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WO2024119769A9 - Preparation and use of car-nk cell with enhanced capacity to infiltrate tumor sites - Google Patents

Preparation and use of car-nk cell with enhanced capacity to infiltrate tumor sites Download PDF

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Publication number
WO2024119769A9
WO2024119769A9 PCT/CN2023/101475 CN2023101475W WO2024119769A9 WO 2024119769 A9 WO2024119769 A9 WO 2024119769A9 CN 2023101475 W CN2023101475 W CN 2023101475W WO 2024119769 A9 WO2024119769 A9 WO 2024119769A9
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Prior art keywords
car
cells
nucleic acid
construct
acid molecule
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French (fr)
Chinese (zh)
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WO2024119769A1 (en
Inventor
张彩
胡渊
陈敏华
王烃
谢思奇
伏永玲
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Shanghai NK Cell Tech Co Ltd
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Shanghai NK Cell Tech Co Ltd
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001166—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/001168—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention belongs to the field of biomedicine, and specifically, the present invention relates to the preparation of CAR-NK cells and their use in tumor treatment.
  • CAR-T chimeric antigen receptor T
  • GVHD graft-versus-host disease
  • CAR-NK cells have the advantage of good safety over CAR-T cells, and generally do not cause side effects such as cytokine storm and GVHD; NK cells do not require antigen presentation and are not restricted by MHC, and can play a direct role in killing tumor cells; CAR-NK cells can identify and kill tumors with multiple recognition mechanisms such as CAR dependence and NKR dependence, and have a wide anti-tumor spectrum. Therefore, CAR-NK cells have broad application prospects in anti-tumor treatment and have become a hot spot in the field of cell immunotherapy research and development.
  • NK cells have difficulty infiltrating into solid tumors.
  • NK cell infusion in patients with solid tumors An important factor affecting the clinical results of NK cell infusion in patients with solid tumors is the failure of sufficient NK cells to migrate to the tumor site to kill the tumor. Effectively transporting NK cells to the tumor site is the key to the success of cancer immunotherapy. Therefore, improving the ability of NK cells to infiltrate into solid tumors has become an important research and development strategy for gene-modified NK cells.
  • Chemokines and chemokine receptors play an important role in the migration of lymphocytes such as T or NK cells to tumors. Lymphocytes can be attracted and recruited by chemokines secreted by tumors through chemokine receptors on the cell surface and guided into the tumor site.
  • CXCR2 is a receptor for multiple chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL7 and CXCL8).
  • chemokines are highly expressed in a variety of solid tumors such as liver cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, pancreatic cancer, ovarian cancer, etc., and are related to tumor proliferation, angiogenesis, invasion and metastasis, and the formation of an immunosuppressive microenvironment.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • CXCR2 is usually only expressed in memory B cells and certain myeloid cells (such as neutrophils and basophils), and human T cells and NK cells do not express it. Therefore, it is difficult for T cells and NK cells to infiltrate the tumor site. Therefore, if CXCR2 is overexpressed in lymphocytes, it is expected to effectively promote the aggregation of T cells or NK cells to the tumor site, and effectively improve the anti-tumor effect of T cells or NK cells. And the inventors found that when CAR-lymphocytes co-express CXCR2 receptors, compared with co-expressing CXCR6 receptors, their chemotaxis and targeting ability to tumors are stronger, and their ability to kill tumor cells is stronger.
  • the present invention proposes a CAR-lymphocyte, whose surface single-chain antibody can recognize tumor antigens of all solid tumors and hematological tumors and can express CXCR2 receptors intracellularly, thereby enhancing the ability of lymphocytes to migrate to the tumor site and effectively improving the killing effect of lymphocytes on tumors.
  • the present invention provides a construct.
  • the construct comprises a first nucleic acid and a second nucleic acid.
  • the first nucleic acid encodes a chimeric antigen receptor
  • the second nucleic acid encodes a chemokine receptor.
  • the construct can encode a chimeric antigen receptor and a chemokine receptor at the same time.
  • the construct of the embodiment of the present invention is introduced into lymphocytes, and the chimeric antigen receptor and the chemokine receptor are expressed on the surface of the lymphocytes.
  • the chimeric antigen receptor can enable lymphocytes to target tumor antigens and localize to the surface of cells expressing the antigen.
  • the chemokine receptor can increase the infiltration ability of lymphocytes to the tumor site, effectively improving the tumor-killing effect of lymphocytes.
  • the above construct may further include at least one of the following additional technical features:
  • the chimeric antigen receptor includes: an extracellular region, the extracellular region includes a single-chain antibody and a CD8 hinge region, the single-chain antibody includes a heavy chain variable region and a light chain variable region, the extracellular region single-chain antibody can specifically recognize tumor antigens, and the C-terminus of the single-chain antibody is connected to the N-terminus of the CD8 hinge region.
  • transmembrane region wherein the transmembrane region includes a CD8 transmembrane region, wherein the N-terminus of the CD8 transmembrane region is connected to the C-terminus of the CD8 hinge region of the extracellular region and is embedded in the cell membrane of the cell;
  • the intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, and the intracellular region includes a 4-1BB co-stimulatory factor domain and a CD3 ⁇ intracellular signal segment.
  • the tumor antigen is selected from embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., including at least one of MSLN (mesothelin), HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, PSCA, PSMA, GD2, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, ROR1, CD19, CD20, CD22, CD30, CD33, and BCMA.
  • the chemokine receptor includes at least one selected from CXCR2, CXCR6, CXCR1, CXCR3, CXCR4, CXCR5, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, CX3CR1, and XCR1.
  • the chemokine is CXCR2.
  • the expression of CXCR2 makes lymphocytes more capable of infiltrating into the tumor.
  • the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.1; the CD8 transmembrane region has an amino acid sequence as shown in SEQ ID NO.2; the 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO.3; the CD3 ⁇ intracellular signal segment has an amino acid sequence as shown in SEQ ID NO.4; the CD8 hinge region has an amino acid sequence as shown in SEQ ID NO:5; the CXCR2 has an amino acid sequence as shown in SEQ ID NO.6; and the CXCR6 has an amino acid sequence as shown in SEQ ID NO.7.
  • the first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the chemokine receptor in a transgenic cell, and the chimeric antigen receptor and the chemokine receptor are in a non-fused form.
  • the chimeric antigen receptor and the chemokine receptor after expression exist separately and can independently exert their respective functions of targeting and chemotactic tumor cells.
  • the first nucleic acid molecule and the second nucleic acid molecule are disposed on the same carrier.
  • the first nucleic acid molecule and the second nucleic acid molecule are disposed on different carriers.
  • the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the internal ribosome entry site has a nucleotide sequence shown in SEQ ID NO:8.
  • the third nucleic acid molecule is disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a self-cleaving peptide P2A, and the self-cleaving peptide P2A can be cleaved in the transgenic cell.
  • the self-cleaving peptide P2A has an amino acid sequence shown in SEQ ID NO:9.
  • the first promoter is operably connected to the first nucleic acid molecule; similarly, the second promoter is operably connected to the second nucleic acid molecule.
  • the nucleic acid molecule connected to the promoter can independently initiate the transcription process without interfering with the expression of other nucleic acid molecules.
  • the first promoter and the second promoter are independently selected from CMV, EF-1, and RSV promoters.
  • the "construct" described in the present application can be either the target gene sequence or the vector into which the target gene sequence is introduced.
  • the construct is a non-pathogenic viral vector.
  • the non-pathogenic virus is selected from retroviruses, lentiviruses, adenoviruses and other related viruses.
  • the non-pathogenic virus is a lentivirus.
  • the first nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 10;
  • the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:11.
  • the present invention provides an expression vector.
  • the expression vector comprises the construct described above. After the expression vector according to the embodiment of the present invention is introduced into a receptor cell, a chimeric antigen receptor and a chemokine receptor can be synchronously expressed on the membrane surface of the receptor cell.
  • the expression vector is a lentiviral vector.
  • the expression vector is an adenovirus vector, a non-pathogenic vector or a retrovirus vector.
  • the present invention provides a lentiviral vector.
  • the lentiviral vector has a nucleotide sequence shown in SEQ ID NO:12.
  • the chimeric antigen receptor and the chemokine receptor can be synchronously expressed on the membrane surface of the recipient cell.
  • the present invention provides a transgenic cell.
  • the transgenic cell carries the construct, expression vector, lentiviral vector or expresses a chimeric antigen receptor and a chemokine receptor as described above, and the chimeric antigen receptor and the chemokine receptor are in a non-fusion form.
  • the chimeric antigen receptor and the chemokine receptor are as defined in the first aspect of the present invention.
  • the present invention proposes a CAR-lymphocyte.
  • the CAR-lymphocyte carries the construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, and the lentiviral vector described in the third aspect of the present invention; or expresses a chimeric antigen receptor and a chemokine receptor, and the chimeric antigen receptor and the chemokine receptor are in a non-fusion form.
  • the chimeric antigen receptor and the chemokine receptor are as defined in the first aspect of the present invention.
  • the CAR-lymphocytes include at least one selected from NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, CAR-T cells, CAR-NKT cells, CAR- ⁇ T cells, and CAR-macrophages.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises The construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, the lentiviral vector described in the third aspect of the present invention, the transgenic cell described in the fourth aspect of the present invention, and the CAR-lymphocyte described in the fifth aspect of the present invention.
  • the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.
  • the present invention proposes a use of a pharmaceutical composition in the preparation of a drug.
  • the construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, the lentiviral vector described in the third aspect of the present invention, the transgenic cell described in the fourth aspect of the present invention, the CAR-lymphocyte described in the fifth aspect of the present invention, and the pharmaceutical composition described in the sixth aspect of the present invention can be used for immunotherapy of solid tumors or hematological tumors.
  • the solid tumor includes tangible tumors selected from those occurring in internal organs, including at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, and melanoma.
  • the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma in blood cells and hematopoietic system.
  • the present invention proposes a method for treating or preventing solid tumors or hematological tumors.
  • the method comprises administering to the subject at least one of the construct described in the first aspect of the present invention, the expression vector described in the second aspect, the lentiviral vector described in the third aspect, the transgenic cell described in the fourth aspect, the CAR-lymphocyte described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect.
  • a suitable dose of a construct, an expression vector, a lentiviral vector, a transgenic cell, a recombinant cell, a CAR-lymphocyte or a pharmaceutical composition is administered to the subject to inhibit the proliferation of solid tumor or hematological tumor cells.
  • the present invention proposes a construct described in the first aspect, an expression vector described in the second aspect, a lentiviral vector described in the third aspect, a transgenic cell described in the fourth aspect, a CAR-lymphocyte described in the fifth aspect, or a pharmaceutical composition described in the sixth aspect for use in the treatment or prevention of solid tumors or hematological tumors.
  • different treatment methods can be selected for different types of solid tumors or hematological tumors using the above-mentioned products, including one or more combinations of constructs, expression vectors, lentiviral vectors, transgenic cells, recombinant cells, CAR-lymphocytes or pharmaceutical compositions, to improve the pertinence and effectiveness of disease treatment.
  • Figure 1 is a structural schematic diagram of a CAR targeting MSLN according to Example 1 of the present invention and a structural schematic diagram of a CAR connected to P2A and CXCR2 or CXCR6 receptors, wherein SP represents a nucleotide sequence encoding a signal peptide, ⁇ -MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single-chain antibody, CD8 hinge+TM represents a nucleotide sequence encoding a CD8 hinge region and a transmembrane region, and 4-1BB represents a nucleotide sequence encoding The nucleotide sequence of the 4-1BB co-stimulatory factor domain, CD3 ⁇ represents the nucleotide sequence encoding the CD3Z intracellular region, P2A represents the nucleotide sequence encoding the P2A self-cleavage peptide, CXCR2 represents the nucleotide sequence encoding the full length of CXCR2, and CX
  • FIG2 is a graph showing the results of quantitative PCR detection of chemokine expression in pancreatic cancer cells according to Example 2 of the present invention.
  • FIG3 is a graph showing the result of ELISA detection of the secretion level of chemokine CXCL8 in pancreatic cancer tumor cells according to Example 2 of the present invention.
  • FIG4 is a graph showing the result of ELISA detection of the secretion level of chemokine CXCL16 in pancreatic cancer tumor cells according to Example 2 of the present invention.
  • Figure 5 is a graph showing the expression level detection results of CXCR2 in NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 2 of the present invention
  • Figure 6 is a graph showing the expression level detection results of CXCR6 in NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR6-NK-92 cells according to Example 2 of the present invention
  • Figure 7 is a graph showing the results of chemotaxis test of NK-92, anti-MSLN CAR-NK-92, anti-MSLN CAR-CXCR2-NK-92 and anti-MSLN CAR-CXCR6-NK-92 cells according to Example 2 of the present invention;
  • Figure 8 is a graph showing the results of in vitro killing ability test of NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 2 of the present invention
  • Figure 9 is a graph showing the anti-tumor ability test results of NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 3 of the present invention.
  • Figure 10 is a graph showing the results of the detection of NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells infiltrating into the tumor according to Example 3 of the present invention
  • the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • operably linked means that the foreign gene is linked to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their intended functions of regulating the transcription and translation of the foreign gene.
  • Commonly used vectors can be, for example, Virus vectors, plasmids, bacteriophages, etc.
  • the present application constructs a transgenic cell that simultaneously expresses a chimeric antigen receptor and an immunostimulatory molecule, wherein the antigens targeted by the chimeric antigen receptor include embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., including MSLN, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, PSCA, PSMA, GD2, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, ROR1, CD19, CD20, CD22, CD30, C At least one of D33 and BCMA is positioned on the surface of cells expressing the antigen, and the construct can express at least one of CXCR2, CXCR6, CXCR1, CXCR3, CXCR4, CXCR5, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, CX3CR1, and XCR1 receptors,
  • the CAR vector (anti-MSLN-CAR) sequence targeting mesothelin designed by the present invention comprises an extracellular segment (anti-MSLN scFv) targeting and recognizing MSLN, a CD8 hinge region and a transmembrane segment, a 4-1BB intracellular co-stimulatory factor domain and an intracellular signal transduction molecule CD3 ⁇ , and a CXCR2 gene fragment connected by a P2A self-cleaving peptide.
  • the schematic diagram of the gene element structure is shown in Figure 1.
  • anti-MSLN-CAR-CXCR2 vector Construction of anti-MSLN-CAR-CXCR2 vector: The anti-MSLN-CAR gene fragment was amplified using the pSBbi-MSLN CAR-GP plasmid as a template, and the CXCR2 or CXCR6 gene fragment was amplified from the pENTER-CXCR2 or pENTER-CXCR6 vector (Weizhen Biotechnology).
  • the CXCR2 or CXCR6 gene fragment obtained after enzyme digestion was connected to the pSBbi-MSLN CAR-GP plasmid to form anti-MSLN CAR-CXCR2 or anti-MSLN CAR- After the CXCR6 fragment was transformed, identified and sequenced correctly, the anti-MSLN CAR-CXCR2 or anti-MSLN CAR-CXCR6 fragment was amplified and inserted between the AsiSI and MIuI restriction sites of the lentiviral vector pLent-EF1a-P2A-GFP to construct the pLent-EF1a-anti-MSLN CAR-P2A-CXCR2-GFP or pLent-EF1a-anti-MSLN CAR-P2A-CXCR6-GFP vector, and the sequence was verified to be correct by sequencing.
  • N number of cells at the time of infection (approximately 1 ⁇ 105)
  • V volume of diluted virus added
  • NK-92 cells purchased from ATCC
  • NK-92 cells purchased from ATCC
  • ⁇ -MEM medium to resuspend the cells
  • cell density 5 ⁇ 10 5 /mL.
  • Example 2 Expression of CXCR2 or CXCR6 in Anti-MSLN CAR-NK cells, chemotaxis assay and Anti-MSLN CAR-CXCR2-NK cell killing function assay
  • cDNA was extracted from pancreatic cancer cell line Capan-2 cells (purchased from ATCC), and the expression of chemokines CXCL8 (IL-8), CXCL10, CXCL12, CXCL16 and CCL18 was detected by quantitative PCR.
  • the results showed that Capan-2 cells highly expressed CXCL8 and CXCL16 ( Figure 2). Further, the content of CXCL8 and CXCL16 secreted by pancreatic cancer cell line AsPC-1 cells (purchased from ATCC) and Capan-2 cells was detected by ELISA experiment.
  • NK cells The chemotactic ability of NK cells was detected by transwell assay. 3 ⁇ 10 5 NK-92 cells were placed in the upper chamber of the transwell chamber, and 600 ⁇ L of pancreatic cancer Capan-2 cell culture supernatant was added to the lower chamber. After culturing at 37°C for 4 hours, the cells in the lower chamber were collected and counted. The results showed that the migration rate of anti-MSLN CAR-CXCR2-NK-92 cells was 23.38% ⁇ 2.30%, which was significantly higher than that of anti-MSLN CAR-NK-92, anti-MSLN CAR-CXCR6-NK-92 and NK-92 cells (Figure 7).
  • NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells were used as effector cells, and the pancreatic cancer cell line Capan-2 was used as target cells.
  • the effector-target ratio was set to 5:1, 2.5:1 and 1.25:1.
  • the effector cells and target cells were co-incubated for 5 hours, and the LDH release method was used to detect the killing efficiency of the effector cells on the target cells.
  • the results showed that the killing efficiency of anti-MSLN CAR-CXCR2-NK-92 cells and anti-MSLN CAR-NK-92 cells on pancreatic cancer cells Capan-2 was significantly higher than that of unmodified NK-92 cells (Figure 8). This shows that the expression of CXCR2 does not affect the killing effect of CAR-NK92 cells.
  • Example 3 Anti-MSLN CAR-CXCR2NK-92 cells' anti-tumor ability in vivo and their ability to infiltrate into tumors
  • a pancreatic cancer xenograft model was established by subcutaneously implanting pancreatic cancer Capan-2 cells to observe the therapeutic effect of anti-MSLN CAR-CXCR2NK92 cells on pancreatic cancer.
  • Six-week-old female nude mice were selected and implanted with tumors subcutaneously in the armpits.
  • the tumor-bearing dose was 5 ⁇ 10 6 Capan-2 cells per mouse.
  • the mice were first randomly divided into a control group, a NK-92 cell-treated group, and a control group.
  • Treatment group, anti-MSLN CAR-NK-92 cell treatment group and anti-MSLN CAR-CXCR2-NK-92 cell treatment group were first randomly divided into a control group, a NK-92 cell-treated group, and a control group.
  • mice in the treatment group were injected with effector cells 5 ⁇ 10 6 /100 ⁇ L/mouse through the tail vein, and the control group was injected with an equal volume of 1 ⁇ PBS solution, once every week, and IL-2 (5 ⁇ 10 4 IU/mouse) was injected intraperitoneally every 3 days.
  • the tumor volume of the mice was measured every three days, and the treatment was observed for a total of 56 days.
  • the tumor tissue was taken and photographed, and the tumor volume was measured (Figure 9).
  • the results show that the tumor volume of the anti-MSLN CAR-CXCR2-NK-92 cell treatment group was significantly smaller than that of the other three groups, indicating that CAR-NK cells expressing CXCR2 have stronger anti-tumor ability in vivo.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

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Abstract

The present invention relates to preparation and use of a transgenic CAR-NK cell. The transgenic CAR-NK cell expresses a chimeric antigen receptor and a chemokine receptor, the chemokine receptor including at least one of CXCR2, CXCR6, CXCR1, CXCR3, CXCR4, CXCR5, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, CX3CR1, and XCR1.

Description

增强向肿瘤部位浸润能力的CAR-NK细胞制备及应用Preparation and application of CAR-NK cells with enhanced infiltration ability into tumor sites 技术领域Technical Field

本发明属于生物医药领域，具体地，本发明涉及一种CAR-NK细胞的制备及其在肿瘤治疗中的用途。The present invention belongs to the field of biomedicine, and specifically, the present invention relates to the preparation of CAR-NK cells and their use in tumor treatment.

背景技术Background technique

近年来，嵌合抗原受体T(chimeric antigen receptor T，CAR-T)细胞在血液恶性肿瘤治疗中取得了令人瞩目的效果。但是CAR-T细胞在临床应用中易产生细胞因子风暴、神经毒性、移植物抗宿主病(GVHD)等不良反应。另外，目前CAR-T细胞对实体瘤的治疗效果尚不理想，使得CAR-T细胞的临床应用仍面临挑战。CAR-NK细胞较CAR-T细胞具有安全性好的优势，一般不会引起细胞因子风暴和GVHD等副作用；NK细胞不需要抗原递呈、不受MHC限制，即可发挥直接的杀伤肿瘤细胞的作用；CAR-NK细胞可以以CAR依赖和NKR依赖等多种识别机制识别和杀伤肿瘤，抗瘤谱广。因此，CAR-NK细胞在抗肿瘤治疗中具有广泛的应用前景，已成为细胞免疫治疗研发领域的热点。然而，CAR-NK细胞治疗实体瘤面临的难题之一是NK细胞难以浸润到实体瘤内部，影响实体瘤患者NK细胞输注临床结果的重要制约因素是未能有足量的NK细胞迁移至肿瘤部位发挥杀瘤作用，将NK细胞有效运输到肿瘤部位是癌症免疫治疗成功的关键。因此，提高NK细胞向实体瘤内部浸润的能力成为基因修饰NK细胞的重要研发策略。In recent years, chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. However, CAR-T cells are prone to adverse reactions such as cytokine storm, neurotoxicity, and graft-versus-host disease (GVHD) in clinical applications. In addition, the current therapeutic effect of CAR-T cells on solid tumors is not ideal, which makes the clinical application of CAR-T cells still face challenges. CAR-NK cells have the advantage of good safety over CAR-T cells, and generally do not cause side effects such as cytokine storm and GVHD; NK cells do not require antigen presentation and are not restricted by MHC, and can play a direct role in killing tumor cells; CAR-NK cells can identify and kill tumors with multiple recognition mechanisms such as CAR dependence and NKR dependence, and have a wide anti-tumor spectrum. Therefore, CAR-NK cells have broad application prospects in anti-tumor treatment and have become a hot spot in the field of cell immunotherapy research and development. However, one of the challenges faced by CAR-NK cell therapy for solid tumors is that NK cells have difficulty infiltrating into solid tumors. An important factor affecting the clinical results of NK cell infusion in patients with solid tumors is the failure of sufficient NK cells to migrate to the tumor site to kill the tumor. Effectively transporting NK cells to the tumor site is the key to the success of cancer immunotherapy. Therefore, improving the ability of NK cells to infiltrate into solid tumors has become an important research and development strategy for gene-modified NK cells.

趋化因子和趋化因子受体在T或NK细胞等淋巴细胞向肿瘤迁移过程中起着重要作用。淋巴细胞可以通过细胞表面的趋化因子受体被肿瘤分泌的趋化因子吸引招募而被引导进入肿瘤部位。CXCR2为多种趋化因子(CXCL1、CXCL2、CXCL3、CXCL5、CXCL7和CXCL8)的受体。这些趋化因子在肝癌、肾癌、乳腺癌、前列腺癌、肺癌、黑色素瘤、结直肠癌、胰腺癌、卵巢癌等多种实体瘤高表达，且与肿瘤增殖、血管生成、侵袭转移及免疫抑制微环境的形成有关。Chemokines and chemokine receptors play an important role in the migration of lymphocytes such as T or NK cells to tumors. Lymphocytes can be attracted and recruited by chemokines secreted by tumors through chemokine receptors on the cell surface and guided into the tumor site. CXCR2 is a receptor for multiple chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL7 and CXCL8). These chemokines are highly expressed in a variety of solid tumors such as liver cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, pancreatic cancer, ovarian cancer, etc., and are related to tumor proliferation, angiogenesis, invasion and metastasis, and the formation of an immunosuppressive microenvironment.

发明内容Summary of the invention

本发明旨在至少在一定程度上解决相关技术中的技术问题之一。The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

发明人发现，CXCR2通常只表达于记忆B细胞和某些髓系细胞(如中性粒细胞和嗜碱性粒细胞)，人T细胞和NK细胞均不表达，因此，是T细胞和NK细胞难以浸润至肿瘤部位的原因。因此，如果在淋巴细胞中过表达CXCR2有望有效促进T细胞或NK细胞向肿瘤部位的聚集，有效提高T细胞或NK细胞的抗肿瘤效果。且发明人发现，当在CAR-淋巴细胞共表达CXCR2受体时，相比于共表达CXCR6受体，其向肿瘤的趋化和靶向能力更强，对肿瘤细胞的杀伤能力更强。 The inventors found that CXCR2 is usually only expressed in memory B cells and certain myeloid cells (such as neutrophils and basophils), and human T cells and NK cells do not express it. Therefore, it is difficult for T cells and NK cells to infiltrate the tumor site. Therefore, if CXCR2 is overexpressed in lymphocytes, it is expected to effectively promote the aggregation of T cells or NK cells to the tumor site, and effectively improve the anti-tumor effect of T cells or NK cells. And the inventors found that when CAR-lymphocytes co-express CXCR2 receptors, compared with co-expressing CXCR6 receptors, their chemotaxis and targeting ability to tumors are stronger, and their ability to kill tumor cells is stronger.

为此，本发明提出了一种CAR-淋巴细胞，其表面单链抗体可以识别所有实体瘤和血液瘤的肿瘤抗原并且可以在胞内表达CXCR2受体，增强淋巴细胞向肿瘤部位的迁移能力，有效提高淋巴细胞对于肿瘤的杀伤效果。To this end, the present invention proposes a CAR-lymphocyte, whose surface single-chain antibody can recognize tumor antigens of all solid tumors and hematological tumors and can express CXCR2 receptors intracellularly, thereby enhancing the ability of lymphocytes to migrate to the tumor site and effectively improving the killing effect of lymphocytes on tumors.

因此，在本发明的第一方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体包括第一核酸以及第二核酸。其中，第一核酸编码嵌合抗原受体，第二核酸编码趋化因子受体。Therefore, in the first aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, the construct comprises a first nucleic acid and a second nucleic acid. The first nucleic acid encodes a chimeric antigen receptor, and the second nucleic acid encodes a chemokine receptor.

根据本发明实施例，所述构建体能够同时编码嵌合抗原受体和趋化因子受体。将本发明实施例的构建体导入淋巴细胞，在淋巴细胞表面表达嵌合抗原受体和趋化因子受体。其中，所述嵌合抗原受体能够使得淋巴细胞靶向肿瘤抗原，定位至表达所述抗原的细胞表面。此外，所述趋化因子受体能够增加淋巴细胞向肿瘤部位的浸润能力，有效提高淋巴细胞的杀伤肿瘤效果。According to an embodiment of the present invention, the construct can encode a chimeric antigen receptor and a chemokine receptor at the same time. The construct of the embodiment of the present invention is introduced into lymphocytes, and the chimeric antigen receptor and the chemokine receptor are expressed on the surface of the lymphocytes. Among them, the chimeric antigen receptor can enable lymphocytes to target tumor antigens and localize to the surface of cells expressing the antigen. In addition, the chemokine receptor can increase the infiltration ability of lymphocytes to the tumor site, effectively improving the tumor-killing effect of lymphocytes.

根据本发明的实施例，上述构建体还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above construct may further include at least one of the following additional technical features:

根据本发明的实施例，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体和CD8铰链区，所述单链抗体包括重链可变区和轻链可变区，所述胞外区单链抗体可以特异性识别肿瘤抗原，所述单链抗体的C端与所述CD8铰链区的N端相连。According to an embodiment of the present invention, the chimeric antigen receptor includes: an extracellular region, the extracellular region includes a single-chain antibody and a CD8 hinge region, the single-chain antibody includes a heavy chain variable region and a light chain variable region, the extracellular region single-chain antibody can specifically recognize tumor antigens, and the C-terminus of the single-chain antibody is connected to the N-terminus of the CD8 hinge region.

跨膜区，所述跨膜区包括CD8跨膜区，所述CD8跨膜区的N端与所述胞外区的CD8铰链区的C端相连，并且嵌入到所述细胞细胞膜中；A transmembrane region, wherein the transmembrane region includes a CD8 transmembrane region, wherein the N-terminus of the CD8 transmembrane region is connected to the C-terminus of the CD8 hinge region of the extracellular region and is embedded in the cell membrane of the cell;

胞内区，所述胞内区的N端与所述跨膜区的C端相连，所述胞内区包括4-1BB共刺激因子结构域和CD3ζ胞内信号段。The intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, and the intracellular region includes a 4-1BB co-stimulatory factor domain and a CD3ζ intracellular signal segment.

根据本发明的实施例，所述肿瘤抗原选自胚胎性蛋白、糖蛋白抗原、鳞状细胞抗原等，包括MSLN(间皮素)、HER2、EGFR、GPC3、MUC1、CEA、CLDN 18.2、EpCAM、PSCA、PSMA、GD2、IL-13RA2、B7-H3、CD133、CD70、C-MET、FAP、TROP-2、ROR1、CD19、CD20、CD22、CD30、CD33、BCMA的至少之一。According to an embodiment of the present invention, the tumor antigen is selected from embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., including at least one of MSLN (mesothelin), HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, PSCA, PSMA, GD2, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, ROR1, CD19, CD20, CD22, CD30, CD33, and BCMA.

根据本发明的实施例，所述趋化因子受体包括选自CXCR2、CXCR6、CXCR1、CXCR3、CXCR4、CXCR5、CXCR7、CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR10、CX3CR1、XCR1中的至少之一。According to an embodiment of the present invention, the chemokine receptor includes at least one selected from CXCR2, CXCR6, CXCR1, CXCR3, CXCR4, CXCR5, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, CX3CR1, and XCR1.

根据本发明的实施例，所述趋化因子为CXCR2。CXCR2的表达使得淋巴细胞向肿瘤内部的浸润能力更强。According to an embodiment of the present invention, the chemokine is CXCR2. The expression of CXCR2 makes lymphocytes more capable of infiltrating into the tumor.

根据本发明的实施例，所述单链抗体具有如SEQ ID NO.1所示的氨基酸序列；所述CD8跨膜区具有如SEQ ID NO.2所示的氨基酸序列；所述4-1BB共刺激因子结构域具有如SEQ ID NO.3所示的氨基酸序列；所述CD3ζ胞内信号段具有如SEQ ID NO.4所示的氨基酸序列；所述CD8铰链区具有SEQ ID NO:5所示的氨基酸序列；所述CXCR2具有如SEQ ID NO.6所示的氨基酸序列；所述CXCR6具有如SEQ ID NO.7所示的氨基酸序列。
According to an embodiment of the present invention, the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.1; the CD8 transmembrane region has an amino acid sequence as shown in SEQ ID NO.2; the 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO.3; the CD3ζ intracellular signal segment has an amino acid sequence as shown in SEQ ID NO.4; the CD8 hinge region has an amino acid sequence as shown in SEQ ID NO:5; the CXCR2 has an amino acid sequence as shown in SEQ ID NO.6; and the CXCR6 has an amino acid sequence as shown in SEQ ID NO.7.

根据本发明的实施例，所述第一核酸分子与所述第二核酸分子被设置为转基因细胞中表达所述嵌合抗原受体以及所述趋化因子受体，并且所述嵌合抗原受体以及所述趋化因子受体呈非融合形式。根据本发明的实施例，表达后的所述嵌合抗原受体以及所述趋化因子受体单独存在，可分别独立地发挥各自地靶向和趋化肿瘤细胞的功能。According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the chemokine receptor in a transgenic cell, and the chimeric antigen receptor and the chemokine receptor are in a non-fused form. According to an embodiment of the present invention, the chimeric antigen receptor and the chemokine receptor after expression exist separately and can independently exert their respective functions of targeting and chemotactic tumor cells.

根据本发明的实施例，所述第一核酸分子与所述第二核酸分子设置于同一载体上。According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are disposed on the same carrier.

根据本发明的实施例，所述第一核酸分子与所述第二核酸分子设置于不同的载体上。According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are disposed on different carriers.

根据本发明的实施例，所述内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：8所示的核苷酸序列。

According to an embodiment of the present invention, the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the internal ribosome entry site has a nucleotide sequence shown in SEQ ID NO:8.

根据本发明的实施例，所述第三核酸分子，设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码自剪切肽P2A，所述自剪切肽P2A能够在所述转基因细胞中被切割。According to an embodiment of the present invention, the third nucleic acid molecule is disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a self-cleaving peptide P2A, and the self-cleaving peptide P2A can be cleaved in the transgenic cell.

根据本发明的实施例，所述自剪切肽P2A具有SEQ ID NO：9所示的氨基酸序列。
According to an embodiment of the present invention, the self-cleaving peptide P2A has an amino acid sequence shown in SEQ ID NO:9.

根据本发明的实施例，所述第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；同样的，第二启动子，所述第二启动子与所述第二核酸分子可操作地连接。这样与启动子相连的核酸分子可以单独启动转录过程，与其他核酸分子的表达互不干扰。According to an embodiment of the present invention, the first promoter is operably connected to the first nucleic acid molecule; similarly, the second promoter is operably connected to the second nucleic acid molecule. In this way, the nucleic acid molecule connected to the promoter can independently initiate the transcription process without interfering with the expression of other nucleic acid molecules.

根据本发明的实施例，所述第一启动子和所述第二启动子分别独立地选自CMV,EF-1,RSV启动子。According to an embodiment of the present invention, the first promoter and the second promoter are independently selected from CMV, EF-1, and RSV promoters.

需要说明的是，本申请所述的“构建体”既可以是目的基因序列也可以是导入目的基因序列的载体。It should be noted that the "construct" described in the present application can be either the target gene sequence or the vector into which the target gene sequence is introduced.

根据本发明的实施例，所述构建体是非致病性病毒载体。According to an embodiment of the present invention, the construct is a non-pathogenic viral vector.

根据本发明的实施例，所述非致病性病毒选自反转录病毒、慢病毒和腺病毒等相关病毒。According to an embodiment of the present invention, the non-pathogenic virus is selected from retroviruses, lentiviruses, adenoviruses and other related viruses.

根据本发明的实施例，所述非致病性病毒为慢病毒。According to an embodiment of the present invention, the non-pathogenic virus is a lentivirus.

根据本发明的实施例，所述第一核酸分子具有SEQ ID NO：10所示的核苷酸序列；

According to an embodiment of the present invention, the first nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 10;

根据本发明的实施例，所述第二核酸分子具有SEQ ID NO：11所示的核苷酸序列。

According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:11.

在本发明的第二方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体包括上文所述的构建体。根据本发明实施例的表达载体导入受体细胞后，可在受体细胞的膜表面同步表达嵌合抗原受体以及趋化因子受体。In the second aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the construct described above. After the expression vector according to the embodiment of the present invention is introduced into a receptor cell, a chimeric antigen receptor and a chemokine receptor can be synchronously expressed on the membrane surface of the receptor cell.

根据本发明的实施例，所述表达载体为慢病毒病毒载体。According to an embodiment of the present invention, the expression vector is a lentiviral vector.

根据本发明的实施例，所述表达载体为腺病毒载体、非致病性载体或逆转录病毒载体。According to an embodiment of the present invention, the expression vector is an adenovirus vector, a non-pathogenic vector or a retrovirus vector.

在本发明的第三方面，本发明提出了一种慢病毒载体。根据本发明的实施例，所述慢病毒载体具有SEQ ID NO：12所示的核苷酸序列。

In a third aspect of the present invention, the present invention provides a lentiviral vector. According to an embodiment of the present invention, the lentiviral vector has a nucleotide sequence shown in SEQ ID NO:12.

根据本发明实施例的慢病毒载体导入受体细胞后，可在受体细胞的膜表面同步表达嵌合抗原受体以及趋化因子受体。After the lentiviral vector according to the embodiment of the present invention is introduced into the recipient cell, the chimeric antigen receptor and the chemokine receptor can be synchronously expressed on the membrane surface of the recipient cell.

在本发明的第四方面，本发明提出了一种转基因细胞。根据本发明的实施例，所述转基因细胞携带上文所述的构建体、表达载体、慢病毒载体或表达嵌合抗原受体以及趋化因子受体，并且所述嵌合抗原受体以及所述趋化因子受体呈非融合形式。In a fourth aspect of the present invention, the present invention provides a transgenic cell. According to an embodiment of the present invention, the transgenic cell carries the construct, expression vector, lentiviral vector or expresses a chimeric antigen receptor and a chemokine receptor as described above, and the chimeric antigen receptor and the chemokine receptor are in a non-fusion form.

根据本发明的实施例，所述嵌合抗原受体以及所述趋化因子受体如本发明第一方面所限定的。According to an embodiment of the present invention, the chimeric antigen receptor and the chemokine receptor are as defined in the first aspect of the present invention.

在本发明的第五方面，本发明提出了一种CAR-淋巴细胞。根据本发明的实施例，所述CAR-淋巴细胞携带本发明第一方面所述的构建体、本发明第二方面所述的表达载体、本发明第三方面所述的慢病毒载体；或表达嵌合抗原受体以及趋化因子受体，并且所述嵌合抗原受体以及所述趋化因子受体呈非融合形式。In the fifth aspect of the present invention, the present invention proposes a CAR-lymphocyte. According to an embodiment of the present invention, the CAR-lymphocyte carries the construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, and the lentiviral vector described in the third aspect of the present invention; or expresses a chimeric antigen receptor and a chemokine receptor, and the chimeric antigen receptor and the chemokine receptor are in a non-fusion form.

根据本发明的实施例，所述嵌合抗原受体以及所述趋化因子受体如本发明第一方面所限定的。According to an embodiment of the present invention, the chimeric antigen receptor and the chemokine receptor are as defined in the first aspect of the present invention.

根据本发明的实施例，所述CAR-淋巴细胞包括选自NK-92细胞，外周血NK细胞、脐带血NK细胞、iPSC、CAR-T细胞、CAR-NKT细胞、CAR-γδT细胞、CAR-巨噬细胞中的至少之一。According to an embodiment of the present invention, the CAR-lymphocytes include at least one selected from NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, CAR-T cells, CAR-NKT cells, CAR-γδT cells, and CAR-macrophages.

在本发明的第六方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括 本发明第一方面所述的构建体、本发明第二方面所述的表达载体、本发明第三方面所述的慢病毒载体、本发明第四方面所述的转基因细胞以及本发明第五方面所述的CAR-淋巴细胞。In a sixth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises The construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, the lentiviral vector described in the third aspect of the present invention, the transgenic cell described in the fourth aspect of the present invention, and the CAR-lymphocyte described in the fifth aspect of the present invention.

根据本发明的实施例，所述药物组合物进一步包括：药学上可接受的辅料。According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

在本发明的第七方面，本发明提出了一种药物组合物在制备药物中的用途。根据本发明的实施例，所述本发明第一方面所述的构建体、本发明第二方面所述的表达载体、本发明第三方面所述的慢病毒载体、本发明第四方面所述的转基因细胞、本发明第五方面所述的CAR-淋巴细胞以及本发明第六方面所述的药物组合物可用于实体瘤或血液瘤的免疫治疗。In the seventh aspect of the present invention, the present invention proposes a use of a pharmaceutical composition in the preparation of a drug. According to an embodiment of the present invention, the construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, the lentiviral vector described in the third aspect of the present invention, the transgenic cell described in the fourth aspect of the present invention, the CAR-lymphocyte described in the fifth aspect of the present invention, and the pharmaceutical composition described in the sixth aspect of the present invention can be used for immunotherapy of solid tumors or hematological tumors.

根据本发明的实施例，所述实体瘤包括选自发生在脏器中的有形瘤，包括胰腺癌、卵巢癌、间皮瘤、肝癌、胆管癌、胃癌、结直肠癌、食管癌、肺癌、头颈癌、宫颈癌、脑胶质瘤、肾癌、乳腺癌、前列腺癌、黑色素瘤的至少之一。According to an embodiment of the present invention, the solid tumor includes tangible tumors selected from those occurring in internal organs, including at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, and melanoma.

根据本发明的实施例，所述血液瘤包括选自血细胞和造血系统内的急性髓系白血病、急性淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、何杰金氏淋巴瘤、非何杰金氏淋巴瘤、多发性骨髓瘤的至少之一。According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma in blood cells and hematopoietic system.

在本发明的第八方面，本发明提出了一种治疗或预防实体瘤或血液瘤的方法。根据本发明的实施例，所述方法包括向受试者施用本发明第一方面所述的构建体、第二方面所述的表达载体、第三方面所述的慢病毒载体、第四方面所述的转基因细胞、第五方面所述的CAR-淋巴细胞或第六方面所述的药物组合物中的至少之一。根据本发明的一些具体实施例，通过向受试者施用合适剂量的构建体、表达载体、慢病毒载体、转基因细胞、重组细胞、CAR-淋巴细胞或药物组合物用以抑制实体瘤或血液瘤细胞的增殖。In the eighth aspect of the present invention, the present invention proposes a method for treating or preventing solid tumors or hematological tumors. According to an embodiment of the present invention, the method comprises administering to the subject at least one of the construct described in the first aspect of the present invention, the expression vector described in the second aspect, the lentiviral vector described in the third aspect, the transgenic cell described in the fourth aspect, the CAR-lymphocyte described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect. According to some specific embodiments of the present invention, a suitable dose of a construct, an expression vector, a lentiviral vector, a transgenic cell, a recombinant cell, a CAR-lymphocyte or a pharmaceutical composition is administered to the subject to inhibit the proliferation of solid tumor or hematological tumor cells.

在本发明的第九方面，本发明提出了一种第一方面所述的构建体、第二方面所述的表达载体、第三方面所述的慢病毒载体、第四方面所述的转基因细胞、第五方面所述的CAR-淋巴细胞或第六方面所述的药物组合物在治疗或预防实体瘤或血液瘤中的用途。根据本发明的一些具体实施例，利用上述产品针对不同类型的实体瘤或血液瘤，可以选择使用不同的治疗手段，包括构建体、表达载体、慢病毒载体、转基因细胞、重组细胞、CAR-淋巴细胞或药物组合物中的一种或多种组合，以提高疾病治疗的针对性和有效性。In the ninth aspect of the present invention, the present invention proposes a construct described in the first aspect, an expression vector described in the second aspect, a lentiviral vector described in the third aspect, a transgenic cell described in the fourth aspect, a CAR-lymphocyte described in the fifth aspect, or a pharmaceutical composition described in the sixth aspect for use in the treatment or prevention of solid tumors or hematological tumors. According to some specific embodiments of the present invention, different treatment methods can be selected for different types of solid tumors or hematological tumors using the above-mentioned products, including one or more combinations of constructs, expression vectors, lentiviral vectors, transgenic cells, recombinant cells, CAR-lymphocytes or pharmaceutical compositions, to improve the pertinence and effectiveness of disease treatment.

本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1是根据本发明实施例1的靶向MSLN的CAR的结构模式图以及连接P2A及CXCR2或CXCR6受体的CAR的结构模式图，其中，SP表示编码信号肽的核苷酸序列，α-MSLN-scFv表示编码抗MSLN单链抗体的核苷酸序列，CD8 hinge+TM表示编码CD8铰链区和跨膜区的核苷酸序列，4-1BB表示编码 4-1BB共刺激因子结构域的核苷酸序列，CD3ζ表示编码CD3Z胞内区的核苷酸序列，P2A表示编码P2A自剪切肽的核苷酸序列，CXCR2表示编码CXCR2全长的核苷酸序列，CXCR6表示编码CXCR6全长的核苷酸序列；Figure 1 is a structural schematic diagram of a CAR targeting MSLN according to Example 1 of the present invention and a structural schematic diagram of a CAR connected to P2A and CXCR2 or CXCR6 receptors, wherein SP represents a nucleotide sequence encoding a signal peptide, α-MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single-chain antibody, CD8 hinge+TM represents a nucleotide sequence encoding a CD8 hinge region and a transmembrane region, and 4-1BB represents a nucleotide sequence encoding The nucleotide sequence of the 4-1BB co-stimulatory factor domain, CD3ζ represents the nucleotide sequence encoding the CD3Z intracellular region, P2A represents the nucleotide sequence encoding the P2A self-cleavage peptide, CXCR2 represents the nucleotide sequence encoding the full length of CXCR2, and CXCR6 represents the nucleotide sequence encoding the full length of CXCR6;

图2是根据本发明实施例2的定量PCR检测胰腺癌细胞趋化因子表达的结果图；FIG2 is a graph showing the results of quantitative PCR detection of chemokine expression in pancreatic cancer cells according to Example 2 of the present invention;

图3是根据本发明实施例2的ELISA检测胰腺癌肿瘤细胞中趋化因子CXCL8分泌水平的结果图；FIG3 is a graph showing the result of ELISA detection of the secretion level of chemokine CXCL8 in pancreatic cancer tumor cells according to Example 2 of the present invention;

图4是根据本发明实施例2的ELISA检测胰腺癌肿瘤细胞中趋化因子CXCL16分泌水平的结果图；FIG4 is a graph showing the result of ELISA detection of the secretion level of chemokine CXCL16 in pancreatic cancer tumor cells according to Example 2 of the present invention;

图5是根据本发明实施例2的NK-92、anti-MSLN CAR-NK-92和anti-MSLN CAR-CXCR2-NK-92细胞CXCR2的表达水平检测结果图；Figure 5 is a graph showing the expression level detection results of CXCR2 in NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 2 of the present invention;

图6是根据本发明实施例2的NK-92、anti-MSLN CAR-NK-92和anti-MSLN CAR-CXCR6-NK-92细胞CXCR6的表达水平检测结果图；Figure 6 is a graph showing the expression level detection results of CXCR6 in NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR6-NK-92 cells according to Example 2 of the present invention;

图7是根据本发明实施例2的NK-92、anti-MSLN CAR-NK-92、anti-MSLN CAR-CXCR2-NK-92与anti-MSLN CAR-CXCR6-NK-92细胞趋化能力检测结果图；Figure 7 is a graph showing the results of chemotaxis test of NK-92, anti-MSLN CAR-NK-92, anti-MSLN CAR-CXCR2-NK-92 and anti-MSLN CAR-CXCR6-NK-92 cells according to Example 2 of the present invention;

图8是根据本发明实施例2的NK-92、anti-MSLN CAR-NK-92和anti-MSLN CAR-CXCR2-NK-92细胞体外杀伤能力检测结果图；Figure 8 is a graph showing the results of in vitro killing ability test of NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 2 of the present invention;

图9是根据本发明实施例3的NK-92、anti-MSLN CAR-NK-92和anti-MSLN CAR-CXCR2-NK-92细胞抗肿瘤能力检测结果图；Figure 9 is a graph showing the anti-tumor ability test results of NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 3 of the present invention;

图10是根据本发明实施例3的NK-92、anti-MSLN CAR-NK-92和anti-MSLN CAR-CXCR2-NK-92细胞向肿瘤内部浸润检测结果图；Figure 10 is a graph showing the results of the detection of NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells infiltrating into the tumor according to Example 3 of the present invention;

具体实施方式Detailed ways

下面详细描述本发明的实施例，所述实施例的示例在附图中示出。其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

在对本发明描述的过程中，对于本文中有关的术语进行了解释和说明，这些解释和说明仅仅是为了方便对于方案的理解，并不能看做是对本发明保护方案的限制。In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。常用的载体例如可以为病 毒载体、质粒、噬菌体等等。根据本发明的一些具体实施例的表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前面所述的分离的核酸的表达，进而实现所述分离的核酸编码的蛋白质的体外大量获得。In this article, "operably linked" means that the foreign gene is linked to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors can be, for example, Virus vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into appropriate recipient cells, they can effectively express the aforementioned isolated nucleic acid under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of protein encoded by the isolated nucleic acid.

本申请构建了一种同时表达嵌合抗原受体和免疫刺激分子的转基因细胞，其中，所述嵌合抗原受体靶向的抗原包括胚胎性蛋白、糖蛋白抗原、鳞状细胞抗原等，包括MSLN、HER2、EGFR、GPC3、MUC1、CEA、CLDN 18.2、EpCAM、PSCA、PSMA、GD2、IL-13RA2、B7-H3、CD133、CD70、C-MET、FAP、TROP-2、ROR1、CD19、CD20、CD22、CD30、CD33、BCMA中的至少之一，定位至表达所述抗原的细胞表面，所述构建体能够表达CXCR2、CXCR6、CXCR1、CXCR3、CXCR4、CXCR5、CXCR7、CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR10、CX3CR1、XCR1受体至少之一，提高CAR-淋巴细胞向实体瘤内部浸润的能力，同时增强CAR淋巴细胞的抗肿瘤活性，并应用于实体瘤和血液瘤的治疗。The present application constructs a transgenic cell that simultaneously expresses a chimeric antigen receptor and an immunostimulatory molecule, wherein the antigens targeted by the chimeric antigen receptor include embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., including MSLN, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, PSCA, PSMA, GD2, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, ROR1, CD19, CD20, CD22, CD30, C At least one of D33 and BCMA is positioned on the surface of cells expressing the antigen, and the construct can express at least one of CXCR2, CXCR6, CXCR1, CXCR3, CXCR4, CXCR5, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, CX3CR1, and XCR1 receptors, thereby improving the ability of CAR-lymphocytes to infiltrate solid tumors, while enhancing the anti-tumor activity of CAR lymphocytes, and is used in the treatment of solid tumors and hematological tumors.

下面将更详细地描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention will be described in more detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

需要说明的是，以下实施中所述的“质粒”与“载体”具有相同的意义，可互换使用。It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

实施例1：Anti-MSLN-CAR-CXCR2-NK细胞的制备Example 1: Preparation of Anti-MSLN-CAR-CXCR2-NK cells

1.CAR表达质粒的构建1. Construction of CAR expression plasmid

本发明设计的靶向间皮素的CAR载体(anti-MSLN-CAR)序列，包含靶向识别MSLN的胞外段(anti-MSLN scFv)、CD8铰链(Hinge)区与跨膜段、4-1BB胞内共刺激因子结构域与胞内信号转导分子CD3ζ及由P2A自剪切肽连接的CXCR2基因片段。基因元件结构示意图见图1。The CAR vector (anti-MSLN-CAR) sequence targeting mesothelin designed by the present invention comprises an extracellular segment (anti-MSLN scFv) targeting and recognizing MSLN, a CD8 hinge region and a transmembrane segment, a 4-1BB intracellular co-stimulatory factor domain and an intracellular signal transduction molecule CD3ζ, and a CXCR2 gene fragment connected by a P2A self-cleaving peptide. The schematic diagram of the gene element structure is shown in Figure 1.

anti-MSLN-CAR-CXCR2载体的构建：以pSBbi-MSLN CAR-GP质粒为模板扩增anti-MSLN-CAR基因片段，从pENTER-CXCR2或pENTER-CXCR6载体(维真生物)扩增CXCR2或CXCR6基因片段，酶切后获得的CXCR2或CXCR6基因片段连接到pSBbi-MSLN CAR-GP质粒上，连接成anti-MSLN CAR-CXCR2或anti-MSLN CAR-CXCR6片段，经转化、鉴定、测序正确后，扩增anti-MSLN CAR-CXCR2或anti-MSLN CAR-CXCR6片段并将之插入至慢病毒载体pLent-EF1a-P2A-GFP的AsiSI和MIuI酶切位点之间，构建pLent-EF1a-anti-MSLN CAR-P2A-CXCR2-GFP或pLent-EF1a-anti-MSLN CAR-P2A-CXCR6-GFP载体，测序验证序列正确。Construction of anti-MSLN-CAR-CXCR2 vector: The anti-MSLN-CAR gene fragment was amplified using the pSBbi-MSLN CAR-GP plasmid as a template, and the CXCR2 or CXCR6 gene fragment was amplified from the pENTER-CXCR2 or pENTER-CXCR6 vector (Weizhen Biotechnology). The CXCR2 or CXCR6 gene fragment obtained after enzyme digestion was connected to the pSBbi-MSLN CAR-GP plasmid to form anti-MSLN CAR-CXCR2 or anti-MSLN CAR- After the CXCR6 fragment was transformed, identified and sequenced correctly, the anti-MSLN CAR-CXCR2 or anti-MSLN CAR-CXCR6 fragment was amplified and inserted between the AsiSI and MIuI restriction sites of the lentiviral vector pLent-EF1a-P2A-GFP to construct the pLent-EF1a-anti-MSLN CAR-P2A-CXCR2-GFP or pLent-EF1a-anti-MSLN CAR-P2A-CXCR6-GFP vector, and the sequence was verified to be correct by sequencing.

1.2慢病毒的包装及病毒液浓缩1.2 Lentivirus packaging and virus liquid concentration

取处于对数生长期的293T细胞5×106接种于10cm的培养皿中，加入10mL DMEM培养基，37℃、5％CO2培养箱中培养过夜。待细胞密度达到80％时，更换10mL新鲜的DMEM培养基，继续置于培养箱中培养。配制慢病毒包装体系：将6μg psPAX2质粒，3μg pMD2.G质粒与6μg目的基因载体质粒加入至体积为250μL无血清DMEM培养基中，混合均匀，配制DNA混合液；将15mL加到 体积为235μL的无血清DMEM培养基中，混合均匀。将混合液一次性加入到DNA混合液中，静置混匀，室温孵育15min。将混合液加入293T细胞培养皿中。24h后进行换液，同时观察荧光阳性率。将培养皿放回37℃，5％CO2培养箱中，48h后收取细胞上清，400×g离心5min，去除细胞碎片，将上清用0.45mm的滤头过滤至新的50ml离心管中。加入5×PEG8000溶液，上下颠倒离心管混合均匀，放于4℃冰箱中过夜。将离心管置于4℃，4000×g离心机中离心20min，弃上清，加适量无血清DMEM培养基重悬病毒沉淀，分装至EP管中，放于-80℃冰箱中保存。Take 5×10 6 293T cells in the logarithmic growth phase and inoculate them in a 10 cm culture dish, add 10 mL of DMEM medium, and culture them in a 37°C, 5% CO 2 incubator overnight. When the cell density reaches 80%, replace it with 10 mL of fresh DMEM medium and continue to culture in the incubator. Prepare the lentiviral packaging system: add 6 μg of psPAX2 plasmid, 3 μg of pMD2.G plasmid and 6 μg of target gene vector plasmid to 250 μL of serum-free DMEM medium, mix well, and prepare a DNA mixture; add 15 mL Add to Add 235 μL of serum-free DMEM medium and mix well. Add the mixture to the DNA mixture at one time, let it stand to mix, and incubate at room temperature for 15 minutes. Add the mixture to the 293T cell culture dish. Change the solution after 24 hours, and observe the fluorescence positivity rate at the same time. Put the culture dish back into the 37℃, 5% CO2 incubator, collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove the cell debris, and filter the supernatant with a 0.45mm filter into a new 50ml centrifuge tube. Add 5×PEG8000 solution, mix evenly by turning the centrifuge tube upside down, and place it in a 4℃ refrigerator overnight. Place the centrifuge tube in a 4℃, 4000×g centrifuge for 20 minutes, discard the supernatant, add an appropriate amount of serum-free DMEM medium to resuspend the virus precipitate, divide it into EP tubes, and store it in a -80℃ refrigerator.

1.3慢病毒滴度检测1.3 Lentiviral titer detection

取对数生长期的293T细胞，调整浓度为1×105/mL。取24孔板，每孔加1mL细胞悬液(1×105/孔)，设置3个加入病毒体积梯度。置于37℃、5％CO2培养箱过夜培养。先将浓缩病毒液进行10倍稀释：取1.5mL EP管，吸取60μL病毒浓缩液到EP管中，用540μL DMEM培养基进行稀释，混合均匀。对293T细胞用新鲜DMEM培养基进行换液，分别吸取5μL、50μL与500μL稀释后病毒液加到相应的孔中，做好标记，然后将培养板放回37℃、5％CO2培养箱中。24h后，吸弃孔板中病毒液，再加入1mL新鲜DMEM培养基。72h后，用胰酶消化收获细胞，使用流式仪检测293T细胞GFP表达率，根据下列公式换算病毒滴度：Take 293T cells in the logarithmic growth phase and adjust the concentration to 1×10 5 /mL. Take a 24-well plate, add 1mL of cell suspension (1×10 5 /well) to each well, and set up 3 gradients of virus volume. Place in a 37°C, 5% CO 2 incubator for overnight culture. First dilute the concentrated virus solution 10 times: take a 1.5mL EP tube, pipette 60μL of concentrated virus solution into the EP tube, dilute with 540μL DMEM culture medium, and mix evenly. Replace the 293T cells with fresh DMEM culture medium, pipette 5μL, 50μL and 500μL of diluted virus solution into the corresponding wells, mark them, and then put the culture plate back into the 37°C, 5% CO 2 incubator. After 24h, aspirate the virus solution in the well plate and add 1mL of fresh DMEM culture medium. After 72 hours, the cells were harvested by trypsin digestion, and the GFP expression rate of 293T cells was detected by flow cytometry. The virus titer was calculated according to the following formula:

滴度(TU/mL)＝(C×N×D×1000)/VTiter (TU/mL) = (C×N×D×1000)/V

其中：C＝流式检测的GFP阳性率Where: C = GFP positive rate detected by flow cytometry

N＝感染时细胞的数目(约为1×105)N = number of cells at the time of infection (approximately 1×105)

D＝病毒载体的稀释倍数D = dilution factor of viral vector

V＝加入的稀释病毒的体积数V = volume of diluted virus added

1.4慢病毒感染人NK细胞1.4 Lentivirus infection of human NK cells

取处于生长对数期的NK-92细胞(购自ATCC)，于100×g离心5min收获细胞，加入适量α-MEM培养基重悬细胞，调整细胞密度为5×105个/mL。在24孔板中分别接入5×105个NK-92细胞，病毒浓缩液1mL与鱼精蛋白(购自索莱宝，终浓度8μg/mL)，混合均匀，置于37℃、5％CO2培养箱中培养。24h后，观察细胞状态，换液，将感染的细胞转移入EP管中，100×g离心5min，加入少量新鲜α-MEM培养基重悬细胞，将细胞转入细胞培养瓶中，加入10mL新鲜α-MEM培养基和IL-2(终浓度为200IU/mL)继续培养48h。将细胞转移入流式管中，加入3mL 1×PBS溶液，100×g离心5min，弃上清，弹起细胞沉淀，使用1×PBS溶液再次洗涤一遍。使用流式仪检测GFP的表达率。继续扩大培养，调整感染后NK-92细胞的状态进行扩增。将感染后的NK-92细胞通过流式仪分选GFP阳性的CAR-NK-92细胞，用于后期实验。Take NK-92 cells (purchased from ATCC) in the logarithmic growth phase, harvest the cells by centrifugation at 100×g for 5min, add an appropriate amount of α-MEM medium to resuspend the cells, and adjust the cell density to 5×10 5 /mL. Inoculate 5×10 5 NK-92 cells in 24-well plates, add 1mL of virus concentrate and protamine (purchased from Solebao, final concentration 8μg/mL), mix evenly, and place in a 37°C, 5% CO 2 incubator for culture. After 24h, observe the cell state, change the medium, transfer the infected cells into an EP tube, centrifuge at 100×g for 5min, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the cells into a cell culture flask, add 10mL of fresh α-MEM medium and IL-2 (final concentration of 200IU/mL) and continue to culture for 48h. Transfer the cells into a flow tube, add 3 mL of 1×PBS solution, centrifuge at 100×g for 5 min, discard the supernatant, flick off the cell pellet, and wash again with 1×PBS solution. Use flow cytometry to detect the expression rate of GFP. Continue to expand the culture and adjust the state of the infected NK-92 cells for amplification. Use flow cytometry to sort the infected NK-92 cells for GFP-positive CAR-NK-92 cells for later experiments.

实施例2：Anti-MSLN CAR-NK细胞CXCR2或CXCR6的表达、趋化能力测定以及Anti-MSLN  CAR-CXCR2-NK细胞杀伤功能测定Example 2: Expression of CXCR2 or CXCR6 in Anti-MSLN CAR-NK cells, chemotaxis assay and Anti-MSLN CAR-CXCR2-NK cell killing function assay

2.1胰腺癌肿瘤细胞中高表达趋化因子检测2.1 Detection of highly expressed chemokines in pancreatic cancer cells

取胰腺癌肿瘤细胞系Capan-2细胞(购自ATCC)提取cDNA，通过定量PCR检测趋化因子CXCL8(IL-8)、CXCL10、CXCL12、CXCL16与CCL18的表达，结果显示Capan-2细胞高表达CXCL8与CXCL16(图2)。进一步，通过ELISA实验检测胰腺癌肿瘤细胞系AsPC-1细胞(购自ATCC)与Capan-2细胞分泌CXCL8与CXCL16的含量。结果显示AsPC-1与Capan-2细胞均分泌CXCL8与CXCL16，且Capan-2细胞分泌CXCL8与CXCL16的含量明显高于AsPC-1细胞(图3与图4)。因此，选择CXCL8与CXCL16对应的趋化因子受体CXCR2与CXCR6作为靶点以增强NK细胞向高表达CXCL8和CXCL16趋化因子的胰腺癌等肿瘤的趋化能力。cDNA was extracted from pancreatic cancer cell line Capan-2 cells (purchased from ATCC), and the expression of chemokines CXCL8 (IL-8), CXCL10, CXCL12, CXCL16 and CCL18 was detected by quantitative PCR. The results showed that Capan-2 cells highly expressed CXCL8 and CXCL16 (Figure 2). Further, the content of CXCL8 and CXCL16 secreted by pancreatic cancer cell line AsPC-1 cells (purchased from ATCC) and Capan-2 cells was detected by ELISA experiment. The results showed that both AsPC-1 and Capan-2 cells secreted CXCL8 and CXCL16, and the content of CXCL8 and CXCL16 secreted by Capan-2 cells was significantly higher than that of AsPC-1 cells (Figures 3 and 4). Therefore, the chemokine receptors CXCR2 and CXCR6 corresponding to CXCL8 and CXCL16 were selected as targets to enhance the chemotactic ability of NK cells to pancreatic cancer and other tumors that highly express CXCL8 and CXCL16 chemokines.

2.2 CAR-NK-92细胞CXCR2或CXCR6的表达2.2 Expression of CXCR2 or CXCR6 in CAR-NK-92 cells

流式细胞术分别检测anti-MSLN CAR-CXCR2-NK-92与anti-MSLN CAR-CXCR6-NK-92细胞CXCR2或CXCR6的表达，结果显示，CXCR2在anti-MSLN CAR-CXCR2-NK-92细胞上表达率为99.0％(图5)，CXCR6在anti-MSLN CAR-CXCR6NK-92细胞上表达率为90.5％(图6)，而未修饰的NK-92细胞(不表达CXCR2、CXCR6和CAR的细胞)和anti-MSLN CAR-NK-92细胞基本无CXCR2与CXCR6表达(图5与图6)。说明CXCR2或CXCR6成功表达于NK细胞表面。Flow cytometry was used to detect the expression of CXCR2 or CXCR6 in anti-MSLN CAR-CXCR2-NK-92 and anti-MSLN CAR-CXCR6-NK-92 cells. The results showed that the expression rate of CXCR2 on anti-MSLN CAR-CXCR2-NK-92 cells was 99.0% (Figure 5), and the expression rate of CXCR6 on anti-MSLN CAR-CXCR6NK-92 cells was 90.5% (Figure 6), while unmodified NK-92 cells (cells that do not express CXCR2, CXCR6 and CAR) and anti-MSLN CAR-NK-92 cells had basically no expression of CXCR2 and CXCR6 (Figures 5 and 6). This shows that CXCR2 or CXCR6 is successfully expressed on the surface of NK cells.

2.3 Anti-MSLN CAR-CXCR2NK-92与Anti-MSLN CAR-CXCR6NK-92细胞的趋化能力比较2.3 Comparison of chemotactic ability of Anti-MSLN CAR-CXCR2NK-92 and Anti-MSLN CAR-CXCR6NK-92 cells

通过transwell实验检测NK细胞的趋化能力。在transwell小室上室接入3×105个NK-92细胞，下室加入600μL胰腺癌Capan-2细胞培养上清，37℃培养4h后收集下室中的细胞进行计数。结果显示，anti-MSLN CAR-CXCR2-NK-92细胞的迁移率为23.38％±2.30％，明显高于anti-MSLN CAR-NK-92、anti-MSLN CAR-CXCR6-NK-92和NK-92细胞(图7)。The chemotactic ability of NK cells was detected by transwell assay. 3×10 5 NK-92 cells were placed in the upper chamber of the transwell chamber, and 600 μL of pancreatic cancer Capan-2 cell culture supernatant was added to the lower chamber. After culturing at 37°C for 4 hours, the cells in the lower chamber were collected and counted. The results showed that the migration rate of anti-MSLN CAR-CXCR2-NK-92 cells was 23.38% ± 2.30%, which was significantly higher than that of anti-MSLN CAR-NK-92, anti-MSLN CAR-CXCR6-NK-92 and NK-92 cells (Figure 7).

2.4 Anti-MSLN CAR-CXCR2NK-92细胞的体外杀伤功能2.4 In vitro killing function of Anti-MSLN CAR-CXCR2NK-92 cells

以NK-92、anti-MSLN CAR-NK-92和anti-MSLN CAR-CXCR2-NK-92细胞为效应细胞，以胰腺癌细胞系Capan-2作为靶细胞，设置效靶比为5:1、2.5:1和1.25:1，将效应细胞与靶细胞共孵育5h，LDH释放法检测效应细胞对靶细胞的杀伤效率。结果表明，anti-MSLN CAR-CXCR2-NK-92细胞和anti-MSLN CAR-NK-92细胞对胰腺癌细胞Capan-2的杀伤效率均明显高于未修饰的NK-92细胞(图8)。说明CXCR2的表达不影响CAR-NK92细胞的杀伤效应。NK-92, anti-MSLN CAR-NK-92 and anti-MSLN CAR-CXCR2-NK-92 cells were used as effector cells, and the pancreatic cancer cell line Capan-2 was used as target cells. The effector-target ratio was set to 5:1, 2.5:1 and 1.25:1. The effector cells and target cells were co-incubated for 5 hours, and the LDH release method was used to detect the killing efficiency of the effector cells on the target cells. The results showed that the killing efficiency of anti-MSLN CAR-CXCR2-NK-92 cells and anti-MSLN CAR-NK-92 cells on pancreatic cancer cells Capan-2 was significantly higher than that of unmodified NK-92 cells (Figure 8). This shows that the expression of CXCR2 does not affect the killing effect of CAR-NK92 cells.

实施例3：Anti-MSLN CAR-CXCR2NK-92细胞体内抗肿瘤能力和向肿瘤内部浸润的能力Example 3: Anti-MSLN CAR-CXCR2NK-92 cells' anti-tumor ability in vivo and their ability to infiltrate into tumors

以胰腺癌Capan-2细胞皮下荷瘤建立胰腺癌异种移植模型，观察anti-MSLN CAR-CXCR2NK92细胞对胰腺癌的治疗作用。选择6周龄雌性裸鼠，腋下皮下荷瘤，荷瘤剂量为每只小鼠5×106个Capan-2细胞。两周后，待肿瘤体积达到100mm3左右时开始治疗。首先将小鼠随机分为对照组、NK-92细胞治 疗组、anti-MSLN CAR-NK-92细胞治疗组和anti-MSLN CAR-CXCR2-NK-92细胞治疗组。治疗组小鼠采用尾静脉注射效应细胞5×106/100μL/只，对照组注射等体积的1×PBS溶液，每隔一周注射一次，每隔3天腹腔注射IL-2(5×104IU/只)。每三天测量小鼠肿瘤体积，共观察治疗至56天，取肿瘤组织拍照，并测量肿瘤体积(图9)。结果可见，anti-MSLN CAR-CXCR2-NK-92细胞治疗组的肿瘤体积明显小于其他三组，说明表达CXCR2的CAR-NK细胞具有更强的体内抗肿瘤能力。A pancreatic cancer xenograft model was established by subcutaneously implanting pancreatic cancer Capan-2 cells to observe the therapeutic effect of anti-MSLN CAR-CXCR2NK92 cells on pancreatic cancer. Six-week-old female nude mice were selected and implanted with tumors subcutaneously in the armpits. The tumor-bearing dose was 5×10 6 Capan-2 cells per mouse. Two weeks later, when the tumor volume reached about 100 mm 3 , treatment was started. The mice were first randomly divided into a control group, a NK-92 cell-treated group, and a control group. Treatment group, anti-MSLN CAR-NK-92 cell treatment group and anti-MSLN CAR-CXCR2-NK-92 cell treatment group. The mice in the treatment group were injected with effector cells 5×10 6 /100μL/mouse through the tail vein, and the control group was injected with an equal volume of 1×PBS solution, once every week, and IL-2 (5×10 4 IU/mouse) was injected intraperitoneally every 3 days. The tumor volume of the mice was measured every three days, and the treatment was observed for a total of 56 days. The tumor tissue was taken and photographed, and the tumor volume was measured (Figure 9). The results show that the tumor volume of the anti-MSLN CAR-CXCR2-NK-92 cell treatment group was significantly smaller than that of the other three groups, indicating that CAR-NK cells expressing CXCR2 have stronger anti-tumor ability in vivo.

为进一步明确NK细胞的浸润情况，我们用免疫荧光的方法检测了肿瘤组织中NK细胞浸润情况。如图10所示，结果显示，未治疗组肿瘤组织中几乎没有NK细胞，NK-92治疗组肿瘤组织中有少量NK细胞，anti-MSLN CAR-NK-92细胞治疗组肿瘤组织中相对未治疗和NK92治疗组浸润的NK细胞数量增多，而anti-MSLN CAR-CXCR2-NK-92细胞治疗组肿瘤组织中浸润的NK细胞数量明显高于anti-MSLN CAR-NK-92细胞治疗组。说明表达CXCR2的CAR-NK细胞具有明显增强的向实体肿瘤内部浸润的能力。To further clarify the infiltration of NK cells, we used immunofluorescence to detect the infiltration of NK cells in tumor tissues. As shown in Figure 10, the results showed that there were almost no NK cells in the tumor tissues of the untreated group, a small number of NK cells in the tumor tissues of the NK-92 treatment group, and the number of infiltrating NK cells in the tumor tissues of the anti-MSLN CAR-NK-92 cell treatment group increased compared with the untreated and NK92 treatment groups, while the number of infiltrating NK cells in the tumor tissues of the anti-MSLN CAR-CXCR2-NK-92 cell treatment group was significantly higher than that of the anti-MSLN CAR-NK-92 cell treatment group. This indicates that CAR-NK cells expressing CXCR2 have a significantly enhanced ability to infiltrate into solid tumors.

此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。 Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (32)

一种构建体，其特征在于，包括：A construct, characterized in that it comprises: 第一核酸，所述第一核酸编码嵌合抗原受体；以及a first nucleic acid encoding a chimeric antigen receptor; and 第二核酸，所述第二核酸编码趋化因子受体。A second nucleic acid encoding a chemokine receptor. 根据权利要求1所述的构建体，其特征在于，所述嵌合抗原受体包括：The construct according to claim 1, wherein the chimeric antigen receptor comprises: 胞外区，所述胞外区包括单链抗体和CD8铰链区，所述单链抗体包括重链可变区和轻链可变区，所述胞外区单链抗体可以特异性识别肿瘤抗原，所述单链抗体的C端与所述CD8铰链区的N端相连；An extracellular region, wherein the extracellular region includes a single-chain antibody and a CD8 hinge region, the single-chain antibody includes a heavy chain variable region and a light chain variable region, the single-chain antibody in the extracellular region can specifically recognize a tumor antigen, and the C-terminus of the single-chain antibody is connected to the N-terminus of the CD8 hinge region; 跨膜区，所述跨膜区包括CD8跨膜区，所述CD8跨膜区的N端与所述胞外区的CD8铰链区的C端相连；A transmembrane region, wherein the transmembrane region includes a CD8 transmembrane region, and the N-terminus of the CD8 transmembrane region is connected to the C-terminus of the CD8 hinge region of the extracellular region; 胞内区，所述胞内区的N端与所述跨膜区的C端相连，所述胞内区包括4-1BB共刺激因子结构域和CD3ζ胞内信号段。The intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, and the intracellular region includes a 4-1BB co-stimulatory factor domain and a CD3ζ intracellular signal segment. 根据权利要求2所述的构建体，其特征在于，所述肿瘤抗原选自：胚胎性蛋白、糖蛋白抗原、鳞状细胞抗原等，包括MSLN、HER2、EGFR、GPC3、MUC1、CEA、CLDN 18.2、EpCAM、PSCA、PSMA、GD2、IL-13RA2、B7-H3、CD133、CD70、C-MET、FAP、TROP-2、ROR1、CD19、CD20、CD22、CD30、CD33、BCMA中的至少之一。The construct according to claim 2 is characterized in that the tumor antigen is selected from: embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., including at least one of MSLN, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, PSCA, PSMA, GD2, IL-13RA2, B7-H3, CD133, CD70, C-MET, FAP, TROP-2, ROR1, CD19, CD20, CD22, CD30, CD33, and BCMA. 根据权利要求1所述的构建体，其特征在于，所述趋化因子受体包括选自CXCR2、CXCR6、CXCR1、CXCR3、CXCR4、CXCR5、CXCR7、CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR10、CX3CR1和XCR1中的至少之一。The construct according to claim 1, characterized in that the chemokine receptor includes at least one selected from CXCR2, CXCR6, CXCR1, CXCR3, CXCR4, CXCR5, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, CX3CR1 and XCR1. 根据权利要求1所述的构建体，其特征在于，所述趋化因子为CXCR2。The construct according to claim 1, characterized in that the chemokine is CXCR2. 根据权利要求2或权利要求4任一项所述的构建体，其特征在于，所述单链抗体具有如SEQ ID NO.1所示的氨基酸序列；The construct according to any one of claim 2 or claim 4, characterized in that the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.1; 所述CD8跨膜区具有如SEQ ID NO.2所示的氨基酸序列；The CD8 transmembrane region has an amino acid sequence as shown in SEQ ID NO.2; 所述4-1BB共刺激因子结构域具有如SEQ ID NO.3所示的氨基酸序列；The 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO.3; 所述CD3ζ胞内信号段具有如SEQ ID NO.4所示的氨基酸序列；The CD3ζ intracellular signal segment has an amino acid sequence as shown in SEQ ID NO.4; 所述CD8铰链区具有SEQ ID NO:5所示的氨基酸序列；The CD8 hinge region has the amino acid sequence shown in SEQ ID NO:5; 所述CXCR2具有如SEQ ID NO.6所示的氨基酸序列；The CXCR2 has an amino acid sequence as shown in SEQ ID NO.6; 所述CXCR6具有如SEQ ID NO.7所示的氨基酸序列。The CXCR6 has an amino acid sequence as shown in SEQ ID NO.7. 根据权利要求1所述的构建体，其特征在于，所述第一核酸分子与所述第二核酸分子被设置为转基因细胞中表达所述嵌合抗原受体以及所述趋化因子受体，并且所述嵌合抗原受体以及所述趋化因子受体呈非融合形式。 The construct according to claim 1, characterized in that the first nucleic acid molecule and the second nucleic acid molecule are configured to express the chimeric antigen receptor and the chemokine receptor in a transgenic cell, and the chimeric antigen receptor and the chemokine receptor are in a non-fusion form. 根据权利要求7所述的构建体，其特征在于，所述第一核酸分子与所述第二核酸分子设置于同一载体上。The construct according to claim 7 is characterized in that the first nucleic acid molecule and the second nucleic acid molecule are arranged on the same vector. 根据权利要求7所述构建体，其特征在于，所述第一核酸分子与所述第二核酸分子设置于不同的载体上。The construct according to claim 7 is characterized in that the first nucleic acid molecule and the second nucleic acid molecule are arranged on different vectors. 根据权利要求8所述的构建体，其特征在于，所述构建体进一步包括：The construct according to claim 8, characterized in that the construct further comprises: 内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：8所示的核苷酸序列。An internal ribosome entry site sequence, wherein the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the internal ribosome entry site has a nucleotide sequence shown in SEQ ID NO: 8. 根据权利要求8所述的构建体，其特征在于，所述构建体进一步包括：The construct according to claim 8, characterized in that the construct further comprises: 第三核酸分子，设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码自剪切肽P2A，所述自剪切肽P2A能够在所述转基因细胞中被切割。The third nucleic acid molecule is disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a self-cleaving peptide P2A, which can be cleaved in the transgenic cell. 根据权利要求11所述的构建体，其特征在于，所述自剪切肽P2A具有SEQ ID NO：9所示的氨基酸序列。The construct according to claim 11 is characterized in that the self-cleaving peptide P2A has an amino acid sequence shown in SEQ ID NO: 9. 根据权利要求8或9所述的构建体，其特征在于，进一步包括：The construct according to claim 8 or 9, further comprising: 第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；以及a first promoter operably linked to the first nucleic acid molecule; and 第二启动子，所述第二启动子与所述第二核酸分子可操作地连接。A second promoter is operably linked to the second nucleic acid molecule. 根据权利要求13所述的构建体，其特征在于，所述第一启动子和所述第二启动子分别独立地选自CMV,EF-1,RSV启动子。The construct according to claim 13, characterized in that the first promoter and the second promoter are independently selected from CMV, EF-1, and RSV promoters. 根据权利要求1所述的构建体，其特征在于，所述构建体是非致病性病毒载体。The construct according to claim 1, characterized in that the construct is a non-pathogenic viral vector. 根据权利要求15所述的构建体，其特征在于，所述非致病性病毒选自反转录病毒、慢病毒和腺病毒相关病毒。The construct according to claim 15, characterized in that the non-pathogenic virus is selected from retroviruses, lentiviruses and adenovirus-associated viruses. 根据权利要求16所述的构建体，其特征在于，所述非致病性病毒为慢病毒。The construct according to claim 16, characterized in that the non-pathogenic virus is a lentivirus. 根据权利要求1所述的构建体，其特征在于，所述第一核酸分子具有SEQ ID NO：10所示的核苷酸序列；The construct according to claim 1, characterized in that the first nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 10; 第二核酸分子具有SEQ ID NO：11所示的核苷酸序列。The second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 11. 一种表达载体，其特征在于，包括权利要求1～18任一项所述的构建体。An expression vector, characterized in that it comprises the construct according to any one of claims 1 to 18. 根据权利要求19所述的表达载体，其特征在于，所述表达载体为慢病毒病毒载体。The expression vector according to claim 19, characterized in that the expression vector is a lentiviral vector. 根据权利要求19所述的表达载体，其特征在于，所述表达载体为腺病毒载体、非致病性载体或逆转录病毒载体。The expression vector according to claim 19, characterized in that the expression vector is an adenoviral vector, a non-pathogenic vector or a retroviral vector. 一种慢病毒载体，其特征在于，具有SEQ ID NO：12所示的核苷酸序列。A lentiviral vector, characterized in that it has a nucleotide sequence shown in SEQ ID NO: 12. 一种转基因细胞，其特征在于，携带权利要求1～18任一项所述的构建体、权利要求19～21任一项所述的表达载体或权利要求22所述的慢病毒载体；或 A transgenic cell, characterized in that it carries the construct according to any one of claims 1 to 18, the expression vector according to any one of claims 19 to 21, or the lentiviral vector according to claim 22; or 表达嵌合抗原受体以及趋化因子受体，并且所述嵌合抗原受体以及所述趋化因子受体呈非融合形式，expressing a chimeric antigen receptor and a chemokine receptor, wherein the chimeric antigen receptor and the chemokine receptor are in a non-fused form, 其中，所述嵌合抗原受体以及所述趋化因子受体如权利要求2～6任一项所限定的。Wherein, the chimeric antigen receptor and the chemokine receptor are as defined in any one of claims 2 to 6. 一种CAR-淋巴细胞，其特征在于，所述淋巴细胞携带权利要求1～18任一项所述的构建体、权利要求19～21任一项所述的表达载体或权利要求22所述的慢病毒载体；或A CAR-lymphocyte, characterized in that the lymphocyte carries the construct according to any one of claims 1 to 18, the expression vector according to any one of claims 19 to 21, or the lentiviral vector according to claim 22; or 表达嵌合抗原受体以及趋化因子受体，并且所述嵌合抗原受体以及所述趋化因子受体呈非融合形式，expressing a chimeric antigen receptor and a chemokine receptor, wherein the chimeric antigen receptor and the chemokine receptor are in a non-fused form, 其中，所述嵌合抗原受体以及所述趋化因子受体如权利要求2～6任一项所限定的。Wherein, the chimeric antigen receptor and the chemokine receptor are as defined in any one of claims 2 to 6. 根据权利要求24所述的CAR-淋巴细胞，其特征在于，所述CAR-淋巴细胞包括选自NK-92细胞，外周血NK细胞、脐带血NK细胞、iPSC、CAR-T细胞、CAR-NKT细胞、CAR-γδT细胞、CAR-巨噬细胞中的至少之一。The CAR-lymphocyte according to claim 24, characterized in that the CAR-lymphocyte comprises at least one selected from NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, CAR-T cells, CAR-NKT cells, CAR-γδT cells, and CAR-macrophages. 一种药物组合物，其特征在于，包括：A pharmaceutical composition, characterized in that it comprises: 权利要求1～18任一项所述的构建体、权利要求19～21任一项所述的表达载体、权利要求22所述的慢病毒载体、权利要求23所述的转基因细胞或权利要求24～25任一项所述的CAR-淋巴细胞。The construct of any one of claims 1 to 18, the expression vector of any one of claims 19 to 21, the lentiviral vector of claim 22, the transgenic cell of claim 23, or the CAR-lymphocyte of any one of claims 24 to 25. 根据权利要求26所述的药物组合物，其特征在于，所述药物组合物进一步包括：药学上可接受的辅料。The pharmaceutical composition according to claim 26, characterized in that the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient. 权利要求1～18任一项所述的构建体、权利要求19～21任一项所述的表达载体、权利要求22所述的慢病毒载体、权利要求23所述的转基因细胞、权利要求24～25任一项所述的CAR-淋巴细胞或权利要求26～27任一项所述的药物组合物在制备药物中的用途，所述药物用于实体瘤或血液瘤的免疫治疗。Use of the construct of any one of claims 1 to 18, the expression vector of any one of claims 19 to 21, the lentiviral vector of claim 22, the transgenic cell of claim 23, the CAR-lymphocyte of any one of claims 24 to 25, or the pharmaceutical composition of any one of claims 26 to 27 in the preparation of a drug for immunotherapy of solid tumors or hematological tumors. 根据权利要求28所述的用途，其特征在于，所述实体瘤包括选自发生在脏器中的有形瘤，包括胰腺癌、卵巢癌、间皮瘤、肝癌、胆管癌、胃癌、结直肠癌、食管癌、肺癌、头颈癌、宫颈癌、脑胶质瘤、肾癌、乳腺癌、前列腺癌、黑色素瘤中的至少之一。The use according to claim 28 is characterized in that the solid tumor includes a tangible tumor selected from those occurring in internal organs, including at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, and melanoma. 根据权利要求28所述的用途，其特征在于，所述血液瘤包括选自血细胞和造血系统内的急性髓系白血病、急性淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、何杰金氏淋巴瘤、非何杰金氏淋巴瘤、多发性骨髓瘤中的至少之一。The use according to claim 28 is characterized in that the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma in blood cells and hematopoietic system. 一种治疗或预防实体瘤或血液瘤的方法，其特征在于，包括向受试者施用权利要求1～18任一项所述的构建体、权利要求19～21任一项所述的表达载体、权利要求22所述的慢病毒载体、权利要求23所述的转基因细胞、权利要求24～25任一项所述的CAR-淋巴细胞或权利要求26～27任一项所述的药物组合物中的至少之一。A method for treating or preventing solid tumors or hematological tumors, characterized in that it comprises administering to a subject at least one of the construct according to any one of claims 1 to 18, the expression vector according to any one of claims 19 to 21, the lentiviral vector according to claim 22, the transgenic cell according to claim 23, the CAR-lymphocyte according to any one of claims 24 to 25, or the pharmaceutical composition according to any one of claims 26 to 27. 权利要求1～18任一项所述的构建体、权利要求19～21任一项所述的表达载体、权利要求22所 述的慢病毒载体、权利要求23所述的转基因细胞、权利要求24～25任一项所述的CAR-淋巴细胞或权利要求26～27任一项所述的药物组合物在治疗或预防实体瘤或血液瘤中的用途。 The construct according to any one of claims 1 to 18, the expression vector according to any one of claims 19 to 21, the Use of the lentiviral vector according to claim 24, the transgenic cell according to claim 23, the CAR-lymphocyte according to any one of claims 24 to 25, or the pharmaceutical composition according to any one of claims 26 to 27 in the treatment or prevention of solid tumors or hematological tumors.
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