质的铰链区或其部分。在某些实施方案中，所述铰链结构域包含CD8α的铰链区或其部分，例如含有CD8α的铰链区的至少15个(例如20、25、30、35或40个)连续氨基酸的片段。在某些示例性实施方案中，所述间隔结构域包含SEQ ID NO:62所示的氨基酸序列。
IV.信号肽
在某些实施方案中，本发明的CAR可进一步在其N端包含信号肽。通常，信号肽是将与其连接的序列靶向至所需位点的多肽序列。在某些实施方案中，所述信号肽可以将与其连接的CAR靶向至细胞的分泌途径，并允许该CAR进一步整合并锚定到脂质双分子层中。可用于CAR的信号肽是本领域技术人员已知的。在某些实施方案中，所述信号肽包含重链信号肽(例如IgG1的重链信号肽)、粒细胞-巨噬细胞集落刺激因子受体2(GM-CSFR2)信号肽、IL2信号肽、或CD8α信号肽。在某些优选的实施方案中，所述信号肽选自CD8α信号肽。在某些示例性实施方案中，所述信号肽包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方案中，本发明的CAR与另外的生物活性分子共表达。所述另外的生物活性分子可以有其专有的信号肽，为与上一段的信号肽区别，此信号肽命名为信号肽-2。信号肽-2引导另外的生物活性分子转运到细胞内特定的位点或细胞膜外。所述信号肽-2可与上一段所述的信号肽相同或不同。优选地，所述信号肽-2可与上一段所述的信号肽 不同。
V.胞内信号传导结构域
本发明的CAR中所包含的胞内信号传导结构域参与将本发明的CAR与MSLN的结合所产生的信号传导进免疫效应细胞内部，激活表达CAR的免疫效应细胞的至少一种正常效应子功能，或增强表达CAR的免疫效应细胞的至少一种细胞因子的分泌(例如IL-2，IFN-γ)。
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和/或共刺激信号传导结构域。
在某些实施方案中，所述初级信号传导结构域可以是包含免疫受体酪氨酸活化基序(ITAM)的任何胞内信号传导结构域。在某些实施方案中，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)。在某些实施方案中，所述初级信号传导结构域包含选自下列的蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b或CD66d。在某些实施方案中，所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域。
在某些实施方案中，所述共刺激信号传导结构域可以是来自共刺激分子的胞内信号传导结构域。在某些实施方案中，所述共刺激信号传导结构域包含选自下列的蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)或DAP10。
在某些实施方案中，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域、或CD137(4-1BB)的胞内信号传导结构域、或二者片段的组合。
在某些实施方案中，所述胞内信号传导结构域包含一个共刺激信号传导结构域。在某些实施方案中，所述胞内信号传导结构域包含两个或更多个共刺激信号传导结构域。在此类实施方案中，所述两个或更多个共刺激信号传导结构域可以是相同的，也可以是不同的。
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信号传导结构域。所述初级信号传导结构域以及至少一个共刺激信号传导结构域可以以任意顺序串联至跨膜结构域的羧基端。
在某些实施方案中，所述胞内信号传导结构域可包含CD3ζ的胞内信号传导结构域和CD137的胞内信号传导结构域。在某些示例性实施方案中，所述CD3ζ的胞内信号传导 结构域包含SEQ ID NO:68所示的氨基酸序列。在某些示例性实施方案中，所述CD137的胞内信号传导结构域包含SEQ ID NO:66所示的氨基酸序列。
在某些示例性实施方案中，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO：70所示序列。
VI.全长CAR
本发明提供了能够特异性地结合MSLN的嵌合抗原受体，所述嵌合抗原受体从其N端至C端依次包含抗MSLN结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域。在某些优选实施方案中，其中所述胞内信号传导结构域从N端到C端为共刺激信号传导结构域和初级信号传导结构域。
在某些实施方案中，所述间隔结构域包含CD8(例如CD8α)的铰链区，其具有SEQ ID NO：62所示序列。在某些实施方案中，所述跨膜结构域包含CD8(例如CD8α)的跨膜区，其具有SEQ ID NO：64所示序列。
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中初级信号传导结构域包含CD3ζ的胞内信号传导结构域，其具有SEQ ID NO：68所示序列。共刺激信号传导结构域包含CD137的胞内信号传导结构域，其具有SEQ ID NO：66所示序列。在某些优选的实施方案中，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO：70所示序列。
在某些优选的实施方案中，所述嵌合抗原受体从其N端至C端依次包含所述信号肽、抗MSLN结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域(从N端到C端为共刺激信号传导结构域和初级信号传导结构域)。
在某些示例性实施方案中，所述信号肽包含IgG1的重链信号肽或CD8α信号肽。在某些示例性实施方案中，所述信号肽包含CD8α信号肽，其具有SEQ ID NO：60所示序列。
在某些示例性实施方案中，本发明的CAR包含选自下列的氨基酸序列：
(1)SEQ ID NO:83所示的氨基酸序列，
(2)与SEQ ID NO:83所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，并且所述序列基本保留了其所源自的氨基酸序列的至少一种生物学活性(例如，能够以非MHC限制 的方式将免疫效应细胞的特异性和反应性指向表达MSLN的细胞的能力)；或者，
(3)与SEQ ID NO:83所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些示例性实施方案中，本发明的CAR包含选自下列的氨基酸序列：
(1)SEQ ID NO:88所示的氨基酸序列，
(2)与SEQ ID NO:88所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，并且所述序列基本保留了其所源自的氨基酸序列的至少一种生物学活性(例如，能够以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达MSLN的细胞的能力)；或者，
(3)与SEQ ID NO:88所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些示例性实施方案中，本发明的CAR包含选自下列的氨基酸序列：
(1)SEQ ID NO:90所示的氨基酸序列，
(2)与SEQ ID NO:90所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，并且所述序列基本保留了其所源自的氨基酸序列的至少一种生物学活性(例如，能够以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达MSLN的细胞的能力)；或者，
(3)与SEQ ID NO:90所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
VII.共表达的CAR和另外的生物活性分子
在一些情况下，本发明第五方面所述的CAR还可以与另外的生物活性分子共表达。自裂解肽能阻止翻译过程中氨基酸形成共价键并维持翻译继续进行，这样一来，翻译产物就被“自切割”，从而使特异性地结合MSLN的嵌合抗原受体和另外的生物活性分子分离。因此，当本发明第五方面所述的CAR还可以与另外的生物活性分子共表达时，能够特异性地结合MSLN的嵌合抗原受体成为独立的具有胞外抗原结合结构域、间隔结构域、 跨膜结构域以及胞内信号传导结构域的CAR，而另外的生物活性分子则能够分泌到细胞外或者表达成膜嵌合型的多肽或蛋白。随着表达特异性地结合MSLN-CAR的免疫细胞在肿瘤微环境的扩增和富集，另外的生物活性分子富集在肿瘤微环境，与抗MSLN-CAR协同发挥抗肿瘤效应。
在某些实施方案中，所述另外的生物活性分子选自下列组分的一种或一种以上：特异性地结合免疫检查点的抗体或其抗原结合片段(例如，抗PD-1、PD-L1、CTLA-4、或LAG-3抗体或其抗原结合片段)、细胞因子(例如，IL-15、IL-7、IL-12、IL-18、IL-21)、或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18、mIL-21)。
在某些实施方案中，编码抗MSLN-CAR的核酸序列通过自裂解肽的核酸序列与另外的生物活性分子的核酸序列连接。抗MSLN-CAR可以在另外的生物活性分子的N端或C端。在某些示例的实施方案中，抗MSLN-CAR在另外的生物活性分子的5’端。任何能够引起融合蛋白裂解成两个独立的蛋白的自裂解肽可以应用到本发明中。在某些示例的实施方案中，所述自裂解肽为P2A，优选地具有SEQ ID NO:72所示的序列，其核苷酸序列可以根据基因重组的需要进行优化。在此类实施方案中，包含CAR和另外的生物活性分子的融合蛋白具有如下的结构：
N’-信号肽--特异地结合人MSLN的胞外抗原结合结构域--间隔结构域—跨膜结构域-胞内信号传导结构域-自裂解肽-信号肽-2--另外的生物活性分子-C’。其中信号肽-2与N’的信号肽相同或不同。
在某些实施方案中，所述另外的生物活性分子在其临近的上游具有与MSLN-CAR的N端不同的信号肽。在某些示例的实施方案中，所述信号肽-2为人IL2信号肽，具有如SEQ ID NO：74所示的序列。
在某些实施方案中，当本发明的CAR与多于一个的所述另外的生物活性分子共同表达时，编码所述多于一个的另外的生物活性分子的多个核酸序列之间通过编码自裂解肽(例如P2A)的序列连接。
在某些实施方案中，所述另外的生物活性分子为抗PD-1线性抗体，优选地，所述线性抗体具有如SEQ ID NO：77所示的序列。
在某些实施方案中，所述另外的生物活性分子为膜嵌合型IL-15，优选地，所述膜嵌合型IL-15具有如SEQ ID NO：81所示的序列。
嵌合抗原受体的制备
生成嵌合抗原受体以及包含该嵌合抗原受体的免疫效应细胞(例如T细胞)的方法是本领域已知的，可包括用至少一种编码CAR的多核苷酸转染细胞，并在细胞中表达多核苷酸。例如，可将编码本发明的CAR的核酸分子包含于表达载体(例如，慢病毒载体)中，所述表达载体能够在宿主细胞例如T细胞中表达，以制造所述CAR。
因此，本发明第六方面提供了一种分离的核酸分子，其包含编码第五方面所述的嵌合抗原受体的核苷酸序列。
本领域技术人员理解，由于遗传密码的简并性，编码一种本发明的嵌合抗原受体的核苷酸序列可以具有多种不同的序列。因此，除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括作为彼此的简并形式且编码相同氨基酸序列的所有核苷酸序列。
在某些示例性实施方案中，所述编码第五方面所述的嵌合抗原受体的核苷酸序列选自：(1)SEQ ID NO:84所示的序列或其简并变体；(2)与(1)所述的序列相比基本上相同的序列，例如，与(1)所述相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)所述的序列相比具有一个或更多个核苷酸取代的序列；并且所述序列基本保留了其所源自的核苷酸序列的至少一种生物学活性(例如，能够编码具有以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达MSLN的细胞的能力)。
在某些示例性实施方案中，所述编码第五方面所述的嵌合抗原受体的核苷酸序列选自：(1)SEQ ID NO:89所示的序列或其简并变体；(2)与(1)所述的序列相比基本上相同的序列，例如，与(1)所述相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)所述的序列相比具有一个或更多个核苷酸取代的序列；并且所述序列基本保留了其所源自的核苷酸序列的至少一种生物学活性(例如，能够编码具有以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达MSLN的细胞的能力)。
在某些示例性实施方案中，所述编码第五方面所述的嵌合抗原受体的核苷酸序列选自：(1)SEQ ID NO:91所示的序列或其简并变体；(2)与(1)所述的序列相比基本上相同的序列，例如，与(1)所述相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、 至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)所述的序列相比具有一个或更多个核苷酸取代的序列；并且所述序列基本保留了其所源自的核苷酸序列的至少一种生物学活性(例如，能够编码具有以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达MSLN的细胞的能力)。
如上文中所述的，本发明的CAR还可以与另外的生物活性分子共表达，以协同发挥抗肿瘤作用。
因此，本发明第七方面还提供了一种核酸构建体，其包含编码第五方面所述的嵌合抗原受体的第一核酸序列，并且进一步包含编码另外的生物活性分子的第二核酸序列。
在某些实施方案中，所述第二核酸序列所编码的另外的生物活性分子具有抗肿瘤活性。
在某些实施方案中，所述第二核酸序列所编码的另外的生物活性分子选自下列组分的一种或一种以上：免疫检查点抑制剂(例如，抗PD-1、PD-L1、CTLA-4、或LAG-3抗体或其抗原结合片段)、细胞因子(例如，IL-15、IL-7、IL-12、IL-18、或IL-21)、或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18、或mIL-21)。
在某些实施方案中，所述第二核苷酸序列所编码的另外的生物活性分子在其N端进一步包含信号肽-2。在某些实施方案中，所述信号肽-2不同于所述第一核酸序列所编码的嵌合抗原受体中所包含的信号肽。在某些实施方案中，所述另外的生物活性分子N端的信号肽-2是IL2信号肽，IL2信号肽是指IL2天然基因序列中所包含的信号肽序列，优选地，所述IL2天然基因为人IL2天然基因，所述IL2信号肽是人IL2信号肽。在某些示例性实施方案中，所述IL2信号肽包含如SEQ ID NO：74所示的序列。
在某些实施方案中，所述第一核苷酸序列位于所述第二核苷酸序列的上游。
在某些实施方案中，所述第一核酸序列和第二核酸序列通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接。在某些实施方案中，所述自裂解肽是P2A(例如，如SEQ ID NO：72所示)。在某些示例性实施方案中，所述编码自裂解肽的序列连接至所述第一核苷酸序列的3’端，并且连接至所述第二核苷酸序列的5’端。
在某些实施方案中，所述另外的生物活性分子选自的免疫检查点抑制剂为抗PD-1或PD-L1抗体或其抗原结合片段(例如scFv)。
在某些实施方案中，所述抗PD-1或PD-L1抗体或其抗原结合片段包含如下任一组的重链可变区和/或轻链可变区：(1)Nivolumab或其变体的重链可变区和/或轻链可变区，(2)Pembrolizumab或其变体的重链可变区和/或轻链可变区，(3)Atezolizumab或其变体的重链可变区和/或轻链可变区，(4)Durvalumab或其变体的重链可变区和/或轻链可变区，(5)Avelumab或其变体的重链可变区和/或轻链可变区，(6)具有SEQ ID NO:79所示序列或其变体的VH和/或具有SEQ ID NO:80所示序列或其变体的VL。所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些实施方案中，所述抗PD-1或PD-L1抗体或其抗原结合片段为单链抗体(例如scFv)。
在某些实施方案中，所述另外的生物活性分子包含抗PD-1单链抗体，所述抗PD-1单链抗体具有SEQ ID NO:79所示序列VH和/或具有SEQ ID NO:80所示序列的VL。在某些实施方案中，所述VH和VL通过连接子连接。在某些实施方案中，所述连接子包含如(G mS) n所示的序列，其中m选自1-6的整数。在某些实施方案中，m为3、4、或5；n选自1-10的整数。在某些实施方案中，n为2、3、4、5、或6。
在某些实施方案中，所述抗PD-1单链抗体包含选自下列的氨基酸序列：(1)SEQ ID NO:77所示的氨基酸序列；(2)与SEQ ID NO:77所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列；(3)与SEQ ID NO:77所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些示例性实施方案中，第七方面所述的核酸构建体从其5’端至3’端依次包含：所述第一核酸序列、编码自裂解肽的核苷酸序列、编码信号肽-2的核苷酸序列、编码免疫检查点抑制剂的核苷酸序列。在某些示例性实施方案中，第七方面所述的核酸构建体包含选自下列的核苷酸序列：(1)SEQ ID NO:85所示的核苷酸序列或其简并变体；(2)与(1)中所述的序列相比基本上相同的序列，例如与(1)中所述的序列相比具有至少50％、至 少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
在某些实施方案中，所述另外的生物活性分子选自的膜嵌合型多肽为mIL-15。在某些实施方案中，所述膜嵌合型多肽mIL-15包含选自下列的氨基酸序列：(1)SEQ ID NO：81所示的序列；(2)与SEQ ID NO:81所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列；(3)与SEQ ID NO:81所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
在某些示例性实施方案中，第七方面所述的核酸构建体从其5’端至3’端依次包含：所述第一核酸序列、编码自裂解肽的核苷酸序列、编码膜嵌合型多肽的核苷酸序列。在某些示例性实施方案中，第七方面所述的核酸构建体包含选自下列的核苷酸序列：(1)SEQ ID NO:86所示的核苷酸序列或其简并变体；(2)与(1)所述的序列相比基本相同的序列，例如与(1)所述的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
在某些实施方案中，所述第二核酸序列所编码的另外的生物活性分子包含至少两种选自以下的组分：免疫检查点抑制剂(例如，抗PD-1、PD-L1、CTLA-4、或LAG-3抗体或其抗原结合片段)、细胞因子(例如，IL-15、IL-7、IL-12、IL-18、或IL-21)、或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18、或mIL-21)。
在某些实施方案中，所述第二核酸序列中所包含的编码所述至少两种组分的核苷酸序列彼此之间通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接，在某些实施方案中，所述自裂解肽是P2A(例如，如SEQ ID NO：72所示)。
在某些实施方案中，所述第二核酸序列所编码的另外的生物活性分子包含：(i)抗 PD-1抗体或其抗原结合片段(例如scFv)和(ii)mIL-15。
在某些示例性实施方案中，第七方面所述核酸构建体从其5’端至3’端依次包含所述第一核酸序列、编码自裂解肽的核苷酸序列、编码信号肽-2的核苷酸序列、编码抗PD-1抗体或其抗原结合片段的核苷酸序列、编码自裂解肽的核苷酸序列、编码mIL-15的核苷酸序列。在某些示例性实施方案中，第七方面所述核酸构建体包含选自下列的核苷酸序列：(1)SEQ ID NO:87所示的核苷酸序列或其简并变体；(2)与(1)所述的序列相比基本相同的序列，例如与(1)所述的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
本发明第八方面提供了一种载体，其包含第六所述的分离的核酸分子，或第七方面所述的核酸构建体。
在某些实施方案中，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，病毒载体。
在某些实施方案中，所述载体是表达载体。
在某些实施方案中，所述载体是游离型载体。
在某些实施方案中，所述载体是病毒载体。
在某些示例性实施方案中，所述病毒载体是慢病毒载体、腺病毒载体或逆转录病毒载体。
在某些实施方案中，所述载体是游离型或非整合病毒载体，例如整合缺陷型逆转录病毒或慢病毒。
本发明第九方面提供了一种宿主细胞，其包含如上第六方面所述的分离的核酸分子、第七方面所述的核酸构建体或第八方面所述的载体。可以通过各种合适的方式将如上所述的载体引入宿主细胞，例如磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、电穿孔、TALEN方法、ZFN方法、非病毒载体介导的转染(例如脂质体)或病毒载体介导的转染(如慢病毒感染，逆转录病毒感染，腺病毒感染)，以及其他用于转移入宿主细胞的物理、化学或生物学手段，如转座子技术，CRISPR-Cas9等技术。
在某些实施方案中，所述宿主细胞包含第六方面所述的分离的核酸分子或包含所 述核酸分子的载体，所述宿主细胞表达本发明的嵌合抗原受体。
在某些实施方案中，所述宿主细胞包含第七方面所述的核酸构建体或包含所述核酸构建体的载体，所述宿主细胞表达本发明的嵌合抗原受体以及另外的生物活性分子。
在某些实施方案中，所述宿主细胞选自哺乳动物(如人)的免疫细胞。在某些实施方案中，所述免疫细胞来源于患者或健康供体。在某些实施方案中，所述免疫细胞选自T淋巴细胞、自然杀伤(NK)细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合；优选地，所述免疫细胞来源于T淋巴细胞或NK细胞。
本发明第十方面提供了制备表达本发明的嵌合抗原受体的细胞的方法，其包括：(1)提供宿主细胞；(2)将如第六方面所述的分离的核酸分子或包含所述核酸分子的载体引入所述宿主细胞，以获得能够表达所述嵌合抗原受体的宿主细胞。还提供了共表达本发明的嵌合抗原受体以及另外的生物活性分子的细胞的方法，其包括：(1)提供宿主细胞；(2)将第七方面所述的核酸构建体或包含所述核酸构建体的载体引入所述宿主细胞，获得能够共表达所述嵌合抗原受体和另外的生物活性分子的宿主细胞。
在某些实施方案中，所述宿主细胞选自免疫细胞，例如T淋巴细胞、NK细胞、单核细胞、树突状细胞、巨噬细胞及其任意组合。在某些实施方案中，所述免疫细胞选自T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及这些细胞的任意组合。
在某些实施方案中，在步骤(1)中，所述免疫细胞提供自患者或者健康供体，并且经过预处理；所述预处理包括免疫细胞的分选、激活和/或增殖；在某些实施方案中，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞。
在某些实施方案中，在步骤(2)中，将核酸分子或载体通过病毒感染引入免疫细胞。在某些实施方案中，在步骤(2)中将核酸分子或载体通过非病毒载体转染的方式引入免疫细胞，如通过转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法、电穿孔方法、磷酸钙转染、DEAE-葡聚糖介导的转染或显微注射等方法。
在某些实施方案中，在步骤(2)之后，所述方法还包括：扩增步骤(2)获得的免疫细胞。
经改造的免疫细胞
通过本发明提供的上述制备方法可将来源于患者或健康供体的免疫细胞改造为表达特异性结合MSLN的CAR以及可选的另外的生物活性分子的免疫细胞。
因此，本发明第十一方面还提供了一种经改造的免疫细胞，其表达本发明的特异性结合MSLN的CAR。
在某些实施方案中，所述经改造的免疫细胞包含第六方面所述的分离的核酸分子或包含所述核酸分子的载体。
在某些实施方案中，所述经改造的免疫细胞还表达另外的生物活性分子。在某些实施方案中，所述经改造的免疫细胞包含第七方面所述的核酸构建体或包含所述核酸构建体的载体。在一些情况下，本发明所述经改造的免疫细胞在表达第五方面所述的CAR的同时，还可以共表达另外的生物活性分子，其能够特异性地结合MSLN的嵌合抗原受体成为独立的具有胞外抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域的CAR，而另外的生物活性分子则能够分泌到细胞外或者表达成膜嵌合型的多肽或蛋白。随着表达特异性地结合MSLN-CAR的免疫细胞在肿瘤微环境的扩增和富集，另外的生物活性分子富集在肿瘤微环境，与抗MSLN-CAR协同发挥抗肿瘤效应。
在某些实施方案中，所述免疫细胞来源于患者或健康供体的T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合。这些免疫细胞被通过第十方面所提供的方法导入第六方面所述的分离的核酸分子、第七方面所述的核酸构建体或第八方面所述的载体从而制备为经改造的免疫细胞。
在某些实施方案中，经改造的免疫细胞可以具有MSLN靶点以外的结合特异性，例如：经改造的免疫细胞，还表达不特异性针对MSLN的CAR；优选地，所述不特异性针对MSLN的CAR具有针对选自下列靶点的特异性：CD19、GPC3、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2。
在某些实施方案中，经改造的免疫细胞的免疫排除有关的基因(例如，TRAC、TRBC、B2M、HLA-A、HLA-B或HLA-C)和免疫共抑制通路或信号分子的基因(例如，PD-1、CTLA-4或LAG-3)中的一种或两种靶基因的转录或表达被抑制，使得靶基因介导的信号传导在所述的经改造的免疫细胞中被阻断；优选地，所述靶基因的转录或表达被抑制采用的方法选自基因敲除(例如，CRISPR、CRISPR/Cas9)、同源重组、干扰RNA。
免疫细胞组合物
在第十二方面，本发明还提供了免疫细胞组合物，所述免疫细胞组合物包括前述经改造的免疫细胞，以及可选的未改造和/或未成功改造的免疫细胞，这些未改造和/或未成 功改造的免疫细胞不表达特异性针对MSLN的CAR。限制于当前的技术水平及一些未知的原因，并不是所有免疫细胞经过改造都能表达特异性针对MSLN的CAR。而且不表达CAR的免疫细胞也有一定的生物学活性，因此免疫细胞组合物可以含有表达和不表达特异性针对MSLN的CAR的免疫细胞，该免疫细胞组合物依然能够满足临床应用的需求。
在某些实施方案中，经改造的表达特异性针对MSLN的CAR的免疫细胞占免疫细胞组合物总细胞数的大约10％-100％，优选地40％-80％。
在某些实施方案中，免疫细胞组合物被培养成免疫细胞系，因此，另一方面，本发明还提供了含有免疫细胞组合物的免疫细胞系。
在另一个方面，本发明提供了制备特异性结合MSLN的嵌合抗原受体，或用于制备表达所述嵌合抗原受体的细胞或者共表达所述嵌合抗原受体以及另外的生物活性分子的免疫细胞的试剂盒。在某些实施方案中，所述试剂盒包括如第六方面所述的分离的核酸分子、第七方面所述的核酸构建体或第八方面所述的载体，或第九方面所述的宿主细胞，和必要的溶剂，如无菌水，生理盐水，或细胞培养液，如LB培养液，如EliteCell原代T淋巴细胞培养体系(产品编号：PriMed-EliteCell-024)，以及可选的，还包括使用说明书。
在另一个方面，本发明提供了前述试剂盒用于制备能够特异性结合MSLN的嵌合抗原受体、或表达所述嵌合抗原受体的细胞、或共表达所述嵌合抗原受体以及另外的生物活性分子的免疫细胞的应用。
药物组合物
在第十三方面，本发明提供了一种药物组合物，其含有本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体(包括双特异性嵌合抗原受体或与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述药物组合物还包含另外的药学活性剂，例如具有抗肿瘤活性的药物(例如anti-PD1抗体、anti-PD-L1抗体、anti-CTLA-4抗体、培美曲塞、顺铂、紫杉醇、吉西他滨、卡培他滨或FOLFIRINOX)。
在某些实施方案中，本发明第一方面所述的抗体或其抗原结合片段、第五方面所述 的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物与所述另外的药学活性剂可以同时、分开或相继施用。
在某些实施方案中，本发明的药物组合物包含：第六方面所述的分离的核酸分子、第七方面所述的核酸构建体或第八方面所述的载体、或第九方面所述的宿主细胞。
在某些实施方案中，本发明的药物组合物包含：本发明的经改造的免疫细胞或免疫细胞组合物。
本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而 发生变化。在某些实施方案中，本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物通过静脉注射或推注给予。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物。“预防有效量”是指，足以预防，阻止，或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
治疗方法及用途
在另一方面，本发明提供了一种用于在受试者(例如人)中预防和/或治疗与间皮素的表达相关的疾病的方法，所述方法包括向有此需要的受试者施用有效量的本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物。
在某些实施方案中，所述与间皮素的表达相关的疾病选自增生性疾病，例如肿瘤。在某些实施方案中，所述与间皮素的表达相关的疾病是与间皮素的表达相关的非肿瘤相关的适应症。
在某些实施方案中，所述肿瘤是MSLN阳性肿瘤。在某些实施方案中，所述肿瘤选自实体瘤(例如恶性胸膜间皮瘤、胰腺癌、肺癌(例如肺鳞癌)、乳腺癌、卵巢癌(例如 卵巢上皮癌))。
在某些实施方案中，所述方法包括向所述受试者施用有效量的第一方面所述的抗体或其抗原结合片段。
在某些实施方案中，所述方法包括向所述受试者施用有效量的第五方面所述的嵌合抗原受体、第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第八方面所述的载体、第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物。在某些实施方案中，所述宿主细胞是免疫细胞(例如人免疫细胞)。
在某些实施方案中，所述方法包括以下步骤：(1)提供所述受试者所需的免疫细胞(例如T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞、或这些细胞的任意组合)；(2)将包含本发明第六方面所述的分离的核酸分子、第七方面所述的核酸构建体或第八方面所述的载体导入步骤(1)所述的免疫细胞，以获得表达所述嵌合抗原受体以及任选的另外的生物活性分子的细胞；(3)将步骤(2)中获得的免疫细胞施用至所述受试者以进行治疗。
在某些实施方案中，所述方法通过剂量分次，例如一次，两次，三次或更多次分开施用部分剂量，向所述受试者施用表达本发明的CAR的免疫细胞，例如在治疗的第一天施用总剂量的第一百分比，在随后的(例如第二，第三，第四，第五，第六或第七天或更晚)治疗日施用总剂量的第二百分比，例如在随后的(例如第三，第四，第五，第六，第七，第八，第九，第十天或更晚)治疗日施用总剂量的第三百分比(例如，剩余百分比)。
在某些实施方案中，在治疗的第一天施用总剂量的10％的细胞，在第二天施用总剂量的30％的细胞，并且在第三天施用总剂量的剩余60％的细胞。
在某些实施方案中，在治疗的第一天施用总剂量的50％的细胞，在随后的(例如第二，第三，第四，第五，第六或第七或更晚)治疗日施用总剂量的50％的细胞。在某些实施方案中，在治疗的第一天施用总剂量的1/3的细胞，在随后的(例如第二，第三，第四，第五，第六或第七天或更晚)治疗日施用总剂量的1/3的细胞，在随后的(例如第三，第四，第五，第六，第七，第八，第九，第十天或更晚)施用总剂量的1/3的细胞。
在某些实施方案中，总细胞剂量包含1×10 7至10×10 8个CAR阳性免疫细胞，例如包含(1-5)×10 7至(5-10)×10 8个CAR阳性免疫细胞。
在某些实施方案中，医师可以根据病人的状态，肿瘤的大小和阶段，或联合治疗的药物等临床情况来调节剂量或治疗方案。
在某些实施方案中，将本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物与另外的试剂联合施用。在某些实施方案中，所述另外的试剂包括(i)增加包含CAR核酸或CAR多肽的细胞(例如表达本发明的CAR的免疫细胞，本发明的经改造的免疫细胞或免疫细胞组合物)的功效的作用剂；(ii)改善与施用包含CAR核酸或CAR多肽的细胞(例如表达本发明的CAR的免疫细胞，本发明的经改造的免疫细胞或免疫细胞组合物)相关的一种或多种副作用的作用剂；(iii)具有抗肿瘤活性的另外的药学活性剂。这些试剂可以在施用本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物之前、同时或之后施用。
在某些实施方案中，以上所述方法还包括向所述受试者施用第二疗法，所述第二疗法可以是已知用于肿瘤的任何疗法，例如手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其任意组合。
在某些实施方案中，所述第二疗法可以与以上所述的方法分开或联合应用；或，所述第二疗法可以与以上所述的方法同时或相继应用。
在某些实施方案中，所述受试者可以为哺乳动物，例如人。
在另一个方面，提供了本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物在制备用于预防和/或治疗肿瘤的药物中的用途。前述治疗方法中的剂量，剂型，给药途径，适应症，联合治疗等各个方面都可以应用到所述药物的用途中。
缩略词
CAR        嵌合抗原受体
CDR        免疫球蛋白可变区中的互补决定区
CDR-H1     免疫球蛋白重链可变区中的互补决定区1
CDR-H2     免疫球蛋白重链可变区中的互补决定区2
CDR-H3     免疫球蛋白重链可变区中的互补决定区3
CDR-L1     免疫球蛋白轻链可变区中的互补决定区1
CDR-L2     免疫球蛋白轻链可变区中的互补决定区2
CDR-L3     免疫球蛋白轻链可变区中的互补决定区3
FR         抗体构架区：抗体可变区中除CDR残基以外的氨基酸残基
VH         抗体重链可变区
VL         抗体轻链可变区
Kabat      由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
IMGT       基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system
Figure PCTCN2021123047-appb-000001
(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
Chothia    由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
IL-2       白细胞介素2
IFN        干扰素
PCR        聚合酶链式反应
FACS       流式细胞荧光分选
K D         平衡解离常数
kon        结合速率常数
kdis       解离速率常数
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子生物学、微生物学、细胞生物学、生物化学、免疫学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”指能够通过位于免疫球蛋白分子可变区的至少一个抗原识别位点特异性结合靶(如碳水化合物、多核苷酸、脂质、多肽等)的免疫球蛋白分子。如本文所用，该术语不仅包括完整的多克隆或单克隆抗体，而且包括其片段(例如Fab、Fab'、F(ab')2、Fv)、单链(例如scFv，di-scFv，(scFv) 2)和结构域抗体(包括例如鲨鱼和骆驼抗体)、以及包括抗体的融合蛋白、以及包括抗原识别位点的任何其它修饰构型的免疫球蛋白分子。本发明的抗体不受任何特定的产生抗体的方法限制。抗体包括任何类型的抗体，例如IgG、IgA或IgM(或其亚类)，并且抗体不需要属于任何特定的类型。取决于抗体重链恒定区的氨基酸序列，免疫球蛋白可以分配到不同的类型。有五种主要类型的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中几种可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类型的免疫球蛋白的重链恒定区分别被称为α、δ、ε、γ和μ。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。不同类型的免疫球蛋白的亚基结构和三维构型是众所周知的。重链恒定区由4个结构域(CH1、hinge region、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。
抗体的VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDRs和4个FRs组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDRs，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语抗体的“抗原结合片段”是指抗体的片段的多肽，例如全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括骆驼Ig、Ig NAR、Fab片段、Fab'片段、F(ab)' 2片段、F(ab)' 3片段、Fd、Fv、scFv、di-scFv、(scFv) 2、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(“dsFv”)和单结构域抗体(sdAb，纳米抗体)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“骆驼Ig”或“骆驼VHH”是指重链抗体的最小已知抗原结合单位(Koch-Nolte等人,FASEB J.,21:3490-3498(2007))。“重链抗体”或“骆驼抗体”是指含有两个VH结构域并且不含轻链的抗体(Riechmann L.等人,免疫学方法杂志 (J.Immunol.Methods)231:25-38(1999)；WO94/04678；WO94/25591；美国专利第6,005,079号)。
如本文中所使用的，术语“IgNAR”或“免疫球蛋白新抗原受体”是指来自鲨鱼免疫组库的由一个可变新抗原受体(VNAR)结构域和五个恒定新抗原受体(CNAR)结构域的同源二聚体组成的一类抗体。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDRs赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDRs)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer  Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在某些实施方案中，VH和VL结构域可以以任何合适的排列彼此相对定位。例如，包含NH 2-VH-VH-COOH、NH 2-VL-VL-COOH的scFv。所述scFv可以形成任何工程上可能的结构，单链抗体(scFv)，串联抗体(tandem di-scFvs)，双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白，骆驼Ig、IgNAR等。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv) 2，其指的是两个或两个以上单个scFv并联而形成抗体。
如本文中所使用的，术语“双功能抗体”是指具有两个抗原结合位点的抗体片段，所述片段在同一多肽链(VH-VL)中包含连接到轻链可变结构域(VL)的重链可变结构域(VH)。通过使用过短以使得同一链上的两个结构域之间不能配对的连接子，迫使结构域与另一链的互补结构域配对，并且产生两个抗原结合位点。双功能抗体可以是二价的或双特异性的。双功能抗体更全面描述于例如EP 404,097；WO 1993/01161；Hudson等人,自然医学(Nat.Med.)9:129-134(2003)；和Hollinger等人,PNAS USA 90:6444-6448(1993)中。三功能抗体和四功能抗体也描述于Hudson等人,自然医学9:129-134(2003)中。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力(Holt,L.等人,生物技术趋势(Trends in Biotechnology),21(11):484-490，2003)。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，表述“特异性结合”或“特异性针对”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(K D)表示。在本发明中，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数K D(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量K D、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。载体可以包括在细胞中直接自主复制的序列，或可以包括足以允许整合到宿主细胞DNA中的序列。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及病毒载体等。病毒载体的非限制性实例包括，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。 一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“游离型载体”中游离型是指载体能够复制而不整合到宿主的染色体DNA中并且不由分裂宿主细胞逐渐丧失，还意指所述载体在染色体外或游离地复制。
如本文中所使用的，术语“病毒载体”广泛用以指包括典型地促进核酸分子转移或整合到细胞的基因组中的病毒衍生的核酸元件的核酸分子(例如转移质粒)，或介导核酸转移的病毒颗粒。除了核酸之外，病毒颗粒典型地将包括各种病毒组分并且有时还包括宿主细胞组分。
术语“病毒载体”可以指能够将核酸转移到细胞中的病毒或病毒颗粒，或指转移的核酸本身。病毒载体和转移质粒含有主要衍生自病毒的结构和/或功能遗传元件。
如本文中所使用的，术语“逆转录病毒载体”是指含有主要衍生自逆转录病毒的结构和功能遗传元件或其部分的病毒载体或质粒。
如本文中所使用的，术语“慢病毒载体”是指含有主要衍生自慢病毒的结构和功能遗传元件或其部分(包括LTR)的病毒载体或质粒。在某些实施方案中，术语“慢病毒载体”、“慢病毒表达载体”可以用以指慢病毒转移质粒和/或感染性慢病毒颗粒。在本文提及元件(例如克隆位点、启动子、调节元件、异源核酸等)时，应理解，这些元件的序列以RNA形式存在于本发明的慢病毒颗粒中并且以DNA形式存在于本发明的DNA质粒中。
如本文中所使用的，“整合缺陷型”逆转录病毒或慢病毒是指具有不能将病毒基因组整合到宿主细胞的基因组中的整合酶的逆转录病毒或慢病毒。在某些实施方案中，整合酶蛋白突变以特异性降低其整合酶活性。整合缺陷型慢病毒载体可以通过修饰编码整合酶蛋白的pol基因，产生编码整合缺陷型整合酶的突变pol基因而获得。所述整合缺陷型病毒载体已经描述于专利申请WO 2006/010834中，所述专利申请以全文引用的方式并入本文中。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞，免疫细胞(如T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞等)。宿主细胞可以包括单个细胞或细胞群体。
如本文中所使用的，术语“嵌合抗原受体”或“CAR”是指包含至少一个细胞外抗原结合结构域，间隔结构域，跨膜结构域和细胞质信号传导结构域(本文也称为“胞内信号传导结构域”)的重组多肽构建体，其将针对目的抗原(例如MSLN)的基于抗体的特异性与免疫效应细胞活化胞内结构域组合以展现针对表达该目的抗原(例如MSLN)细胞的特异性免疫活性。在本发明中，表述“表达CAR的免疫效应细胞”是指表达CAR并且具有由该CAR的靶向结构域决定的抗原特异性的免疫效应细胞。制造CAR(例如，用于癌症治疗)的方法是本领域已知的，可参见例如，Park等人,Trends Biotechnol.,29:550-557,2011；Grupp等人,N Engl J Med.,368:1509-1518,2013；Han等人,J.Hema tol.Oncol.,6:47,2013；PCT专利公开文本WO2012/079000、WO2013/059593；和美国专利公开文本2012/0213783，其全部通过引用整体并入本文。
如本文中所使用的，术语“胞外抗原结合结构域”是指能够特异性结合目的抗原或受体的多肽。该结构域将能够与细胞表面分子相互作用。例如，可以选择胞外抗原结合结构域来识别作为与特定疾病状态相关的靶细胞细胞表面标志物的抗原。
如本文中所使用的，术语“胞内信号传导结构域”是指传导效应信号功能信号并引导细胞进行专门的功能的蛋白质部分。因此，胞内信号传导结构域具有激活表达CAR的免疫效应细胞的至少一种正常效应子功能的能力。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
如本文中所使用的，术语“初级信号传导结构域”是指能够以刺激方式或以抑制方式调节TCR复合物的初级活化的蛋白质部分。以刺激方式作用的初级信号传导结构域通常含有已知为基于免疫受体酪氨酸的活化基序(ITAM)的信号传导基序。含有特别用于本发明中的初级信号传导结构域的ITAM的非限制性实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。
如本文中所使用的，术语“共刺激信号传导结构域”是指共刺激分子的胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体以外的在结合到抗原后提供T淋巴细胞的高效活化和功能所需的第二信号的细胞表面分子。所述共刺激分子的非限制性实例包括CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)、DAP10。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如 Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：无菌水，生理盐水，pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，术语“受试者”是指包括其中可以引出免疫应答的活生物体。在某些实施方式中，所述受试者(例如人)患有肿瘤(例如与MSLN相关的肿瘤)，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中使用的，术语“免疫细胞”是指涉及免疫反应例如涉及促进免疫效应子功能的细胞。免疫细胞的实例包括T细胞(例如α/βT细胞和γ/δT细胞)、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞和骨髓来源巨噬细胞。
本发明所述的免疫细胞可以是自身的/自体的(“自我”)或非自身的(“非自我”，例如同种异体的、同基因的或异基因的)。如本文中使用的，“自身的”是指来自同一受试者的细胞；“同种异体的”是指与比较细胞遗传不同的同一物种的细胞；“同基因的”是指与比较细胞遗传相同的来自不同受试者的细胞；“异基因的”是指与比较细胞来自不同物种的细胞。在优选实施例中，本发明的细胞是同种异体的。
可用于本文所述的CAR的示例性免疫细胞包括T淋巴细胞和/或NK细胞。术语“T细胞”或“T淋巴细胞”是本领域公知的并且意图包括胸腺细胞、未成熟的T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。T细胞可以是T辅助(Th)细胞，例如T辅助1(Th1)或T辅助2(Th2)细胞。T细胞可以是辅助T细胞(HTL；CD4T细胞)CD4T细胞、细胞毒性T细胞(CTL；CD8T细胞)、CD4CD8T细胞、CD4CD8T细胞或任何其它T细胞子组。在某些实施方案中，T细胞可以包括原初T细胞和记忆T细胞。
本领域技术人员将理解，其它细胞也可以用作具有如本文所述的CAR的免疫细胞。具体来说，免疫细胞还包括NK细胞、单核细胞、巨噬细胞或树突状细胞、NKT细胞、嗜中性白细胞和巨噬细胞。免疫细胞还包括免疫细胞的祖细胞，其中所述祖细胞可以在体内或体外经诱导以分化成免疫细胞。因此，在某些实施方案中，免疫细胞包括免疫细胞的祖细胞，例如含于衍生自脐血、骨髓或流动周边血液的CD34+细胞群体内的造血干细胞(HSC)，其在受试者中投与后分化成成熟免疫细胞，或其可以在体外经诱导以分化成成熟免疫细胞。
如本文中使用的，术语“经改造的免疫细胞”是指，表达本文所述的任何一种CAR， 或导入了本文所述的任何一种分离的核酸或载体的免疫细胞。可以用多种方法将编码CAR多肽的多核苷酸引入细胞后，也可以在细胞中原位合成CAR多肽。或者，可以在细胞外生产CAR多肽，然后将其引入细胞。将多核苷酸构建体引入细胞的方法是本领域已知的。在一些实施方案中，可以使用稳定的转化方法将多核苷酸构建体整合到细胞的基因组中。在其他实施方案中，瞬时转化方法可用于瞬时表达多核苷酸构建体，并且多核苷酸构建体未整合到细胞的基因组中。在其它实施方案中，可以使用病毒介导的方法。多核苷酸可以通过任何合适的方法引入细胞，例如重组病毒载体(例如逆转录病毒、腺病毒)，脂质体等。瞬时转化方法包括，例如但不限于显微注射、电穿孔或微粒轰击。多核苷酸可以包括在载体中，例如质粒载体或病毒载体。
如本文中使用的，术语“免疫效应子功能”是指免疫效应细胞的增强或促进对靶细胞的免疫攻击(例如对靶细胞的杀伤，或者抑制其生长或增殖)的功能或反应。例如，T细胞的效应子功能可以是溶细胞活性或辅助活性，包括细胞因子的分泌。
发明的有益效果
相比于手术、放疗和化疗等传统肿瘤治疗方法，嵌合抗原受体(CAR)-T细胞疗法被认为是最有前途的癌症治疗方法之一，然而，由于实体瘤复杂的肿瘤微环境极大限制了CAR-T细胞疗法在实体瘤中的治疗效果，如何增强CAR-T细胞疗法在实体瘤中的疗效是目前研究的重点。
本发明提供了靶向MSLN的包含本发明的抗体或其抗原结合片段的嵌合抗原受体，表达本发明的嵌合抗原受体的免疫效应细胞相比于已知靶向MSLN的CAR-T具有提高的效应子功能(例如，肿瘤杀伤活性以及释放细胞因子活性)。此外，本发明还通过共表达本发明的CAR和PD-1抗体，阻断PD-1与PD-L1的结合，使T细胞恢复活性，从而增强免疫应答；以及通过共表达mIL-15，促进T、NK细胞的增殖和活化，增强CAR-T细胞的肿瘤杀伤效果。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
附图说明
图1显示了实施例2中构建的嵌合抗原受体的结构。其中，抗MSLN结合结构域为scFv，分别选自G5、G9、G16。
图2显示了G5-CAR-T、G9-CAR-T、G16-CAR-T对NCI-H226靶细胞的杀伤活性测定结果。
图3显示了G16-CAR-T细胞经NCI-H226靶细胞激活后IL2、TNF-α、IFN-γ分泌水平检测结果。
图4显示了G5-CAR-T、G9-CAR-T、G16-CAR-T对SKOV-3靶细胞的杀伤活性测定结果。
图5显示了G5-CAR-T、G9-CAR-T、G16-CAR-T细胞经SKOV-3靶细胞激活后IL2、TNF-α、IFN-γ分泌水平检测结果。
图6显示了G16-CAR-T、G16-PD1-CAR-T、G16-mIL15-CAR-T、G16-PD1-mIL15-CAR-T对SKOV-3靶细胞的杀伤活性测定结果。
图7显示了G16-CAR-T对阴性A431细胞的杀伤活性测定结果。
图8显示了G16-CAR-T经A431阴性细胞处理后IL2、TNF-α、IFN-γ分泌水平检测结果。
图9显示了小鼠体内G16-CAR-T对PANC1靶细胞的杀伤能力。
图10显示了小鼠体内G16-CAR-T、G16-PD1-CAR-T、G16-PD1-mIL15-CAR-T对SKOV-3靶细胞的杀伤能力。
序列信息
本发明涉及的序列信息提供如下：
Figure PCTCN2021123047-appb-000002
Figure PCTCN2021123047-appb-000003
Figure PCTCN2021123047-appb-000004
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：结合人类MSLN的特异性单链抗体(scFv)的制备
1)噬菌体库筛选MSLN-scfv
收集健康人的外周血，分离PBMC细胞，提取细胞的总RNA，并反转录出cDNA。采用cDNA和扩增抗MSLN抗体重链可变区和轻链可变区的引物构建噬菌体抗体库。
用0.05％PBST洗涤磁珠；将1.33ml全人源噬菌体文库与50ul磁珠混匀，将混合物轻轻滚动10分钟后用磁力分离器分离磁珠，将上清液转移至新的磁珠中，滚动30分钟后分离磁珠。第三次与上述步骤相同，滚动时间为60分钟。将上清吸取到新的离心管中，加入10ug生物素化MSLN、磁珠，结合30min，再将离心管置于磁力架上2min。转移上清到15ml离心管中，4℃保存。将磁珠洗涤14次，前4次使用mPBST，后换为PBST，每次1ml洗涤液。最后用1ml 100mM三乙胺洗脱磁珠上结合的噬菌体，然后用100ul 1M Tris-HCl(pH7.5)中和洗脱液。取10ul噬菌体，用90ul PBS稀释。取10ul稀释液加入90ul SS320细胞(OD600＝0.5-0.6)中进行扩增噬菌体，并进行滴度测定。将扩增的噬菌体转移至50ml离心管中，9000g,4℃离心15min。将上清液转移至新的离心管中，添加20％PEG6000/2.5M NaCl，充分混合后在冰上放置2h。12000g离心30min，弃上清，并用0.5ml PBST重悬噬菌体沉淀，10000g离心8分钟，将上清转移到新的试管中，取一部分进行滴度测定，剩余进行第2轮、第3轮筛选。
2)ELISA检测单克隆噬菌体
在微量滴定板中生产单克隆噬菌体：将单个菌落接种到96深孔板中，每孔含300 ul 2YT培养基，10ug/ml Tet，37℃ 250rpm培养5-6小时。从各孔里取150ul培养物添加等体积无菌50％甘油，-80℃保存。向每孔添加25ul辅助噬菌体(2.5x10 9)，37℃孵育30分钟。补加150ul 2YT培养基和200ug/ml Amp、20ug/ml Tet、100ug/ml卡那霉素，2mM IPTG，32℃，250rpm过夜培养。第二天将深孔板3200g离心15分钟，将上清液转移至新的96孔板，4℃保存。
ELISA检测：用PBS将生物素标记的MSLN(MSN-H826x，ACROBiosystems)稀释到100ug/ml-8ug/ml，包被在链霉亲和素96孔板中，4℃过夜。用300ul PBS清洗孔1次。200ul mPBST(2％牛奶)37℃封闭1小时。去封闭液，向每孔中加入80ul噬菌体上清液和80ul mPBST，室温静置1小时。PBST洗板5次，加入100ul anti-M13-HRP(在mPBST中稀释至0.4ug/ml)，室温静置1小时。PBST洗板5次，加入100ul TMB，室温反应3min，再加入100ul终止液(0.2M H 2SO 4)。使用酶联免疫检测仪检测450nm吸光值。
MSLN scFv测序：根据ELISA检测结果选取158个单克隆进行测序，获取scfv序列，用于测序的正向和反向引物分别为：PKLT1F(SEQ ID NO:92)；PKLT1R(SEQ ID NO:93)。使用Sequcher软件分析序列结果，最终获得3个候选scfv，分别命名为：G16、G5和G9。
表1：scFv的可变区及CDR序列
Figure PCTCN2021123047-appb-000005
Figure PCTCN2021123047-appb-000006
3)scFv-Fc的构建与抗体聚体分析
将3个候选scFv序列与Fc(human IgG1)序列连接构建在TGEX-KAL载体中，然后转染expi293细胞进行表达和纯化scFv-Fc蛋白，Fc(human IgG1)序列如SEQ ID NO：94所示。SEC分析实验结果表明，3个候选序列单体峰(主峰)面积占比大于85％。
表2：候选scFv-Fc蛋白的SEC数据
Figure PCTCN2021123047-appb-000007
注：ND表示未检测到。
4)MSLN细胞结合试验
为鉴定MSLN scFv-Fc蛋白结合亲和力，将MSLN scFv-Fc蛋白梯度稀释后对选择3种表达MSLN的细胞系进行染色，通过流式细胞术检测细胞结合能力。结果如下表所示，抗MSLN抗体G9、G16对3种MSLN阳性细胞的的EC50为2-23nM，因此与MSLN具有良好的结合亲和力。
表3：候选scFv-Fc对3种MSLN阳性细胞的亲和力测定结果
Figure PCTCN2021123047-appb-000008
实施例2：慢病毒质粒的构建及病毒包装
2.1慢病毒质粒的构建：
(1)嵌合抗原受体各部分的连接顺序
首先，基于上述实施例中的scFv序列，进一步构建CAR。以CD137的胞内结构域和CD3Zeta的ITAM区作为激活信号，与上述scFv进行融合，同时加上信号肽，CD8 铰链区，CD8跨膜区，构建嵌合抗原受体表达载体，构建的嵌合抗原受体结构如下表所示。
表4：基础嵌合抗原受体的结构
Figure PCTCN2021123047-appb-000009
在上述基础CAR结构基础上，通过编码P2A自裂解肽的序列连接编码一个或多个其他生物活性分子的序列(例如PD-1scFv和/或mIL-15，多个生物活性分子之间可进一步通过P2A自裂解肽核苷酸序列连接)，以获得共表达CAR。上述共表达CAR在细胞中表达时，P2A序列所连接的生物活性分子分泌到CAR-T细胞外或嵌合表达在CAR-T细胞膜上，协同发挥抗肿瘤作用。例如，由于MSLN CAR-T特异性地与肿瘤细胞结合，G16-PD1-CAR-T活化后通过分泌抗PD-1抗体减轻或消除免疫抑制，增加抗肿瘤效果；G16-mIL15-CAR-T活化后，CAR-T细胞表达膜嵌合的IL-15，膜嵌合的IL-15通过刺激分泌一些细胞因子增加抗肿瘤效果并使抗肿瘤效果更持久。共表达CAR的编码核酸序列结构如下表所示。
表5：共表达CAR结构和编码核酸序列
Figure PCTCN2021123047-appb-000010
以上所构建的基础CAR和共表达CAR的结构可参见图1。
(2)以上结构中scFv和mIL15核苷酸序列密码子优化后委外合成及构建到Lenti-4-EF1a载体，挑取单克隆进行培养及保种，最终提取质粒进行测序，将测序正确的菌液用于制备慢病毒质粒。
2.2病毒包装：
将以上构建的CAR慢病毒质粒与转染试剂混合液逐滴加入到293T(ATCC)细胞中，轻轻晃动培养皿，充分混匀。将培养皿置于37℃、5％CO 2培养箱；培养6～8小时后，将含有转染试剂的培养基去掉，更换为新鲜的完全培养基。收集连续培养48小时、72小时后的含有病毒的培养基上清，加入PEG后4℃过夜沉淀，4000×g 4℃离心1小时。离心结束后，在生物安全柜中，小心将离心管中的液体吸去，加入300μL病毒冻存液将沉淀重悬，将病毒置于-80℃保存。
实施例3：CAR-T细胞制备
1)原代T细胞分离：
(1)采用淋巴细胞分离液(GE)分离得到人的PBMC细胞，置于37℃，5％CO 2的培养箱中培养，加入100μl/mL的CD3抗体和CD28抗体，充分混匀后，室温孵育15分钟。
(2)取出磁珠，用移液枪上下吹打至少5次，充分混匀。
(3)吸取50μl磁珠/mL至上述样品中，充分混匀后，室温孵育10分钟。
(4)添加完全培养基至管内总体积为2.5mL，将管子(开盖)插入磁极中，室温静置5分钟。
(5)孵育完毕后，管子继续留在磁极中，轻轻倒置，将管内的细胞倒出。
(6)将细胞重悬于X-vivo 15培养基中，并添加10％FBS，300U/mL IL-2,5ng/mL IL-15和10ng/mL IL-7。
2)T细胞的激活：
调整细胞密度至1×10 6细胞/mL，加入细胞因子及抗体复合物(按终浓度为300U/mL的IL-2、10ng/mL IL-7、5ng/mL IL-15、500ng/mL Anti-CD3(OKT3)、2μg/mL Anti-CD28配置)，连续培养48小时。
3)病毒感染：
(1)按照MOI＝20，计算所需要的病毒量。计算公式如下：所需病毒量(mL)＝(MOI*细胞数量)/病毒滴度
(2)从-80℃冰箱取出病毒后，迅速在37℃水浴锅中融化。在六孔板中加入上述计算所得的病毒量，添加终浓度为5μg/mL的DEAE，充分混匀后，使用封口膜将六孔板四边密封，800×g离心1小时。
(3)离心结束后，撕掉封口膜，将六孔板置于37℃5％CO 2的培养箱中，继续培养24小时。
(4)250×g离心10分钟，去掉含有病毒的培养基上清，用新鲜培养基重悬细胞沉淀，将细胞转移至新的六孔板中，继续培养3-6天备用。
实施例4：CAR-T细胞的阳性率检测
编码CAR的核酸序列在启动子的驱动下表达，使用生物素标记的MSLN抗原对慢病毒转染的T细胞进行标记，随后用荧光标记的链霉亲和素检测,并通过流式进行测定，反映CAR在T细胞表面的表达水平。通过如上方法检测实施例3获得的CAR-T细胞的CAR阳性率进行检测，FACS检测结果如下表所示。结果显示，所有CAR-T细胞的CAR阳性率均大于20％，表明慢病毒转染效应细胞后，成功表达了CAR，成功构建了表达MSLN-CAR嵌合抗原受体T细胞。
表6：CAR的阳性率检测结果
嵌合抗原受体 CAR阳性率
G5 50.45
G9 38.04
G16 41.47
实施例5：CAR-T对NCI-H226靶细胞的杀伤活性评价
5.1 CAR-T对NCI-H226靶细胞裂解能力评价
通过慢病毒转导的方法将Luciferase基因整合到NCI-H226细胞基因组中，获得能稳定表达Luciferase的NCI-H226人肺鳞癌细胞(NCI-H226-luc)。使用0.25％胰酶消化NCI-H226-luc细胞，含10％FBS的1640培养基终止消化，离心后，重悬细胞，调整细胞密度至1×10 5个/mL，按照100μL/孔的量接种靶细胞NCI-H226-luc于96孔板中，5％CO 2 37℃培养箱静置30min。分别收集G5-CAR-T、G9-CAR-T、G16-CAR-T，离心收集并用10％FBS的1640培养基重悬CAR-T细胞，以G5-CAR-T、G9-CAR-T、G16-CAR-T以及未转染CAR的空白T细胞作为效应细胞，然后按照1:1、0.5:1、0.25:1的E/T(效应细胞/靶细胞)比例加入到含有NCI-H226-luc的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO 2 37℃培养箱中培养18～24h。培养结束后，将孔板从培养箱中取出，加入20ul荧光检测试剂，使用酶标仪检测荧光读值。
CAR-T的杀伤活性检测结果如图2，均能发挥对肿瘤细胞的裂解生物活性。G16- CAR-T在效应细胞/靶细胞比例为1时，对肿瘤细胞的裂解率高达98％。G9-CAR-T和G5-CAR-T在效应细胞/靶细胞比例为1时，对肿瘤细胞的裂解率达约80％。
5.2细胞因子释放测定
收集NCI-H226细胞，使用培养基调整细胞密度至1×10 5个/mL，按照100μL/孔的量接种靶细胞于96孔板中，并用培养基重悬G16-CAR-T细胞，以G16-CAR-T以及未转染CAR的空白T细胞作为效应细胞，然后按照1:1的E/T(效应细胞/靶细胞)比例加入到含有靶细胞的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO 237℃培养箱中培养过夜。培养结束后，将孔板从培养箱中取出，离心，取上清，使用ELISA试剂盒(IL2、TNF-α、IFN-γ)检测CAR-T细胞的细胞因子释放。
检测结果如图3所示，G16-CAR-T能明显增强IL2、TNF-α、IFN-γ的分泌或释放，具有良好的增强免疫的活性。
实施例6：CAR-T对SKOV-3靶细胞的杀伤活性评价
6.1 CAR-T对SKOV-3靶细胞裂解能力评价
通过慢病毒转导的方法将Luciferase基因整合到SKOV-3细胞基因组中，获得能够稳定表达Luciferase的人卵巢癌SKOV-3细胞(SKOV-3-luc)。使用0.25％胰酶消化SKOV-3-luc细胞，含10％FBS的McCoy’s5A培养基培养基终止消化，离心后，重悬细胞，调整细胞密度至1×10 5个/mL，按照100μL/孔的量接种靶细胞SKOV-3-luc于96孔板中，5％CO 2 37℃培养箱静置30min。分别收集G5-CAR-T、G9-CAR-T、G16-CAR-T，离心收集并用10％FBS的McCoy’s5A培养基重悬CAR-T细胞，以G5-CAR-T、G9-CAR-T、G16-CAR-T以及未转染CAR的空白T细胞作为效应细胞，然后按照1:1、0.5:1、0.25:1的E/T(效应细胞/靶细胞)比例加入到含有SKOV-3-luc的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO 2 37℃培养箱中培养18-24h。培养结束后，将孔板从培养箱中取出，加入20ul荧光检测试剂，使用酶标仪检测荧光读值。
CAR-T的杀伤活性检测结果如图4，均能发挥对肿瘤细胞的裂解生物活性。G16-CAR-T在效应细胞/靶细胞比例为1时，对肿瘤细胞的裂解率高达95％。G9-CAR-T和G5-CAR-T在效应细胞/靶细胞比例为1时，对肿瘤细胞的裂解率达约50％。
6.2细胞因子释放测定
收集SKOV-3-luc细胞，使用培养基调整细胞密度至1×10 5个/mL，按照100μL/孔的量接种靶细胞于96孔板中，并用培养基重悬G5-CAR-T、G9-CAR-T、G16-CAR-T细胞，以G5-CAR-T、G9-CAR-T、G16-CAR-T以及未转染CAR的空白T细胞作为效应细胞，然后按照1:1的E/T(效应细胞/靶细胞)比例加入到含有靶细胞的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO 2 37℃培养箱中培养过夜。培养结束后，将孔板从培养箱中取出，离心，取上清，使用ELISA试剂盒(IL2、TNF-α、IFN-γ)检测CAR-T细胞的细胞因子释放。
检测结果如图5所示，G5-CAR-T、G9-CAR-T、G16-CAR-T均能够在肿瘤细胞刺激下促进TNF-α、IFN-γ等细胞因子的分泌或释放，尤其是G16-CAR-T能明显增强IL2、TNF-α、IFN-γ的分泌或释放，具有良好的增强免疫的活性。
6.3共表达PD-1抗体和/或mIL-15的CAR-T对SKOV-3靶细胞的杀伤活性评价
使用0.25％胰酶消化SKOV-3-luc细胞，含10％FBS的McCoy’s5A培养基培养基终止消化，离心后，重悬细胞，调整细胞密度至1×10 5个/mL，按照100μL/孔的量接种靶细胞SKOV-3-luc于96孔板中，5％CO 2 37℃培养箱静置30min。收集G16-CAR-T、G16-PD1-CAR-T、G16-mIL15-CAR-T、G16-PD1-mIL15-CAR-T，离心收集并用10％FBS的McCoy’s5A培养基重悬CAR-T细胞，以G16-CAR-T、G16-PD1-CAR-T、G16-mIL15-CAR-T、G16-PD1-mIL15-CAR-T以及未转染CAR的空白T细胞作为效应细胞，然后按照1:1、0.5:1、0.25:1的E/T(效应细胞/靶细胞)比例加入到含有SKOV-3-luc的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO 2 37℃培养箱中培养18-24h。培养结束后，将孔板从培养箱中取出，加入20ul荧光检测试剂，使用酶标仪检测荧光读值。
CAR-T的杀伤活性检测结果如图6，均能发挥对肿瘤细胞的裂解生物活性。G16-CAR-T、G16-PD1-CAR-T、G16-PD1-mIL15-CAR-T对肿瘤细胞的裂解生物活性相当，在效应细胞/靶细胞比例为1时，对肿瘤细胞的裂解率高达98％。G16-mIL15-CAR-T在效应细胞/靶细胞比例为1时，对肿瘤细胞的裂解率达约73％。
实施例7：CAR-T对MSLN阴性A431细胞的杀伤活性评价
7.1 CAR-T对A431细胞裂解能力评价
通过慢病毒转导的方法将Luciferase基因整合到MSLN阴性的A431细胞基因组中，获得能稳定表达Luciferase的A431人皮肤鳞癌细胞(A431-luc)。使用0.25％胰酶消化A431-luc细胞，含10％FBS的1640培养基终止消化，离心后，重悬细胞，调整细胞密度至1×10 5个/mL，按照100μL/孔的量接种靶细胞A431-luc于96孔板中，5％CO 2 37℃培养箱静置30min。收集G16-CAR-T，离心收集并用10％FBS的1640培养基重悬CAR-T细胞，以G16-CAR-T以及未转染CAR的空白T细胞作为效应细胞，然后按照0.5:1、0.25:1、0.125:1的E/T(效应细胞/靶细胞)比例加入到含有A431-luc的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO 2 37℃培养箱中培养18～24h。培养结束后，将孔板从培养箱中取出，加入20ul荧光检测试剂，使用酶标仪检测荧光读值。
CAR-T的杀伤活性检测结果如图7，结果表明，G16-CAR-T对MSLN阴性的A431细胞无杀伤活性，表明G16-CAR-T对MSLN阴性细胞无脱靶杀伤。
7.2细胞因子释放测定
收集A431-luc细胞，使用培养基调整细胞密度至1×10 5个/mL，按照100μL/孔的量接种靶细胞于96孔板中，并用培养基重悬G16-CAR-T以及未转染CAR的空白T细胞作为效应细胞，然后按照1:1的E/T(效应细胞/靶细胞)比例加入到含有靶细胞的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO 2 37℃培养箱中培养过夜。培养结束后，将孔板从培养箱中取出，离心，取上清，使用ELISA试剂盒(IL2、TNF-α、IFN-γ)检测CAR-T细胞的细胞因子释放。
检测结果如图8所示，结果表明，对于MSLN阴性的A431-luc细胞，G16-CAR-T的IL2、TNF-α、IFN-γ均无分泌，表明MSLN阴性细胞对G16-CAR-T无激活功能。
实施例8：体内模型评估CAR-T细胞对靶细胞的杀伤能力
8.1体内模型评估CAR-T细胞对PANC1靶细胞的杀伤能力
6只B-NDG小鼠右侧或右侧肩胛处皮下接种5×10 6个PANC1肿瘤细胞，待肿瘤平均体积达到100～150mm 3时，随机分为2组，每只小鼠腹腔给予环磷酰胺100mg/kg，次日尾静脉回输5×10 6个G16-CAR-T以及未转染CAR的空白T细胞。每周两次用游标卡尺测量肿瘤直径和称量小鼠体重，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
CAR-T细胞对靶细胞的杀伤能力结果如图9，与阴性对照组相比，G16-CAR-T对肿瘤细胞有良好的抑制作用。所有治疗组在观察期内均无动物死亡及显著动物体重降低，未见明显的药物毒性反应，治疗期间小鼠耐受性良好。
8.2体内模型评估CAR-T细胞对SK-OV-3靶细胞的杀伤能力
SKOV-3细胞体外单层培养，培养条件为McCoy's 5A培养基中加10％胎牛血清，于37℃、含5％CO 2空气的培养箱中培养。一周2-3次用胰酶-EDTA进行消化处理传代。收获对数生长期细胞，计数，接种。12只B-NDG小鼠右侧或右侧肩胛处皮下接种1×10 7个SKOV-3肿瘤细胞，待肿瘤平均体积达到～100mm 3时，随机分为4组，每只小鼠腹腔给予环磷酰胺100mg/kg，次日尾静脉回输5×10 6个G16-CAR-T、G16-PD1-CAR-T、G16-PD1-mIL15-CAR-T以及未转染CAR的空白T细胞。每周两次用游标卡尺测量肿瘤直径和称量小鼠体重，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
CAR-T细胞对靶细胞的杀伤能力结果如图10，与阴性对照组相比，G16-CAR-T、G16-PD1-CAR-T、G16-PD1-mIL15-CAR-T均有良好的抑瘤趋势。到第18天时，与阴性对照组相比，所有治疗组的抑制肿瘤效果显著。所有治疗组在观察期内均无动物死亡及显著动物体重降低，未见明显的药物毒性反应，治疗期间小鼠耐受性良好。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (41)

  1. 能够特异性结合MSLN的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (1a)如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：3或其变体的CDR-H1；序列为SEQ ID NO：4或其变体的CDR-H2；序列为SEQ ID NO：5或其变体的CDR-H3；和/或，如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：6或其变体的CDR-L1；序列为SEQ ID NO：7或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；
    或
    (1b)如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：9或其变体的CDR-H1；序列为SEQ ID NO：10或其变体的CDR-H2；序列为SEQ ID NO：11或其变体的CDR-H3；和/或，如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；
    或
    (1c)如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：14或其变体的CDR-H1；序列为SEQ ID NO：15或其变体的CDR-H2；序列为SEQ ID NO：5或其变体的CDR-H3；和/或，如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：6或其变体的CDR-L1；序列为SEQ ID NO：7或其变体的CDR-L2；序列为SEQ ID NO：8或其变体的CDR-L3；
    或
    (2a)如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：18或其变体的CDR-H1；序列为SEQ ID NO：19或其变体的CDR-H2；序列为SEQ ID NO：20或其变体的CDR-H3；和/或，如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：21或其变体的CDR-L1；序列为SEQ ID NO：22或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；
    或
    (2b)如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：24或其变体的CDR-H1；序列为SEQ ID NO：25或其变体的CDR-H2；序列为SEQ ID NO： 26或其变体的CDR-H3；和/或，如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；
    或
    (2c)如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：29或其变体的CDR-H1；序列为SEQ ID NO：30或其变体的CDR-H2；序列为SEQ ID NO：20或其变体的CDR-H3；和/或，如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：21或其变体的CDR-L1；序列为SEQ ID NO：22或其变体的CDR-L2；序列为SEQ ID NO：23或其变体的CDR-L3；
    或
    (3a)如下三个根据Kabat编号系统所定义的重链CDRs：序列为SEQ ID NO：33或其变体的CDR-H1；序列为SEQ ID NO：34或其变体的CDR-H2；序列为SEQ ID NO：35或其变体的CDR-H3；和/或，如下三个根据Kabat编号系统所定义的轻链CDRs：序列为SEQ ID NO：36或其变体的CDR-L1；序列为SEQ ID NO：37或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；
    或
    (3b)如下三个根据IMGT编号系统所定义的重链CDRs：序列为SEQ ID NO：39或其变体的CDR-H1；序列为SEQ ID NO：40或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，如下三个根据IMGT编号系统所定义的轻链CDRs：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；
    或
    (3c)如下三个根据Chothia编号系统所定义的重链CDRs：序列为SEQ ID NO：44或其变体的CDR-H1；序列为SEQ ID NO：45或其变体的CDR-H2；序列为SEQ ID NO：35或其变体的CDR-H3；和/或，如下三个根据Chothia编号系统所定义的轻链CDRs：序列为SEQ ID NO：36或其变体的CDR-L1；序列为SEQ ID NO：37或其变体的CDR-L2；序列为SEQ ID NO：38或其变体的CDR-L3；
    其中，(1a)、(1b)、(1c)、(2a)、(2b)、(2c)、(3a)、(3b)、(3c)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段还包括来自人的免疫球蛋白的构架区(FRs)；
    优选地，所述抗体或其抗原结合片段特异性结合人MSLN。
  2. 权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (1)包含如SEQ ID NO:1所示的序列或其变体的VH和/或包含如SEQ ID NO:2所示的序列或其变体的VL；
    (2)包含如SEQ ID NO:16所示的序列或其变体的VH和/或包含如SEQ ID NO:17所示的序列或其变体的VL；或
    (3)包含如SEQ ID NO:31所示的序列或其变体的VH和/或包含如SEQ ID NO:32所示的序列或其变体的VL；
    其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)如下六个根据Kabat编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：3的CDR-H1；序列为SEQ ID NO：4的CDR-H2；序列为SEQ ID NO：5的CDR-H3；序列为SEQ ID NO：6的CDR-L1；序列为SEQ ID NO：7的CDR-L2；序列为SEQ ID NO：8的CDR-L3；或
    (b)如下六个根据IMGT编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：9的CDR-H1；序列为SEQ ID NO：10的CDR-H2；序列为SEQ ID NO：11的CDR-H3；序列为SEQ ID NO：12的CDR-L1；序列为SEQ ID NO：13的CDR-L2；序列为SEQ ID NO：8的CDR-L3；或
    (c)如下六个根据Chothia编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：14的CDR-H1；序列为SEQ ID NO：15的CDR-H2；序列为SEQ ID NO：5的CDR-H3；序列为SEQ ID NO：6的CDR-L1；序列为SEQ ID NO：7的CDR-L2；序列 为SEQ ID NO：8的CDR-L3；
    优选地，所述抗体或其抗原结合片段包含：
    包含如SEQ ID NO:1所示的序列或其变体的VH和/或包含如SEQ ID NO:2所示的序列或其变体的VL；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  4. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)如下六个根据Kabat编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：18的CDR-H1；序列为SEQ ID NO：19的CDR-H2；序列为SEQ ID NO：20的CDR-H3；序列为SEQ ID NO：21的CDR-L1；序列为SEQ ID NO：22的CDR-L2；序列为SEQ ID NO：23的CDR-L3；或
    (b)如下六个根据IMGT编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：24的CDR-H1；序列为SEQ ID NO：25的CDR-H2；序列为SEQ ID NO：26的CDR-H3；序列为SEQ ID NO：27的CDR-L1；序列为SEQ ID NO：28的CDR-L2；序列为SEQ ID NO：23的CDR-L3；或
    (c)如下六个根据Chothia编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：29的CDR-H1；序列为SEQ ID NO：30的CDR-H2；序列为SEQ ID NO：20的CDR-H3；序列为SEQ ID NO：21的CDR-L1；序列为SEQ ID NO：22的CDR-L2；序列为SEQ ID NO：23的CDR-L3；
    优选地，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:16所示的序列或其变体的VH和/或包含如SEQ ID NO:17所示的序列或其变体的VL；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  5. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)如下六个根据Kabat编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：33的CDR-H1；序列为SEQ ID NO：34的CDR-H2；序列为SEQ ID NO：35的CDR-H3；序列为SEQ ID NO：36的CDR-L1；序列为SEQ ID NO：37的CDR-L2；序列为SEQ ID NO：38的CDR-L3；或
    (b)如下六个根据IMGT编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：39的CDR-H1；序列为SEQ ID NO：40的CDR-H2；序列为SEQ ID NO：41的CDR-H3；序列为SEQ ID NO：42的CDR-L1；序列为SEQ ID NO：43的CDR-L2；序列为SEQ ID NO：38的CDR-L3；或
    (c)如下六个根据Chothia编号系统所定义的重链和轻链的CDRs：序列为SEQ ID NO：44的CDR-H1；序列为SEQ ID NO：45的CDR-H2；序列为SEQ ID NO：35的CDR-H3；序列为SEQ ID NO：36的CDR-L1；序列为SEQ ID NO：37的CDR-L2；序列为SEQ ID NO：38的CDR-L3；
    优选地，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:31所示的序列或其变体的VH和/或包含如SEQ ID NO:32所示的序列或其变体的VL；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  6. 权利要求1-5任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自骆驼Ig、IgNAR、Fab片段、Fab'片段、F(ab)' 2片段、F(ab)' 3片段、单链抗体(例如scFv、di-scFv或(scFv) 2)、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(dsFv)和单结构域抗体(sdAb，纳米抗体)。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段是单链抗体，例如scFv、di-scFv或(scFv) 2；
    优选地，所述单链抗体从其N端至C端依次包括：
    (1)包含如SEQ ID NO:1所示的序列或其变体的VH-连接子-包含如SEQ ID NO:2所示的序列或其变体的VL；
    (2)包含如SEQ ID NO:16所示的序列或其变体的VH-连接子-包含如SEQ ID NO:17所示的序列或其变体的VL；
    (3)包含如SEQ ID NO:31所示的序列或其变体的VH-连接子-包含如SEQ ID NO:32所示的序列或其变体的VL；
    (4)包含如SEQ ID NO:2所示的序列或其变体的VL-连接子-包含如SEQ ID NO:1所示的序列或其变体的VH；
    (5)包含如SEQ ID NO:17所示的序列或其变体的VL-连接子-包含如SEQ ID NO:16所示的序列或其变体的VH；或
    (6)包含如SEQ ID NO:32所示的序列或其变体的VL-连接子-包含如SEQ ID NO:31所示的序列或其变体的VH；其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；
    优选地，所述单链抗体的VH和VL通过连接子连接；优选地，所述连接子为多肽；优选地，所述连接子包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2；更优选地，所述连接子具有SEQ ID NO：52的序列；
    更优选地，所述单链抗体包含选自下列的氨基酸序列：(1)SEQ ID NOs:54、56、58任一项所示的氨基酸序列；(2)与SEQ ID NOs:54、56、58任一项所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列；或(3)与SEQ ID NOs:54、56、58任一项所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合 片段进一步包含重链恒定区(CH)和轻链恒定区(CL)；
    优选地，所述重链恒定区选自IgG、IgM、IgE、IgD和IgA；
    优选地，所述轻链恒定区选自κ或λ。
  9. 分离的核酸分子，其包含编码权利要求1-8任一项所述的抗体或其抗原结合片段的核苷酸序列；
    优选地，所述分离的核酸分子包含选自下列的核苷酸序列：(1)SEQ ID NO：55、57和59任一项所示的核苷酸序列；(2)与SEQ ID NO：55、57和59任一项所示的核苷酸序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
  10. 载体，其包含权利要求9所述的分离的核酸分子；
    优选地，所述载体选自DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体或病毒载体；
    优选的，所述载体是表达载体；
    优选地，所述载体是游离型载体；
    优选地，所述载体是病毒载体；更优选地，所述病毒载体是慢病毒载体、腺病毒载体或逆转录病毒载体。
  11. 宿主细胞，其包含权利要求9所述的分离的核酸分子，或权利要求10所述的载体。
  12. 制备权利要求1-8任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求11所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  13. 能够特异性结合MSLN的嵌合抗原受体(CAR)，其包含抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，其中所述抗原结合结构域包含权利要求1-8任一项所述的抗体或其抗原结合片段；
    优选地，所述抗原结合结构域包含所述抗体或其抗原结合片段作为第一结合结构域，并且进一步包含不结合MSLN的第二结合结构域；更优选地，所述第二结合结构域结合的抗原选自：CD19、GPC3、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2；
    优选地，所述抗体或其抗原结合片段是单链抗体，例如scFv、di-scFv或(scFv) 2；
    优选地，所述抗体或其抗原结合片段的VH和VL通过连接子连接；优选地，所述连接子包含一个或几个(例如1个、2个或3个)如(G mS) n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2；更优选地，所述连接子具有SEQ ID NO：52的序列。
  14. 权利要求13所述的嵌合抗原受体，其中，所述抗原结合结构域包含选自下列的氨基酸序列：(1)SEQ ID NOs:54、56、58任一项所示的氨基酸序列；(2)与SEQ ID NOs:54、56、58任一项所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列；或(3)与SEQ ID NOs:54、56、58任一项所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  15. 权利要求13或14所述的嵌合抗原受体，其中，所述跨膜结构域选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD28、CD45、CD3ε、CD3ζ、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD152、CD154和PD-1；
    优选地，所述跨膜结构域选自下列蛋白的跨膜区：CD8α、CD4、PD-1、CD152和CD154；
    优选地，所述跨膜结构域包含序列如SEQ ID NO：64所示的CD8α跨膜区。
  16. 权利要求13-15任一项所述的嵌合抗原受体，其中所述间隔结构域位于抗原结合结构域与跨膜结构域之间，所述间隔结构域选自铰链结构域和/或免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区；
    优选地，所述铰链结构域包含CD8α、PD-1、CD152或CD154的铰链区；更优选地，所述铰链结构域包含序列如SEQ ID NO：62所示的CD8α铰链区。
  17. 权利要求13-16任一项所述的嵌合抗原受体，其中，所述胞内信号传导结构域包含初级信号传导结构域和/或共刺激信号传导结构域；
    优选地，所述胞内信号传导结构域从N端到C端依次包含共刺激信号传导结构域和初级信号传导结构域；
    优选地，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信号传导结构域；
    优选地，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)；
    优选地，所述初级信号传导结构域包含选自以下蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b或CD66d；更优选地，所述初级信号传导结构域包含序列如SEQ ID NO：68所示的CD3ζ胞内信号传导结构域；
    优选地，所述共刺激信号传导结构域包含选自下列蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)或DAP10；
    优选地，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域或CD137(4-1BB)的胞内信号传导结构域或二者片段的组合；更优选地，所述共刺激信号传导结构域包含序列如SEQ ID NO：66所示的CD137(4-1BB)胞内信号传导结构域；
    更优选地，所述胞内信号传导结构域序列包含如SEQ ID NO：70所示的序列。
  18. 权利要求13-17任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体进一步在其N端包含信号肽；
    优选地，所述信号肽包含重链信号肽(例如IgG1的重链信号肽)、粒细胞-巨噬细胞集落刺激因子受体2(GM-CSFR2)信号肽、IL2信号肽、或CD8α信号肽；更优选地，所述信号肽包含如SEQ ID NO：60所示的序列。
  19. 权利要求13-18任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体从其N端至C端依次包含所述信号肽、抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传 导结构域；
    优选地，所述信号肽包含IgG1的重链信号肽或CD8α信号肽(例如，如SEQ ID NO：60所示序列的信号肽)；
    优选地，所述间隔结构域包含CD8(例如CD8α)的铰链区(例如，如SEQ ID NO：62所示序列的铰链区)；
    优选地，所述跨膜结构域包含CD8(例如CD8α)的跨膜区(例如，如SEQ ID NO：64所示序列的跨膜区)；
    优选地，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO：68所示序列)，所述共刺激信号传导结构域包含CD137(4-1BB)的胞内信号传导结构域(例如，如SEQ ID NO：66所示序列)；更优选地，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO：70所示序列；
    优选地，所述嵌合抗原受体包含选自下列的氨基酸序列：(1)SEQ ID NOs:83、88、90任一项所示的氨基酸序列；(2)与SEQ ID NOs:83、88、90任一项所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；或者，(3)与SEQ ID NOs:83、88、90任一项所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  20. 分离的核酸分子，其包含编码权利要求13-19任一项所述的嵌合抗原受体的核苷酸序列；
    优选地，所述分离的核酸分子包含选自下列的核苷酸序列：(1)SEQ ID NOs:84、89、91任一项所示的序列或其简并变体；(2)与(1)中任一项所示序列相比基本上相同的序列(例如，与(1)中任一项所示的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)中任一项所示的序列相比具有一个或更多个核苷酸取代的序列)。
  21. 核酸构建体，其包含：
    (1)编码权利要求13-19任一项所述的嵌合抗原受体的第一核酸序列；和
    (2)编码另外的生物活性分子的第二核酸序列；
    优选地，所述第二核苷酸序列所编码的另外的生物活性分子选自下列组分的一种或一种以上：免疫检查点抑制剂(例如，抗PD-1、PD-L1、CTLA-4、或LAG-3抗体或其抗原结合片段)、细胞因子(例如，IL-15、IL-7、IL-12、IL-18、或IL-21)、或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18、或mIL-21)；
    优选地，所述第二核苷酸序列所编码的另外的生物活性分子在其N端进一步包含信号肽-2；优选地，所述信号肽-2不同于所述第一核酸序列所编码的嵌合抗原受体中所包含的信号肽；优选地，所述另外的生物活性分子N端的信号肽-2是IL2信号肽(例如，如SEQ ID NO：74所示)。
  22. 权利要求21所述的核酸构建体，其中，所述第一核酸序列和第二核酸序列通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接；
    优选地，所述自裂解肽是P2A(例如，如SEQ ID NO：72所示)。
  23. 权利要求21或22所述的核酸构建体，其中，所述另外的生物活性分子选自的免疫检查点抑制剂为抗PD-1或PD-L1抗体或其抗原结合片段；
    优选地，所述抗PD-1或PD-L1抗体或其抗原结合片段包含如下任一组的重链可变区和/或轻链可变区：(1)Nivolumab或其变体的重链可变区和/或轻链可变区，(2)Pembrolizumab或其变体的重链可变区和/或轻链可变区，(3)Atezolizumab或其变体的重链可变区和/或轻链可变区，(4)Durvalumab或其变体的重链可变区和/或轻链可变区，(5)Avelumab或其变体的重链可变区和/或轻链可变区，(6)具有SEQ ID NO:79所示序列或其变体的VH和/或具有SEQ ID NO:80所示序列或其变体的VL；其中，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；
    优选地，所述抗PD-1或PD-L1抗体或其抗原结合片段为单链抗体(例如scFv)；
    优选地，所述另外的生物活性分子包含抗PD-1单链抗体，所述抗PD-1单链抗体包含选自下列的氨基酸序列：(1)SEQ ID NO:77所示的氨基酸序列；(2)与SEQ ID NO:77所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列；(3)与SEQ ID NO:77所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；
    优选地，所述核酸构建体从其5’端至3’端依次包含：所述第一核酸序列、编码自裂解肽的核苷酸序列、编码信号肽-2的核苷酸序列、编码免疫检查点抑制剂的核苷酸序列；优选地，所述核酸构建体包含选自下列的核苷酸序列：(1)SEQ ID NO:85所示的核苷酸序列或其简并变体；(2)与(1)中所述的序列相比基本上相同的序列，例如与(1)中所述的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
  24. 权利要求21或22所述的核酸构建体，其中，所述另外的生物活性分子选自的膜嵌合型多肽为mIL-15；
    优选地，所述膜嵌合型多肽mIL-15包含选自下列的氨基酸序列：(1)SEQ ID NO：81所示的序列；(2)与SEQ ID NO:81所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列；(3)与SEQ ID NO:81所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；
    优选地，所述核酸构建体从其5’端至3’端依次包含：所述第一核酸序列、编码自裂解肽的核苷酸序列、编码膜嵌合型多肽的核苷酸序列；优选地，所述核酸构建体包含选自下列的核苷酸序列：(1)SEQ ID NO:86所示的核苷酸序列或其简并变体；(2)与(1)所述的序列相比基本相同的序列，例如与(1)所述的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少 91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
  25. 权利要求21-24任一项所述的核酸构建体，其中，所述第二核酸序列所编码的另外的生物活性分子包含至少两种选自以下的组分：免疫检查点抑制剂(例如，抗PD-1、PD-L1、CTLA-4、或LAG-3抗体或其抗原结合片段)、细胞因子(例如，IL-15、IL-7、IL-12、IL-18、或IL-21)、或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18、或mIL-21)；
    优选地，所述第二核酸序列中所包含的编码所述至少两种组分的核苷酸序列彼此之间通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接；优选地，所述自裂解肽是P2A(例如，如SEQ ID NO：72所示)；
    优选地，所述第二核酸序列所编码的另外的生物活性分子包含：(i)抗PD-1抗体或其抗原结合片段(例如scFv)和(ii)mIL-15；
    优选地，所述核酸构建体从其5’端至3’端依次包含所述第一核酸序列、编码自裂解肽的核苷酸序列、编码信号肽-2的核苷酸序列、编码抗PD-1抗体或其抗原结合片段的核苷酸序列、编码自裂解肽的核苷酸序列、编码mIL-15的核苷酸序列；优选地，所述核酸构建体包含选自下列的核苷酸序列：(1)SEQ ID NO:87所示的核苷酸序列或其简并变体；(2)与(1)所述的序列相比基本相同的序列，例如与(1)所述的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
  26. 载体，其包含权利要求20所述的分离的核酸分子，或权利要求21-25任一项所述的核酸构建体；
    优选地，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，或病毒载体；
    优选的，所述载体是表达载体；
    优选地，所述载体是游离型载体；
    优选地，所述载体是病毒载体；更优选地，所述病毒载体是慢病毒载体，腺病毒载体或逆转录病毒载体。
  27. 经改造的免疫细胞，其表达权利要求13-19任一项所述的嵌合抗原受体(CAR)；优选地，所述经改造的免疫细胞包含权利要求20所述的分离的核酸分子或包含所述分离的核酸分子的载体。
  28. 权利要求27所述的经改造的免疫细胞，其进一步表达另外的生物活性分子，所述另外的生物活性分子选自以下组分的一种或一种以上：特异性地结合免疫检查点的抗体或其抗原结合片段(例如，抗PD-1、PD-L1、CTLA-4、或LAG-3抗体或其抗原结合片段)、细胞因子(例如，IL-15、IL-7、IL-12、IL-18、IL-21)、或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18、mIL-21)；
    优选地，所述经改造的免疫细胞包含权利要求21或22所述的核酸构建体或包含所述核酸构建体的载体。
  29. 权利要求28所述的经改造的免疫细胞，其表达权利要求13-19任一项所述的嵌合抗原受体以及抗PD-1或PD-L1抗体或其抗原结合片段；
    优选地，所述经改造的免疫细胞包含权利要求23所述的核酸构建体或包含所述核酸构建体的载体。
  30. 权利要求28所述的经改造的免疫细胞，其表达权利要求13-19任一项所述的嵌合抗原受体以及mIL-15；
    优选地，所述经改造的免疫细胞包含权利要求24所述的核酸构建体或包含所述核酸构建体的载体。
  31. 权利要求28所述的经改造的免疫细胞，其表达权利要求13-19任一项所述的嵌合抗原受体、以及另外的生物活性分子，所述另外的生物活性分子包含至少两种选自以下的组分：免疫检查点抑制剂(例如，抗PD-1、PD-L1、CTLA-4、或LAG-3抗体或其抗原结合片段)、细胞因子(例如，IL-15、IL-7、IL-12、IL-18、或IL-21)、或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18、或mIL-21)；
    优选地，所述经改造的免疫细胞包含权利要求25所述的核酸构建体或包含所述核酸构建体的载体。
  32. 权利要求27-31任一项所述的经改造的免疫细胞，其中，所述免疫细胞来源于T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合；优选地，所述免疫细胞得自患者；可选地，所述免疫细胞得自健康供体；优选地，所述免疫细胞来源于T淋巴细胞或NK细胞。
  33. 权利要求27-32任一项所述的经改造的免疫细胞，其中，所述免疫细胞还表达不特异性针对MSLN的CAR；优选地，所述不特异性针对MSLN的CAR具有针对选自下列靶点的特异性：CD19、GPC3、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2。
  34. 权利要求27-33任一项所述的经改造的免疫细胞，其中，所述经改造的免疫细胞的免疫排除有关的基因(例如，TRAC、TRBC、B2M、HLA-A、HLA-B或HLA-C)和免疫共抑制通路或信号分子的基因(例如，PD-1、CTLA-4或LAG-3)中的一种或两种靶基因的转录或表达被抑制；优选地，所述靶基因的转录或表达被抑制采用的方法选自基因敲除(例如，CRISPR、CRISPR/Cas9)、同源重组、干扰RNA。
  35. 制备权利要求27-33任一项所述的经改造的免疫细胞的方法，其包括：(1)提供来自患者或者健康供体的免疫细胞；(2)将权利要求20所述的分离的核酸分子或包含其的载体引入步骤(1)所述的免疫细胞，以获得能够表达MSLN特异性结合嵌合抗原受体的免疫细胞；或者，将权利要求21-25任一项所述的核酸构建体或包含其的载体引入步骤(1)所述的免疫细胞，以获得能够共表达MSLN特异性结合嵌合抗原受体和另外的生物活性分子的免疫细胞；
    优选地，在步骤(1)中，所述免疫细胞经预处理，所述预处理包括免疫细胞的分选、激活和/或增殖；更优选地，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞；
    优选地，在步骤(2)中将所述核酸分子或载体通过病毒感染引入免疫细胞；
    优选地，在步骤(2)中将所述核酸分子或载体通过非病毒载体转染的方式引入免疫细胞，如通过磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法或电穿孔方法；
    优选地，在步骤(2)之后还包括扩增步骤(2)获得的免疫细胞的步骤。
  36. 免疫细胞组合物，包括权利要求27-34任一项所述的经改造的免疫细胞；可选地，所述组合物还包括未改造和/或未成功改造的免疫细胞；优选地，所述经改造的免疫细胞数占所述免疫细胞组合物细胞总数的10％-100％，更优选40％-80％。
  37. 试剂盒，所述试剂盒包括权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9所述的分离的核酸分子，或权利要求10所述的载体，或权利要求11所述的宿主细胞，或权利要求20所述的分离的核酸分子，或权利要求21-25任一项所述的核酸构建体，或权利要求26所述的载体；
    优选地，所述试剂盒包括权利要求20所述的分离的核酸分子，或权利要求21-25任一项所述的核酸构建体，或权利要求26所述的载体；优选地，所述试剂盒用于制备权利要求13-19任一项所述的嵌合抗原受体(CAR)或权利要求27-34任一项所述的经改造的免疫细胞或权利要求36所述的免疫细胞组合物。
  38. 药物组合物，其含有权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9所述的分离的核酸分子，或权利要求10所述的载体，或权利要求11所述的宿主细胞，或权利要求20所述的分离的核酸分子，或权利要求21-25任一项所述的核酸构建体，或权利要求26所述的载体，或权利要求27-34任一项所述的经改造的免疫细胞，或权利要求36所述的免疫细胞组合物，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物还包含另外的药学活性剂，例如具有抗肿瘤活性的药物；优选地，所述另外的药学活性剂包括anti-PD1抗体、anti-PD-L1抗体、anti-CTLA-4抗体、培美曲塞、顺铂、紫杉醇、吉西他滨、卡培他滨或FOLFIRINOX；
    可选地，所述药物组合物包含的抗体或其抗原结合片段、或分离的核酸分子、或载体、或宿主细胞、或核酸构建体、或经改造的免疫细胞、或免疫细胞组合物，可与所述另外的药学活性剂同时、分开或相继施用。
  39. 权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9所述的分离的核酸分子，或权利要求10所述的载体，或权利要求11所述的宿主细胞，或权利要求20所述的分离的核酸分子，或权利要求21-25任一项所述的核酸构建体，或权利要求26所 述的载体，或权利要求27-34任一项所述的经改造的免疫细胞，或权利要求36所述的免疫细胞组合物，或权利要求38所述的药物组合物，在制备用于预防和/或治疗与间皮素的表达相关的疾病的药物中的用途；
    优选地，所述与间皮素的表达相关的疾病选自增生性疾病，例如肿瘤，或是与间皮素的表达相关的非肿瘤相关的适应症；
    优选地，所述肿瘤是MSLN阳性肿瘤；
    优选地，所述肿瘤选自实体瘤；优选地，所述实体瘤选自恶性胸膜间皮瘤、胰腺癌、肺癌(例如肺鳞癌)、乳腺癌、卵巢癌(例如卵巢上皮癌)。
  40. 用于在受试者(例如人)中预防和/或治疗与间皮素的表达相关的疾病的方法，所述方法包括向有此需要的受试者施用有效量的权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9所述的分离的核酸分子，或权利要求10所述的载体，或权利要求11所述的宿主细胞，或权利要求20所述的分离的核酸分子，或权利要求21-25任一项所述的核酸构建体，或权利要求26所述的载体，或权利要求27-34任一项所述的经改造的免疫细胞，或权利要求36所述的免疫细胞组合物，或权利要求38所述的药物组合物；
    优选地，所述与间皮素的表达相关的疾病选自增生性疾病，例如肿瘤，或是与间皮素的表达相关的非肿瘤相关的适应症；
    优选地，所述肿瘤是MSLN阳性肿瘤；
    优选地，所述肿瘤选自实体瘤；优选地，所述实体瘤选自恶性胸膜间皮瘤、胰腺癌、肺癌(例如肺鳞癌)、乳腺癌、卵巢癌(例如卵巢上皮癌)；
    优选地，所述方法还包括向所述受试者施用第二疗法，所述第二疗法选自手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其任意组合。
  41. 权利要求40所述的方法，其中，所述方法包括向所述受试者施用权利要求27-34任一项所述的经改造的免疫细胞，或权利要求36所述的免疫细胞组合物，或权利要求38所述的药物组合物；
    优选地，所述方法包括以下步骤：(1)提供受试者所需的免疫细胞；(2)(2)将权利要求20所述的分离的核酸分子、或权利要求21-25任一项所述的核酸构建体或包含所述分离的核酸分子或核酸构建体的载体引入步骤(1)所述的免疫细胞；(3)将步骤(2)中获得的免 疫细胞施用至所述受试者；
    可选地，在步骤(3)中，所述免疫细胞的总剂量包含1×10 7至10×10 8个CAR阳性细胞；
    优选地，在步骤(3)中，所述免疫细胞的总剂量分次施用至所述受试者。
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116003638A (zh) * 2023-01-20 2023-04-25 北京基因启明生物科技有限公司 一种有效杀伤胆管型肝癌的CAR-iNKT细胞技术
CN116785415A (zh) * 2023-05-04 2023-09-22 北京百普赛斯生物科技股份有限公司 一种特异性针对免疫细胞激活/增殖刺激的产品、制备及应用
WO2024012395A1 (en) * 2022-07-11 2024-01-18 Nona Biosciences (Suzhou) Co., Ltd. Anti-mesothelin antibodies
WO2024026358A1 (en) * 2022-07-27 2024-02-01 Teneobio, Inc. Mesothelin binding proteins and uses thereof
US11981745B2 (en) 2021-10-06 2024-05-14 Link Immunotherapeutics, Inc. Anti-mesothelin antigen-binding molecules and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116987192B (zh) * 2023-09-26 2023-12-15 旭和（天津）医药科技有限公司 抗人b淋巴细胞刺激因子受体baffr的抗原结合多肽及其用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106467573A (zh) * 2015-08-21 2017-03-01 科济生物医药（上海）有限公司 抗间皮素全人抗体以及靶向间皮素的免疫效应细胞
US20170267755A1 (en) * 2016-03-16 2017-09-21 Sri International Isolated anti-mesothelin antibodies, conjugates and uses thereof
CN110746505A (zh) * 2018-07-23 2020-02-04 上海细胞治疗集团有限公司 特异性结合间皮素的单克隆抗体及嵌合抗原受体
CN111560072A (zh) * 2020-07-16 2020-08-21 上海恒润达生生物科技有限公司 抗人msln抗体以及靶向msln的免疫效应细胞

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106467573A (zh) * 2015-08-21 2017-03-01 科济生物医药（上海）有限公司 抗间皮素全人抗体以及靶向间皮素的免疫效应细胞
US20170267755A1 (en) * 2016-03-16 2017-09-21 Sri International Isolated anti-mesothelin antibodies, conjugates and uses thereof
CN110746505A (zh) * 2018-07-23 2020-02-04 上海细胞治疗集团有限公司 特异性结合间皮素的单克隆抗体及嵌合抗原受体
CN111560072A (zh) * 2020-07-16 2020-08-21 上海恒润达生生物科技有限公司 抗人msln抗体以及靶向msln的免疫效应细胞

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JAFARZADEH LEILA, ELHAM MASOUMI, KHADIJEH ALISHAH, HAMID REZA MIRZAEI, AREZOO JAMALI, KEYVAN FALLAH-MEHRJARDI, HOSEIN ROSTAMIAN, M: "Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR) Expressing T Cell", IRANIAN JOURNAL OF ALLERGY ASTHMA AND IMMUNOLOGY, vol. 19, no. 3, 23 June 2020 (2020-06-23), pages 264 - 275, XP055919450, DOI: 10.18502/ijaai.v19i3.3454 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11981745B2 (en) 2021-10-06 2024-05-14 Link Immunotherapeutics, Inc. Anti-mesothelin antigen-binding molecules and uses thereof
WO2024012395A1 (en) * 2022-07-11 2024-01-18 Nona Biosciences (Suzhou) Co., Ltd. Anti-mesothelin antibodies
WO2024026358A1 (en) * 2022-07-27 2024-02-01 Teneobio, Inc. Mesothelin binding proteins and uses thereof
CN116003638A (zh) * 2023-01-20 2023-04-25 北京基因启明生物科技有限公司 一种有效杀伤胆管型肝癌的CAR-iNKT细胞技术
CN116003638B (zh) * 2023-01-20 2023-09-05 北京基因启明生物科技有限公司 一种有效杀伤胆管型肝癌的CAR-iNKT细胞技术
CN116785415A (zh) * 2023-05-04 2023-09-22 北京百普赛斯生物科技股份有限公司 一种特异性针对免疫细胞激活/增殖刺激的产品、制备及应用
CN116785415B (zh) * 2023-05-04 2024-03-22 北京百普赛斯生物科技股份有限公司 一种特异性针对免疫细胞激活/增殖刺激的产品、制备及应用

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