WO2023036246A1 - 一种转基因免疫效应细胞及其应用 - Google Patents

一种转基因免疫效应细胞及其应用 Download PDF

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WO2023036246A1
WO2023036246A1 PCT/CN2022/117831 CN2022117831W WO2023036246A1 WO 2023036246 A1 WO2023036246 A1 WO 2023036246A1 CN 2022117831 W CN2022117831 W CN 2022117831W WO 2023036246 A1 WO2023036246 A1 WO 2023036246A1
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cells
immune effector
effector cell
chemokine receptor
seq
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PCT/CN2022/117831
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English (en)
French (fr)
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叶立军
彭亮
王先进
黄倩
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深圳市菲鹏生物治疗股份有限公司
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Priority to CN202280005529.5A priority Critical patent/CN116134139A/zh
Publication of WO2023036246A1 publication Critical patent/WO2023036246A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4635—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4636—Immune checkpoint inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/464442—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas

Definitions

  • the invention belongs to the field of biotechnology. More specifically, it relates to a transgenic immune effector cell and its application.
  • chimeric antigen receptor T cell has achieved very good results in the treatment of hematological tumors.
  • the curative effect of BCMA CART on multiple myeloma has reached the objective response rate ( ORR) is 100%, the complete remission rate (CR) is 70.6%, and 5 CART cell products have been launched in the world, all targeting blood tumors.
  • ORR objective response rate
  • CR complete remission rate
  • 5 CART cell products have been launched in the world, all targeting blood tumors.
  • the efficacy of CART in the treatment of solid tumors is not satisfactory.
  • Clinical data show that the clinical efficacy of Mesothelin CART combined with PD1 blockers for mesothelioma is 62.5% and CR is 18.75%. Therefore, the efficacy of CART against solid tumors needs to be improved urgently.
  • Chemokines are small molecule cytokines that can cause cells to undergo chemotactic movement. After binding to chemokine receptors, they can transmit a variety of cellular information. Chemokines can induce leukocytes, especially T lymphocytes, to migrate locally to inflammatory reactions and tumor infiltration. . Compared with hematological tumors, solid tumor cells can secrete chemokines such as CXCL12 and CXCL5 to prevent CART cells from reaching tumor lesions; at the same time, solid tumors rarely secrete CXCR3 and CCR5 ligands that help CART cells to transport. The two factors work together to make It is difficult for CART cells to accurately reach solid tumor sites to exert immune effects. Therefore, how to achieve precise treatment of solid tumors is a major problem in CART cell therapy.
  • the present invention is based on the inventor's discovery and recognition of the following problems:
  • the inventors have developed a transgenic immune effector cell that co-expresses a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule; wherein the chemokine receptor molecule is capable of chemoattracting more chimeric Antigen receptors to the tumor site; in addition, the fusion protein can secrete immune checkpoint antibodies and cytokines, and the dual advantages of immune checkpoint antibodies and cytokines can specifically act on transgenic immune effector cells, reducing the tumor microenvironment’s impact on transgenic cells. While inhibiting immune effector cells, the effect of transgenic immune effector cells is more long-lasting, and cytokines can significantly improve the killing ability of tumor cells, realizing the precise treatment of solid tumors by CART.
  • the present invention provides a transgenic immune effector cell that co-expresses a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule;
  • the chimeric antibody receptor It includes an extracellular region, a transmembrane region and an intracellular region in series; wherein, the extracellular region specifically recognizes tumor antigens, the transmembrane region is embedded in the cell membrane of the transgenic immune effector cell, and the intracellular region Including the intracellular segment of immune co-stimulatory molecules;
  • the fusion protein includes immune checkpoint antibodies and cytokines;
  • the chemokine receptor molecule is a chemokine receptor or a chemokine binding fragment thereof.
  • the present invention proposes a lentivirus carrying the following nucleic acid:
  • a nucleic acid encoding a fusion protein in said transgenic immune effector cell, said fusion protein comprising an immune checkpoint antibody and a cytokine;
  • chemokine receptor molecule in the transgenic immune effector cell, where the chemokine receptor molecule is a chemokine receptor or a chemokine binding fragment thereof.
  • the present invention proposes a construct comprising:
  • the first nucleic acid molecule encodes a chimeric antigen receptor in the transgenic immune effector cell, and the extracellular region of the chimeric antigen receptor specifically recognizes a tumor antigen;
  • a second nucleic acid molecule encoding a fusion protein in the transgenic immune effector cell, the fusion protein comprising an immune checkpoint antibody and a cytokine;
  • a third nucleic acid molecule encodes a chemokine receptor molecule in the transgenic immune effector cell, and the chemokine receptor molecule is a chemokine receptor or a chemokine binding fragment thereof.
  • the present invention proposes a method for preparing the transgenic immune effector cells described in the first aspect, introducing the lentivirus or the construct into the immune effector cells; according to an embodiment of the present invention,
  • the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells; according to an embodiment of the present invention, the immune effector cells are T cells.
  • the present invention proposes the application of the transgenic immune effector cells, the lentivirus or the construct in the preparation of cancer therapeutic drugs.
  • the cancer is a solid tumor.
  • the present invention provides a drug for treating cancer.
  • the transgenic immune effector cells, the lentivirus or the construct are included.
  • the cancer is a solid tumor.
  • the present invention proposes a method for treating cancer, which includes: administering a therapeutically effective amount of the above-mentioned cancer therapeutic drug to a subject in need of treatment.
  • the cancer is a solid tumor.
  • Figure 1 is a schematic structural diagram of the nucleic acid constructed in Example 1 of the present invention; wherein, “Hinge” refers to the hinge region, “Linker” refers to the connecting peptide, scFv represents PD1 scFv, and CAR19 represents the expression of a chimeric antigen receptor targeting CD19
  • CAR19&scFv means the CART19 group expressing scFv
  • CAR19&scFv&IL21 means the CART19 group expressing scFv and IL-21 alone (that is, the scFv and IL-21 are not fused)
  • CAR19&scFv-IL21 means the CART19 expressing the fusion protein scFv-IL-21 Group
  • CAR19&IL21-scFv represents the group of CART19 expressing fusion protein IL-21-scFv.
  • Figure 2 is the detection result of the CAR19 positive rate of D6 after T cell infection in Example 3 of the present invention
  • scFv represents PD1 scFv
  • T cell group represents the ordinary T cell group
  • CAR19 represents the expression of a chimeric antigen receptor targeting CD19
  • CAR19&scFv means the CART19 group expressing scFv
  • CAR19&scFv&IL21 means the CART19 group expressing scFv and IL-21 alone
  • CAR19&scFv-IL21 means the CART19 group expressing fusion protein scFv-IL-21
  • CAR19&IL21-scFv means expressing fusion protein IL - CART19 panel of 21-scFv.
  • Fig. 3 is a schematic structural view of the plasmid constructed in Example 5 of the present invention; wherein, Hinge refers to the hinge region, Linker refers to the connecting peptide, SS1 refers to the MSLN antibody (the tumor antigen is MSLN); Anti PD1scFv (VH+VL) refers to PD1 antibody; Anti PD1-IL-21 refers to a fusion protein composed of PD1 antibody and IL-21.
  • Fig. 4 is a graph showing the percentage results of MSLN antibody and CCR2b positive rate in 293T cells.
  • Figure 5 is a schematic diagram of the vector structure of 89CART cells.
  • Fig. 6 is a result graph of the double positive rate of 89CART cells prepared in the present invention.
  • Fig. 7 is a graph showing the results of migration of 89CART cells prepared by the present invention after being subjected to CCL2 chemotaxis.
  • Fig. 8 is a diagram showing the results of the ability of 89CART cells prepared by the present invention to kill tumor cells in vitro.
  • Figure 9 shows the results of detection of tumor volume changes in mice treated with different T cells.
  • 85 represents 85CART cells
  • 89 represents 89CART cells
  • T cell represents control T cells.
  • Figure 10 is the detection results of the cell suspension and peripheral blood CCL2 concentration in the mouse tumor site.
  • G3-783 represents the mouse numbered 783 in the 89CART cell therapy group
  • G3-788 represents the mouse numbered 788 in the 89CART cell therapy group. mouse.
  • Figure 11 shows the detection results of the ratio of different CART cells in the cell suspension and peripheral blood of the mouse tumor site.
  • the present invention will be further described below in conjunction with specific examples, but the examples do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • the meaning of “multiple” is at least two, such as two, three, four, etc.
  • the meaning of “multiple” and “several” is at least two, such as two, three species, four species, etc., unless otherwise expressly and specifically defined.
  • the invention relates to a transgenic immune effector cell, which co-expresses a chimeric antigen receptor, a fusion protein and a chemokine receptor molecule; a membrane region and an intracellular region; wherein, the extracellular region specifically recognizes a tumor antigen, the transmembrane region is embedded in the cell membrane of the immune effector cell, and the intracellular region includes an intracellular segment of an immune co-stimulatory molecule;
  • the fusion protein includes an immune checkpoint antibody and a cytokine; the chemokine receptor molecule is a chemokine receptor or a chemokine binding fragment thereof.
  • the above-mentioned transgenic immune effector cells co-express chimeric antigen receptors, fusion proteins and chemokine receptor molecules, the fusion proteins can secrete immune checkpoint antibodies and cytokines, and the immune checkpoint antibodies and cytokines
  • the double advantages act specifically on the transgenic immune effector cells, reduce the inhibitory effect of the tumor microenvironment on the transgenic immune effector cells, and at the same time make the effect of the transgenic immune effector cells more long-lasting, and cytokines can significantly improve the killing ability of tumor cells
  • Chemokine receptor molecules significantly increase the ability of immune effector cells to migrate to tumor sites
  • the above-mentioned immune effector cells have a stronger killing effect on tumor cells, and at the same time significantly improve the specificity of the combination of cytokines and immune effector cells, greatly reducing drug toxicity.
  • chemokine receptor molecule belongs to G protein-coupled receptors with seven transmembrane structures, which mainly transduce signals through G protein, and have a certain subfamily specificity in binding to receptors.
  • Chemokine receptor molecules are divided into CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors due to different types of ligands.
  • immune checkpoint refers to a series of molecules expressed on immune cells that can regulate the degree of immune activation, and they play an important role in preventing the occurrence of autoimmunity.
  • Cancer-related immune checkpoints include PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA, TIM3, etc.
  • the term “scFv” refers to "single-chain antibody, which is a genetically engineered antibody, which is composed of an antibody heavy chain variable region (VH) and a light chain variable region (VL) usually by 15-20 amino acids.
  • the single-chain antibody can better retain its affinity activity for the antigen, and has the characteristics of small molecular weight, strong penetrating power and weak antigenicity.
  • the "immune checkpoint antibody” has anti-immune checkpoint activity and can specifically bind to the immune checkpoint.
  • cytokines refers to a class of small molecular proteins with a wide range of biological activities synthesized and secreted by immune cells and some non-immune cells upon stimulation. Cytokines are classified into interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc.
  • the term "chimeric antigen receptor" referred to as CAR is the core component of CART, including the extracellular region that can specifically recognize tumor antigens, the transmembrane region embedded in the cell membrane, and the intracellular region including immune co-stimulatory molecules.
  • the intracellular region of the segment; applying the CAR engineering technology to the CART composed of T cells can be widely used in CART cell therapy.
  • the above-mentioned transgenic immune effector cells further include at least one of the following additional technical features:
  • the chemokine receptor is any one or more of CXC chemokine receptor, CC chemokine receptor, CX3C chemokine receptor or XC chemokine receptor.
  • the CXC chemokine receptor is any one or more of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 or CXCR7.
  • the CC chemokine receptor is any one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 or CCR11.
  • the CX3C chemokine receptor is CX3CR1.
  • the XC chemokine receptor is XCR1.
  • the chemokine receptor molecule is CCR2b, and the amino acid sequence of the chemokine receptor molecule is shown in SEQ ID NO:5.
  • the immune co-stimulatory molecule is selected from any one or more of 4-1BB, CD28, CD3, OX-40, CD40L, CD27, CD30, or their derivatives.
  • the immune co-stimulatory molecule is 4-1BB.
  • the immune checkpoint is selected from any one or more of PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA or TIM3.
  • the immune checkpoint is PD1.
  • the cytokine is an interleukin; the cytokine is selected from IL-21, IL-23, IL-2, IL-7, IL-9, IL-12, IL-15 or IL Any one or more of -18.
  • the cytokine is IL-21.
  • the fusion protein further includes a linker peptide; the immune checkpoint antibody is linked to the cytokine through the linker peptide.
  • Short peptides commonly used in the field containing G and S amino acids can be used as the connecting peptides of the present invention.
  • short peptide refers to a peptide with no more than 50 amino acid residues, such as 4-50, such as 4-30, such as 4-20, or It can also be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , or 20.
  • the immune checkpoint antibody is PD1 antibody
  • the cytokine is IL-21.
  • the C-terminus of the PD1 antibody is connected to the N-terminus of the connecting peptide, and the N-terminus of the IL-21 is connected to the C-terminus of the connecting peptide.
  • the N-terminus of the PD1 antibody is connected to the C-terminus of the connecting peptide, and the C-terminus of the IL-21 is connected to the N-terminus of the connecting peptide.
  • the fusion protein obtained by the fusion connection of PD1 antibody and IL-21 can significantly reduce the expression of PD1 on the surface of immune effector cells, and the PD1 antibody specifically binds to PD1 on the surface of immune effector cells, so that IL-21 also specifically acts In the immune effector cells, it effectively reduces the toxicity caused by the combination of IL-21 and the IL-21 receptor on the surface of other cells, and the killing effect of the immune effector cells on the tumor is more significant.
  • PD1 is mainly expressed on the surface of T cells, mainly CD8+ T cells.
  • the fusion protein secreted by the transgenic immune effector cells, its PD1 antibody and IL-21 selectively bind to the surface of T cells and CART cells, on the one hand, block the PD1/PD-L1 signaling pathway, on the other hand, make IL-21 -21 specifically acts on T cells and CART cells to perform its dual functions; at the same time, the fusion protein greatly increases the half-life of the drug due to its larger molecular weight.
  • the amino acid sequence of the connecting peptide is as shown in SEQ ID NO:7.
  • the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3 or SEQ ID NO: 9.
  • the fusion protein PD1 scFv-IL-21 has the amino acid sequence shown in SEQ ID NO: 3
  • the fusion protein IL-21-PD1 scFv has the amino acid sequence shown in SEQ ID NO: 9
  • PD1 scFv represents the PD1 antibody
  • IL- 21 indicates that the cytokine is IL-21
  • the connection sequence in the fusion protein PD1 scFv-IL-21 is that the C-terminal of PD1 scFv is connected with the N-terminal of IL-21
  • the connection sequence in the fusion protein IL-21-PD1 scFv is IL-21
  • the C-terminus of -21 is connected to the N-terminus of PD1 scFv.
  • the extracellular region includes an antibody that specifically recognizes a tumor antigen and the hinge region of CD8; the transmembrane region includes a transmembrane segment of CD8; the immune co-stimulatory molecule is 4-1BB, and the cellular
  • the inner region includes the intracellular segment of 4-1-BB and the CD3 Zeta chain; the CD8 hinge region of the extracellular region is connected to the transmembrane region, and the transmembrane region is connected to the 4-1-BB of the intracellular region.
  • the intracellular segments of BB are connected.
  • the tumor antigen is any one or more of MSLN, GD2, GPC3, CD19, EGFR VIII, GUCY2C, HER2, MUC16 or Claudin 18.2.
  • the tumor antigen is MSLN or CD19.
  • amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1 or SEQ ID NO: 11.
  • the chimeric antigen receptor, fusion protein and chemokine receptor molecule are linked by a 2A peptide.
  • the 2A peptide is selected from any one or more of P2A, T2A, F2A or E2A.
  • the chimeric antigen receptor is linked to the fusion protein via P2A, and the fusion protein is linked to the chemokine receptor molecule via T2A.
  • the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells.
  • the immune effector cells are T cells.
  • T cells mentioned in the present invention refer to T lymphocytes, which can recognize antigens and secrete lymphokines. When T lymphocytes receive antigens exposed by antigenic determinants, they will differentiate into effector T cells.
  • the chemokine receptor molecule is CCR2b
  • the immune costimulatory molecule is 4-1BB
  • the immune checkpoint is PD1
  • the tumor antigen is MSLN
  • the cytokine is IL-21
  • the immune effector cell is T
  • the CCR2b in chimeric antigen receptor T (CART) cells can chemoattract more CARTs to the tumor site, and the PD1 antibody in the fusion protein can relieve the immunosuppression of CARTs, and IL-21 can significantly increase the immune suppression of CARTs. tumor cell killing ability.
  • the present invention also relates to a lentivirus carrying the following nucleic acid:
  • nucleic acid encoding the chimeric antigen receptor specifically recognizes tumor antigens
  • nucleic acid encoding the fusion protein the fusion protein comprising immune checkpoint antibodies and A cytokine
  • 3) a nucleic acid encoding said chemokine receptor molecule which is a chemokine receptor or a chemokine-binding fragment thereof.
  • the above-mentioned lentivirus according to the embodiment of the present invention is introduced into the recipient cells to obtain the transgenic immune effector cells, which can express and secrete fusion proteins including immune checkpoint antibodies and cytokines, chimeric antigen receptors, etc. reduce the expression of immune checkpoints on the surface of immune effector cells, reduce the inhibitory effect of the tumor microenvironment on immune effector cells, and attract more chimeric antigen receptors to the tumor site, making immune effector cells
  • the tumor-killing effect is more significant and long-lasting, and the safety is high.
  • nucleic acid is generally RNA or DNA, and the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • DNA is preferably used when it is ligated into a vector.
  • the term "lentivirus” is a viral vector, and the lentiviral vector can effectively integrate foreign genes or foreign shRNA into the host chromosome, so as to achieve the effect of persistent expression of the target sequence.
  • the above-mentioned lentivirus further includes at least one of the following additional technical features:
  • the tumor antigen is MSLN or CD19
  • the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1 or SEQ ID NO: 11
  • the nucleic acid encoding the chimeric antigen receptor Has the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12.
  • the immune checkpoint antibody is PD1 antibody
  • the cytokine is IL-21
  • the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3 or SEQ ID NO: 9, encoding the
  • the nucleic acid of the fusion protein has a nucleotide sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 10.
  • the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3, and the nucleic acid encoding the fusion protein has the nucleotide sequence shown in SEQ ID NO: 4.
  • the chemokine receptor molecule is CCR2.
  • CCR2 is a specific receptor for monocyte chemoattractant protein-1 (CCL2), and also a receptor for CCL7, CCL8, CCL11, CCL12, and CCL13, including two subtypes of CCR2a and CCR2b , the two are derived from different cuts of the same gene, only the hydroxyl end is different, but CCR2b is the main functional formation.
  • CCL2 monocyte chemoattractant protein-1
  • the chemokine receptor molecule is CCR2b.
  • the amino acid sequence of the chemokine receptor molecule is shown in SEQ ID NO: 5, and the nucleic acid encoding the chemokine receptor molecule has the nucleotides shown in SEQ ID NO: 6 sequence.
  • the lentivirus carries the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10.
  • the present invention also relates to a construct comprising:
  • the first nucleic acid molecule encodes the chimeric antigen receptor, and the extracellular region of the chimeric antigen receptor specifically recognizes a tumor antigen;
  • a second nucleic acid molecule encoding the fusion protein, the fusion protein comprising an immune checkpoint antibody and a cytokine;
  • a third nucleic acid molecule encodes the chemokine receptor molecule, and the chemokine receptor molecule is a chemokine receptor or a chemokine binding fragment thereof.
  • the above-mentioned construct according to the embodiment of the present invention is introduced into the recipient cells to obtain the transgenic immune effector cells, which can express chimeric antigen receptors on the surface of the transgenic immune effector cells and secrete fusion proteins and chemokine receptor molecules,
  • the killing effect on tumor cells is more significant, durable and safe.
  • the above construct further includes at least one of the following additional technical features:
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule respectively express chimeric antigen receptor, fusion protein and chemokine receptor molecule in the immune effector cells.
  • the chimeric antigen receptor, fusion protein and chemokine receptor molecules are in unfused form.
  • the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells.
  • the immune effector cells are T cells.
  • the construct further includes: a first promoter operably linked to the first nucleic acid molecule.
  • the first promoter is selected from any one or more of U6, H1, CMV, EF-1, LTR or RSV promoters.
  • the vector for the construct is a non-pathogenic viral vector.
  • the non-pathogenic viral vector is selected from any one or more of retroviral vectors, lentiviral vectors or adeno-associated viral vectors.
  • the non-pathogenic viral vector is a lentiviral vector; the nucleic acid encoding the lentiviral vector has the nucleotide sequence shown in SEQ ID NO:8.
  • the present invention also relates to a method for preparing the immune effector cells by introducing the lentivirus or the construct into the immune effector cells.
  • lentivirus or the above-mentioned construct according to the embodiment of the present invention into immune effector cells, co-express and secrete chimeric antigen receptors, fusion proteins and chemokine receptor molecules in the cells, and reduce the expression of immune checkpoints and tumor microbiology.
  • the inhibitory effect of the environment on immune effector cells can kill tumor cells more significantly and for a long time.
  • the immune effector cells are selected from any one or more of T cells, NK cells, NKT cells, macrophages or CIK cells.
  • the immune effector cells are T cells.
  • the present invention also relates to the application of the immune effector cells, the lentivirus or the construct in the preparation of cancer therapeutic drugs.
  • the present invention also relates to a drug for treating cancer, comprising the immune effector cells, the lentivirus or the construct.
  • the cancer is a solid tumor.
  • the present invention also relates to a method of treating cancer, comprising: administering to a subject in need thereof a therapeutically effective amount of a cancer treatment drug as described above.
  • the cancer is a solid tumor.
  • the aforementioned solid tumors include, but are not limited to, ovarian cancer and pancreatic cancer.
  • the inventors have developed a transgenic immune effector cell that co-expresses a chimeric antigen receptor, a fusion protein, and a chemokine receptor molecule; wherein the chemokine receptor molecule is capable of chemoattracting more chimeric Antigen receptors go to the tumor site, so that the migration ability of immune effector cells to the tumor site is significantly improved; in addition, the fusion protein can secrete immune checkpoint antibodies and cytokines, and the dual advantages of immune checkpoint antibodies and cytokines are specific Acting on transgenic immune effector cells, the immune checkpoint antibody in the fusion protein can relieve the immune checkpoint immune suppression of immune effector cells, cytokines can significantly improve the killing ability of tumor cells, and the killing effect is better, long-term and safe, Realize the precise treatment of solid tumors with CART.
  • Example 1 Construction of CART19 (T cells expressing chimeric antigen receptor targeting CD19) lentiviral vector expressing Anti PD1-IL-21 fusion protein and its control vector
  • Amino acid sequence of CAR19 as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 12;
  • Fusion protein PD1 scFv-IL-21 amino acid sequence as shown in SEQ ID NO: 3; nucleotide sequence as shown in SEQ ID NO: 4;
  • Fusion protein IL-21-PD1 scFv amino acid sequence as shown in SEQ ID NO: 9; nucleotide sequence as shown in SEQ ID NO: 10.
  • DMEM medium containing 10% fetal bovine serum was added to a 15mL/150mm2 petri dish , and the virus supernatant was collected 48 hours and 72 hours after transfection, centrifuged at 2000rpm 4°C for 10min to remove cell debris, and then filtered through a 0.45 micron filter For impurities, the filtered virus suspension was centrifuged at 25,000 rpm and 4°C for 2 hours to concentrate the lentivirus, and the concentrated virus was resuspended by adding an appropriate amount of medium and stored at -80°C.
  • the CAR19 positive rate detection results on the sixth day (D6) after T cell infection are shown in Figure 2.
  • the results showed that CAR19 group (66.97%), CAR19&scFv group (65.07%), CAR19&scFv&IL21 group (56.31%), CAR19&scFv-IL21 group (78.1%), CAR19&IL21-scFv group (57.78%), it can be seen from the results that CAR19&scFv-IL21 has the highest infection efficiency.
  • Embodiment 4 CART phenotype identification
  • T, B, and NK cell clusters of each group of cells on D6 after infection were detected by flow cytometry, and the CD4, CD8, and PD1 clusters in T cells were also detected.
  • scFv stands for PD1 scFv
  • T cell group stands for common T cell group
  • CAR19 stands for T cell group expressing chimeric antigen receptor targeting CD19
  • CAR19&scFv stands for CART19 group expressing scFv
  • CAR19&scFv&IL21 stands for scFv alone expression
  • CAR19&scFv-IL21 represents the CART19 group expressing the fusion protein scFv-IL-21
  • CAR19&IL21-scFv represents the CART19 group expressing the fusion protein IL-21-scFv.
  • FIG. 3 The schematic diagram of the structure of the plasmid constructed in the embodiment of the present invention is shown in Figure 3, and the nucleotide sequence of the structure shown in Figure 3 is genetically synthesized, and the nucleotide sequence is constructed on the lentiviral vector according to the restriction site of the lentiviral vector, Primers were designed, and the correctness of the constructed plasmid was verified by sequencing results.
  • MSLN CAR amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2;
  • amino acid sequence of CCR2b chemokine receptor molecule as shown in SEQ ID NO: 5
  • nucleotide sequence as shown in SEQ ID NO: 6.
  • the three constructed plasmids MSLN CAR, MSLN CAR+Anti PD1-IL-21, and MSLN CAR+Anti PD1-IL-21+CCR2b were named PCDHF-85, PCDHF-86, and PCDHF-89 respectively; among them, Anti PD1-IL-21 represents a fusion protein consisting of PD1 antibody and IL-21.
  • PCDHF-85, PCDHF-86, and PCDHF-89 plasmids constructed in Example 5 were packaged with the lentivirus packaging system shown in Table 2. The steps are as follows:
  • Inoculate 293T cells (cell cryopreservation density is 5 ⁇ 10 6 cells/mL) in a 10 cm cell culture dish, add 10 mL of DMEM medium containing 10% FBS (DMEM Gibco, 11995040-1L; FBS Gibco, 10091- 148), 5% CO 2 , cultured in a CO 2 incubator at 37°C for 24 hours;
  • the percentage results of MSLN antibody and CCR2b positive rate in 293T cells are shown in Figure 4, and the titer detection results of each lentivirus are shown in Table 3.
  • the results show that the titers of PCDHF-85, PCDHF-86, and PCDHF-89 lentiviruses were respectively 3.96 ⁇ 10 8 TU/mL, 3.02 ⁇ 10 8 TU/mL, 1.44 ⁇ 10 8 TU/mL.
  • PBMC cells were isolated from 50mL of blood using Ficoll Lymph Separation Solution (Dakota, AS1114546), and positive selection method was used to separate CD3/CD28 antibody-coupled magnetic beads (Dynabeads, CD3/CD28 CTS, Cat. No. 40203D, Lot No. A2-011710E)
  • CD3/CD28 antibody-coupled magnetic beads Distabeads, CD3/CD28 CTS, Cat. No. 40203D, Lot No. A2-011710E
  • Embodiment 8 Chemotaxis of CART cells
  • Chemokine CCL2 can chemoattract cells expressing chemokine receptor CCR2b to migrate to places with high concentration of CCL2, and tumor sites highly express CCL2. Therefore, in order to verify the migration ability of the 89CART cells prepared in Example 7 to the tumor site, human chemokine CCL2 (GenScript, product number: Z02829), X-VIVO 15 hematopoietic stem cell serum-free medium (LONZA, batch number , 8MB230), prepared to a concentration of 30ng/mL, a total of 2.4mL.
  • human chemokine CCL2 GenScript, product number: Z02829
  • X-VIVO 15 hematopoietic stem cell serum-free medium LONZA, batch number , 8MB230
  • OVCAR-3 cells ATCC HTB161
  • T cells, 85CART cells and 89CART cells were cultured (the initial cell density was 2 ⁇ 10 5 cells/mL, and the supernatant was collected after 72 hours of culture, IL-21 ELISA detection kit, Invitrogen, lot number 220657) supernatant The expression level of IL-21 in .
  • Example 10 The performance of CART cells in killing tumor cells in mice
  • ASPC-1-CCL2 cell line Construction of ASPC-1-CCL2 cell line: look up the human CCL2 gene sequence (see SEQ ID NO: 13) from the NCBI database and construct it on the pCDHF vector, named as pCDHF-91 plasmid, and then package the lentivirus (packaging process with reference to Example 6) , ASPC-1 cells (ATCC CRL1682 TM ) were infected with the packaged lentivirus, and the infected cells were cloned on the second day, and the ASPC-1 with high expression of CCL2 was selected for expansion and culture to establish the ASPC-1-CCL2 cell line.
  • ASPC-1 cells ATCC CRL1682 TM
  • ASPC-1-CCL2 cells were subcutaneously inoculated into NPG mice (Biocytogen), 5.0E+06 cells/100 ⁇ l+100 ⁇ l Matrigel (BD of the company, product number 356234) per mouse, and the mice in the three groups were respectively After 7 days, T cells, 85CART cells and 89CART cells (2.0E+06 CAR+cells each) were reinfused into the tail vein, and the changes in the tumor volume of the mice were regularly measured (as shown in Figure 9). The concentration of CCL2 (as shown in Figure 10) and the proportion of CART cells in the tumor site (as shown in Figure 11).

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Abstract

涉及生物技术领域，具体涉及一种转基因免疫效应细胞及其应用。该免疫效应细胞表达的融合蛋白中的免疫检查点抗体能解除免疫检查点对免疫效应细胞的免疫抑制，细胞因子能增强免疫效应细胞的抗肿瘤活性，趋化因子受体分子使得免疫效应细胞向肿瘤部位的迁移能力显著提高；与融合蛋白、以及共表达上述融合蛋白和嵌合抗原受体的细胞相比，该免疫效应细胞对肿瘤细胞具有更强的杀伤效果，药物毒性低，安全性高，有效实现了CAR-T对实体瘤的精准治疗。

Description

一种转基因免疫效应细胞及其应用
优先权声明
本申请要求申请号为202111055628.8，申请日为2021年9月9日，发明名称为一种转基因免疫效应细胞及其应用的中国发明专利申请的优先权，其全部内容通过引用并入本文中。
技术领域
本发明属于生物技术领域。更具体地，涉及一种转基因免疫效应细胞及其应用。
背景技术
目前，嵌合抗原受体T细胞(chimeric antigen receptor T cell，CART)在治疗血液瘤方面已经取得非常好的效果，根据公开数据显示，BCMA CART针对多发性骨髓瘤的疗效已经达到客观缓解率(ORR)为100％，完全缓解率(CR)为70.6％，并且全球已经有5款CART细胞产品上市，均是针对血液瘤。但CART在治疗实体瘤方面的疗效却不尽如人意，临床数据显示针对间皮瘤的Mesothelin CART联合PD1阻断剂的临床疗效ORR为62.5％，CR为18.75％。因此，CART针对实体瘤的疗效亟需提高。
CART细胞在杀伤实体瘤过程中，主要有以下3个方面原因导致CART细胞疗效不佳：1)CART难以大量到达并浸润到肿瘤内部进行杀伤；2)少量浸润到肿瘤内部的CART活性被肿瘤部位高表达的PD1等免疫抑制分子抑制；3)CART细胞与肿瘤细胞接触不足显著影响其增殖和持久性，导致CART细胞在体内容易耗竭。
趋化因子是能使细胞发生趋化运动的小分子细胞因子，与趋化因子受体结合后传递多种细胞信息，趋化因子能诱导白细胞特别是T淋巴细胞向炎症反应和肿瘤浸润局部迁移。与血液瘤相比，实体瘤细胞可分泌CXCL12、CXCL5等趋化因子以阻止CART细胞抵达肿瘤病灶；同时，实体瘤分泌极少帮助CART细胞转运的CXCR3和CCR5配体，两种因素共同作用使得CART细胞很难精准到达实体瘤部位发挥免疫效应。因此，如何实现实体瘤的精准治疗是CART细胞治疗的一大难题。
发明内容
本发明是基于发明人对以下问题的发现和认识作出的：
本发明人开发了一种转基因免疫效应细胞，该免疫效应细胞共表达嵌合抗原受体、融合蛋白和趋化因子受体分子；其中，趋化因子受体分子能趋化更多的嵌合抗原受体到肿瘤部位；另外，融合蛋白能分泌免疫检查点抗体和细胞因子，将免疫检查点抗体和细胞因子的双重优越性专一性的作用于转基因免疫效应细胞，降低肿瘤微环境对转基因免疫效应细胞抑制作用的同时，使转基因免疫效应细胞作用更为长效，且细胞因子能显著提高对肿瘤细胞的杀伤能力，实现CART对实体瘤的精准治疗。
在本发明的第一方面，本发明提出了一种转基因免疫效应细胞，所述转基因免疫效应细胞共表达嵌合抗原受体、融合蛋白和趋化因子受体分子；所述嵌合抗体受体包括依次串联的胞外区、跨膜区和胞内区；其中，所述胞外区特异性识别肿瘤抗原，所述跨膜区嵌入所述转基因免疫效应细胞的细胞膜中，所述胞内区包括免疫共刺激分子的胞内段；所述融合蛋白包括免疫检查点抗体和细胞因子；所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
在本发明的第二方面，本发明提出了一种慢病毒，所述慢病毒携带以下核酸：
1)编码所述转基因免疫效应细胞中的嵌合抗原受体的核酸，所述嵌合抗原受体的胞外区特异性识别肿瘤抗原；
2)编码所述转基因免疫效应细胞中的融合蛋白的核酸，所述融合蛋白包括免疫检查点抗体和细胞因子；
3)编码所述转基因免疫效应细胞中的趋化因子受体分子的核酸，所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
在本发明的第三方面，本发明提出了一种构建体，所述构建体包括：
第一核酸分子，所述第一核酸分子编码所述转基因免疫效应细胞中的嵌合抗原受体，所述嵌合抗原受体的胞外区特异性识别肿瘤抗原；
第二核酸分子，所述第二核酸分子编码所述转基因免疫效应细胞中的融合蛋白，所述融合蛋白包括免疫检查点抗体和细胞因子；
第三核酸分子，所述第三核酸分子编码所述转基因免疫效应细胞中的趋化因子受体分子，所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
在本发明的第四方面，本发明提出了一种制备第一方面所述转基因免疫效应细胞的方法，将所述慢病毒或所述构建体导入免疫效应细胞中；根据本发明的实施例，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的任一种或几种；根据本发明的实施例，所述免疫效应细胞为T细胞。
在本发明的第五方面，本发明提出了所述转基因免疫效应细胞、所述慢病毒或所述构建体在制备癌症治疗药物中的应用。
根据本发明的实施例，所述癌症为实体瘤。
在本发明的第六方面，本发明提出了一种癌症治疗药物。根据本发明的实施例，包括所述转基因免疫效应细胞、所述慢病毒或所述构建体。
根据本发明的实施例，所述癌症为实体瘤。
在本发明的第七方面，本发明提出了一种治疗癌症的方法，其包括：向需要治疗的主体施用治疗有效量的如上所述的癌症治疗药物。
根据本发明的实施例，所述癌症为实体瘤。
附图说明
图1是本发明实施例1构建的核酸的结构示意图；其中，“Hinge”是指铰链区，“Linker”是指连接肽，scFv代表PD1 scFv，CAR19表示表达靶向CD19的嵌合抗原受体的T细胞组，CAR19&scFv表示表达scFv的CART19组，CAR19&scFv&IL21表示单独表达scFv、IL-21的CART19组(即scFv与IL-21未融合)，CAR19&scFv-IL21表示表达融合蛋白scFv-IL-21的CART19组，CAR19&IL21-scFv表示表达融合蛋白IL-21-scFv的CART19组。
图2是本发明实施例3的T细胞侵染后D6的CAR19阳性率检测结果；其中，scFv代表PD1 scFv，T cell组表示普通T细胞组，CAR19表示表达靶向CD19的嵌合抗原受体的T细胞组，CAR19&scFv表示表达scFv的CART19组，CAR19&scFv&IL21表示单独表达scFv、IL-21的CART19组，CAR19&scFv-IL21表示表达融合蛋白scFv-IL-21的CART19组，CAR19&IL21-scFv表示表达融合蛋白IL-21-scFv的CART19组。
图3是本发明实施例5构建的质粒的结构示意图；其中，Hinge是指铰链区，Linker是指连接肽，SS1是指MSLN抗体(肿瘤抗原为MSLN)；Anti PD1scFv(VH+VL)是指PD1抗体；Anti PD1-IL-21是指由PD1抗体和IL-21组成的融合蛋白。
图4是293T细胞MSLN抗体和CCR2b阳性率百分比结果图。
图5是89CART细胞的载体结构示意图。
图6是本发明制备的89CART细胞的双阳率结果图。
图7是本发明制备的89CART细胞受CCL2趋化作用后的迁移情况结果图。
图8是本发明制备的89CART细胞体外杀伤肿瘤细胞的能力结果图。
图9是接受不同T细胞治疗后的小鼠肿瘤体积变化检测结果，图中，85代表85CART 细胞，89代表89CART细胞，T cell代表对照T细胞。
图10是小鼠肿瘤部位的细胞悬液和外周血CCL2浓度检测结果，图中，G3-783代表89CART细胞治疗组编号为783的小鼠，G3-788代表89CART细胞治疗组编号为788的小鼠。
图11是小鼠肿瘤部位的细胞悬液和外周血不同CART细胞的比例检测结果。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
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，四个等，“多种”、“几种”的含义是至少两种，例如两种，三种，四种等，除非另有明确具体的限定。
本发明涉及一种转基因免疫效应细胞，所述转基因免疫效应细胞共表达嵌合抗原受体、融合蛋白和趋化因子受体分子；所述嵌合抗原受体包括依次串联的胞外区、跨膜区和胞内区；其中，所述胞外区特异性识别肿瘤抗原，所述跨膜区嵌入所述免疫效应细胞的细胞膜中，所述胞内区包括免疫共刺激分子的胞内段；所述融合蛋白包括免疫检查点抗体和细胞因子；所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
根据本发明实施例的上述转基因免疫效应细胞共表达嵌合抗原受体、融合蛋白和趋化因子受体分子，融合蛋白能分泌免疫检查点抗体和细胞因子，将免疫检查点抗体和细胞因子的双重优越性专一性的作用于转基因免疫效应细胞，降低肿瘤微环境对转基因免疫效应细胞抑制作用的同时，使转基因免疫效应细胞作用更为长效，细胞因子能显著提高对肿瘤细胞的杀伤能力，趋化因子受体分子使得免疫效应细胞向肿瘤部位的迁移能力显著提高，因此，与单独表达免疫检查点抗体或细胞因子的融合蛋白、以及共表达上述融合蛋白和嵌合抗原受体的细胞相比，上述免疫效应细胞对肿瘤细胞具有更强的杀伤效果，同时显著提高了细胞因子与免疫效应细胞结合的特异性，大大降低了药物毒性。
在本发明中，术语“趋化因子受体分子”属于含7次跨膜结构G蛋白偶联受体，主要通过G蛋白转导信号，其与受体结合具有一定的亚家族特异性。趋化因子受体分子因配体的种类不同分为CXC趋化因子受体、CC趋化因子受体、CX3C趋化因子受体和XC趋化因子 受体。
在本发明中，术语“免疫检查点”是指在免疫细胞上表达，能调节免疫激活程度的一系列分子，它们对防止自身免疫作用的发生起着重要作用。与癌症相关的免疫检查点有PD1、PD-L1、TIGIT、LAG3、CTLA4、BTLA、TIM3等。
在本发明中，术语“scFv”是指“单链抗体，是一种基因工程抗体，是由抗体重链可变区(VH)和轻链可变区(VL)通常通过15-20个氨基酸的短肽(linker)连接而成的抗体。单链抗体能较好的保留其对抗原的亲和活性，并具有分子量小、穿透力强和抗原性弱等特点。
在本发明中，“免疫检查点抗体”具有抗免疫检查点活性，可以与免疫检查点特异性结合。
在本发明中，术语“细胞因子”是指由免疫细胞和某些非免疫细胞经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。细胞因子被分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。
在本发明中，术语“嵌合抗原受体”简称CAR，是CART的核心部件，包括能特异性识别肿瘤抗原的胞外区、嵌入细胞膜中的跨膜区和包括免疫共刺激分子的胞内段的胞内区；将CAR工程技术应用于T细胞组成的CART能广泛应用于CART细胞治疗。
根据本发明的实施例，上述转基因免疫效应细胞还进一步包括如下附加技术特征中的至少之一：
在一些实施方式中，所述趋化因子受体为CXC趋化因子受体、CC趋化因子受体、CX3C趋化因子受体或XC趋化因子受体中的任一种或几种。
在一些实施方式中，所述CXC趋化因子受体为CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6或CXCR7中的任一种或几种。
在一些实施方式中，所述CC趋化因子受体为CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10或CCR11中的任一种或几种。
在一些实施方式中，所述CX3C趋化因子受体为CX3CR1。
在一些实施方式中，所述XC趋化因子受体为XCR1。
在一些实施方式中，所述趋化因子受体分子为CCR2b，所述趋化因子受体分子的氨基酸序列如SEQ ID NO：5所示。
在一些实施方式中，所述免疫共刺激分子选自4-1BB、CD28、CD3、OX-40、CD40L、CD27、CD30、或他们的衍生物中的任一种或几种。
在一些实施方式中，所述免疫共刺激分子为4-1BB。
在一些实施方式中，所述免疫检查点选自PD1、PD-L1、TIGIT、LAG3、CTLA4、BTLA 或TIM3中的任一种或几种。
在一些实施方式中，所述免疫检查点为PD1。
在一些实施方式中，所述细胞因子为白细胞介素；所述细胞因子选自IL-21、IL-23、IL-2、IL-7、IL-9、IL-12、IL-15或IL-18中的任一种或几种。
在一些实施方式中，所述细胞因子为IL-21。
在一些实施方式中，所述融合蛋白还包括连接肽；所述免疫检查点抗体通过连接肽与所述细胞因子相连。本领域常用的含有G和S氨基酸的短肽均可以作为本发明所述连接肽。
在本发明中，术语“短肽”是指氨基酸残基数量不超过50个的肽，例如可以是4-50个，例如还可以是4-30个、例如还可以是4-20个，或者还可以是4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、或20个。
在一些实施方式中，所述免疫检查点抗体为PD1抗体，所述细胞因子为IL-21。
在一些实施方式中，所述PD1抗体的C端与所述连接肽的N端相连，所述IL-21的N端与所述连接肽的C端相连。在一些实施方式中，所述PD1抗体的N端与所述连接肽的C端相连，所述IL-21的C端与所述连接肽的N端相连。
发明人发现，PD1抗体与IL-21融合连接得到的融合蛋白使免疫效应细胞表面的PD1表达量显著降低，PD1抗体特异性结合免疫效应细胞表面的PD1，从而使得IL-21也特异性的作用于免疫效应细胞，有效降低了IL-21与其他细胞表面的IL-21受体结合所造成的毒性，免疫效应细胞对肿瘤的杀伤效果更加显著。
PD1主要表达在T细胞表面，主要是CD8+T细胞。所述转基因免疫效应细胞分泌出的融合蛋白，其PD1抗体和IL-21选择性地结合在T细胞和CART细胞表面，一方面，封闭PD1/PD-L1信号通路，另一方面，又使得IL-21专一性的作用于T细胞和CART细胞，进而行使其双重功能；与此同时，融合蛋白由于分子量变大，大大地提高了药物的半衰期。
在一些实施方式中，所述连接肽的氨基酸序列如SEQ ID NO：7所示。
Figure PCTCN2022117831-appb-000001
在一些实施方式中，所述融合蛋白的氨基酸序列如SEQ ID NO：3或SEQ ID NO：9所示。其中，融合蛋白PD1 scFv-IL-21具有SEQ ID NO：3所示的氨基酸序列，融合蛋白IL-21-PD1 scFv具有SEQ ID NO：9所示的氨基酸序列，PD1 scFv表示PD1抗体，IL-21表示细胞因子为IL-21，融合蛋白PD1 scFv-IL-21中的连接顺序为PD1 scFv的C端与IL-21的N端相连，融合蛋白IL-21-PD1 scFv中的连接顺序为IL-21的C端与PD1 scFv的N端相连。
Figure PCTCN2022117831-appb-000002
Figure PCTCN2022117831-appb-000003
Figure PCTCN2022117831-appb-000004
在一些实施方式中，所述胞外区包括特异性识别肿瘤抗原的抗体和CD8铰链区；所述跨膜区包括CD8的跨膜段；所述免疫共刺激分子为4-1BB，所述胞内区包括4-1-BB的胞内段和CD3 Zeta链；所述胞外区的CD8铰链区与所述跨膜区相连，所述跨膜区与所述胞内区的4-1-BB的胞内段相连。
在一些实施方式中，所述肿瘤抗原为MSLN、GD2、GPC3、CD19、EGFR VIII、GUCY2C、HER2、MUC16或Claudin 18.2中的任一种或几种。
在一些实施方式中，所述肿瘤抗原为MSLN或CD19。
在一些实施方式中，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：1或SEQ ID NO：11所示。
Figure PCTCN2022117831-appb-000005
Figure PCTCN2022117831-appb-000006
Figure PCTCN2022117831-appb-000007
在一些实施方式中，所述嵌合抗原受体、融合蛋白和趋化因子受体分子通过2A肽相连。
在一些实施方式中，所述2A肽选自P2A、T2A、F2A或E2A中的任一种或几种。
在一些实施方式中，所述嵌合抗原受体通过P2A与所述融合蛋白相连，所述融合蛋白通过T2A与所述趋化因子受体分子相连。
在一些实施方式中，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的任一种或几种。
在一些实施方式中，所述免疫效应细胞为T细胞。
本发明中所述T细胞是指T淋巴细胞，具有识别抗原分泌淋巴因子的作用，当T淋巴细胞接受抗原决定簇暴露的抗原时，会分化为效应T细胞。
在一些实施方式中，当所述趋化因子受体分子为CCR2b、免疫共刺激分子为4-1BB、免疫检查点为PD1、肿瘤抗原为MSLN、细胞因子为IL-21、免疫效应细胞为T细胞时，构建得到嵌合抗原受体T(CART)细胞中的CCR2b能趋化更多的CART到肿瘤部位，融合蛋白中的PD1抗体能解除对CART的免疫抑制，同时IL-21显著提高对肿瘤细胞的杀伤能力。
本发明还涉及一种慢病毒，所述慢病毒携带以下核酸：
1)编码所述嵌合抗原受体的核酸，所述嵌合抗原受体的胞外区特异性识别肿瘤抗原；2)编码所述融合蛋白的核酸，所述融合蛋白包括免疫检查点抗体和细胞因子；3)编码所述趋化因子受体分子的核酸，所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
将根据本发明实施例的上述慢病毒导入受体细胞中，得到所述转基因免疫效应细胞，可在转基因免疫效应细胞中表达和分泌包括免疫检查点抗体和细胞因子的融合蛋白、嵌合抗原 受体以及趋化因子受体分子，降低免疫效应细胞表面免疫检查点的表达，降低肿瘤微环境对免疫效应细胞的抑制作用，趋化更多的嵌合抗原受体到肿瘤部位，使得免疫效应细胞对肿瘤的杀伤效果更为显著和长效，安全性高。
本发明中，术语“核酸”通常是RNA或DNA，核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时优选采用DNA。
本发明中，术语“慢病毒”是一种病毒载体，慢病毒载体能将外源基因或外源的shRNA有效地整合到宿主染色体上，从而达到持久性表达目的序列的效果。
根据本发明的实施例，上述慢病毒还进一步包括如下附加技术特征中的至少之一：
在一些实施方式中，所述肿瘤抗原为MSLN或CD19，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：1或SEQ ID NO：11所示，编码所述嵌合抗原受体的核酸具有如SEQ ID NO：2或SEQ ID NO：12所示的核苷酸序列。
Figure PCTCN2022117831-appb-000008
Figure PCTCN2022117831-appb-000009
Figure PCTCN2022117831-appb-000010
在一些实施方式中，所述免疫检查点抗体为PD1抗体，所述细胞因子为IL-21，所述融合蛋白的氨基酸序列如SEQ ID NO：3或SEQ ID NO：9所示，编码所述融合蛋白的核酸具有如SEQ ID NO：4或SEQ ID NO：10所示的核苷酸序列。
Figure PCTCN2022117831-appb-000011
Figure PCTCN2022117831-appb-000012
Figure PCTCN2022117831-appb-000013
在一些实施方式中，所述融合蛋白的氨基酸序列如SEQ ID NO：3所示，编码所述融合蛋白的核酸具有如SEQ ID NO：4所示的核苷酸序列。
在一些实施方式中，所述趋化因子受体分子为CCR2。
在本发明中，术语“CCR2”是单核细胞趋化蛋白-1(CCL2)的特异性受体，同时也是CCL7、CCL8、CCL11、CCL12、CCL13的受体，包括CCR2a和CCR2b两个亚型，两者分别来源于同一基因的不同剪切，仅羟基端不同，但CCR2b是主要的功能形成。
在一些实施方式中，所述趋化因子受体分子为CCR2b。
在一些实施方式中，所述趋化因子受体分子的氨基酸序列如SEQ ID NO：5所示，编码所述趋化因子受体分子的核酸具有如SEQ ID NO：6所示的核苷酸序列。
Figure PCTCN2022117831-appb-000014
Figure PCTCN2022117831-appb-000015
Figure PCTCN2022117831-appb-000016
在一些实施方式中，所述慢病毒携带含有SEQ ID NO：4或SEQ ID NO：10所示的核苷酸序列。
本发明还涉及一种构建体，所述构建体包括：
第一核酸分子，所述第一核酸分子编码所述嵌合抗原受体，所述嵌合抗原受体的胞外区特异性识别肿瘤抗原；
第二核酸分子，所述第二核酸分子编码所述融合蛋白，所述融合蛋白包括免疫检查点抗 体和细胞因子；
第三核酸分子，所述第三核酸分子编码所述趋化因子受体分子，所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
将根据本发明实施例的上述构建体导入受体细胞中，得到所述转基因免疫效应细胞，可在转基因免疫效应细胞表面表达嵌合抗原受体、并分泌融合蛋白以及趋化因子受体分子，对肿瘤细胞的杀伤效果更加显著、持久和安全。
根据本发明的实施例，上述构建体还进一步包括如下附加技术特征中的至少之一：
在一些实施方式中，所述第一核酸分子、第二核酸分子和第三核酸分子在免疫效应细胞中依次分别表达嵌合抗原受体、融合蛋白和趋化因子受体分子。
在一些实施方式中，所述嵌合抗原受体、融合蛋白和趋化因子受体分子呈非融合形式。
在一些实施方式中，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的任一种或几种。
在一些实施方式中，所述免疫效应细胞为T细胞。
在一些实施方式中，所述构建体还包括：第一启动子，所述第一启动子与所述第一核酸分子可操作的连接。
在一些实施方式中，所述第一启动子选自U6、H1、CMV、EF-1、LTR或RSV启动子中的任一种或几种。
在一些实施方式中，所述构建体的载体是非致病性病毒载体。在一些实施方式中，所述非致病性病毒载体选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体中的任一种或几种。
在一些实施方式中，所述非致病性病毒载体为慢病毒载体；编码所述慢病毒载体的核酸具有SEQ ID NO：8所示的核苷酸序列。
Figure PCTCN2022117831-appb-000017
Figure PCTCN2022117831-appb-000018
Figure PCTCN2022117831-appb-000019
Figure PCTCN2022117831-appb-000020
Figure PCTCN2022117831-appb-000021
本发明还涉及一种制备所述免疫效应细胞的方法，将所述慢病毒或所述构建体导入免疫效应细胞中。
将根据本发明实施例的上述慢病毒或上述构建体导入免疫效应细胞，在细胞中共表达和分泌嵌合抗原受体、融合蛋白和趋化因子受体分子，降低免疫检查点的表达和肿瘤微环境对免疫效应细胞的抑制作用，更显著和长效的杀伤肿瘤细胞。
在一些实施方式中，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的任一种或几种。
在一些实施方式中，所述免疫效应细胞为T细胞。
本发明还涉及所述免疫效应细胞、所述慢病毒或所述构建体在制备癌症治疗药物中的应用。
本发明还涉及一种癌症治疗药物，包括所述免疫效应细胞、所述慢病毒或所述构建体。
在一些实施方式中，所述癌症为实体瘤。
本发明还涉及一种治疗癌症的方法，其包括：向需要治疗的主体施用治疗有效量的如上所述的癌症治疗药物。
在一些实施方式中，所述癌症为实体瘤。
上述实体瘤包括但不限于卵巢癌和胰腺癌。
本发明包括以下有益效果：
本发明人开发了一种转基因免疫效应细胞，该免疫效应细胞共表达嵌合抗原受体、融合蛋白和趋化因子受体分子；其中，趋化因子受体分子能趋化更多的嵌合抗原受体到肿瘤部位，使得免疫效应细胞向肿瘤部位的迁移能力显著提高；另外，融合蛋白能分泌免疫检查点抗体和细胞因子，将免疫检查点抗体和细胞因子的双重优越性专一性的作用于转基因免疫效应细胞，融合蛋白中的免疫检查点抗体能解除免疫检查点对免疫效应细胞的免疫抑制，细胞因子能显著提高对肿瘤细胞的杀伤能力，且杀伤效果更佳长效和安全，实现CART对实体瘤的精准治疗。
实施例1 构建表达Anti PD1-IL-21融合蛋白的CART19(表达靶向CD19的嵌合抗原受体的T细胞)慢病毒载体及其对照载体
基因合成图1所示结构的核苷酸序列，按照慢病毒载体酶切位点，将核苷酸片段构建至慢病毒载体上，设计引物，通过测序结果验证载体构建的正确性。
CAR19氨基酸序列：如SEQ ID NO：11所示，核苷酸序列如SEQ ID NO：12所示；
融合蛋白PD1 scFv-IL-21氨基酸序列：如SEQ ID NO：3所示；核苷酸序列如SEQ ID NO：4所示；
融合蛋白IL-21-PD1 scFv氨基酸序列：如SEQ ID NO：9所示；核苷酸序列如SEQ ID NO：10所示。
实施例2 包装并浓缩慢病毒
将293T按照8×10 6个细胞/150mm 2培养皿的密度接种，次日观察细胞的状态，用PEI转染的方法将3代慢病毒包装载体共转至293T，转染完6小时后换液，按照15mL/150mm 2培养皿添加含有10％胎牛血清的DMEM培养基，转染完48小时、72小时收集病毒上清，2000rpm 4℃ 10min离心，去除细胞碎片，之后0.45微米的滤器过滤杂质，过滤后的病毒悬液在25000rpm、4℃的条件下离心2小时浓缩慢病毒，浓缩后的病毒加入适量的培养基重悬，置于-80℃保存。
实施例3 生产CART及对照细胞
抽血20mL，Ficall梯度离心分离PBMC，用Stemcell公司T细胞阴选试剂盒(货号：19051)分离T细胞，分离后的T细胞用添加5％人AB血清及300单位/mL IL-2 X-VIVO 15培养基重悬T细胞至1×10 6个细胞/mL，用含1％FBS X-VIVO 15清洗beads，按照磁珠 ∶T细胞＝2∶1比例加入预先清洗过的磁珠(Cat#40203D，10ML，Life technology)，2-3天后用新鲜的培养基重悬T细胞至3×10 6-5×10 6个细胞/mL，按照MOI＝10值加入慢病毒，同时加入8μg/mL的Polybrene，4-6小时之后，补加培养基稀释细胞至1×10 6个细胞/mL，次日更换新鲜培养基，使细胞浓度维持在0.2×10 6-0.3×10 6 PBMC/mL，之后每隔2-3天更换一次培养基，病毒侵染完72小时后流式分析细胞阳性率。
T细胞侵染后第六天(D6)的CAR19阳性率检测结果如图2所示，结果显示，CAR19组(66.97％)、CAR19&scFv组(65.07％)、CAR19&scFv&IL21组(56.31％)、CAR19&scFv-IL21组(78.1％)、CAR19&IL21-scFv组(57.78％)，由结果可以看出CAR19&scFv-IL21侵染效率最高。
实施例4 CART表型鉴定
侵染后D6的各组细胞，流式细胞术检测其T、B、NK细胞分群，同时检测T细胞中CD4、CD8、PD1的分群。
结果如表1所示，可以看出，CAR19&scFv-IL21组T细胞仅有少量比例的细胞(2.87％)表达PD1分子，提示CAR19&scFv-IL21可能会调控T细胞表面分子PD1的表达，大大降低了T细胞表面PD1表达的比例。
表1
Figure PCTCN2022117831-appb-000022
注：表1中，scFv代表PD1 scFv，T cell组表示普通T细胞组，CAR19表示表达靶向CD19的嵌合抗原受体的T细胞组，CAR19&scFv表示表达scFv的CART19组，CAR19&scFv&IL21表示单独表达scFv、IL-21的CART19组，CAR19&scFv-IL21表示表达融合蛋白scFv-IL-21的CART19组，CAR19&IL21-scFv表示表达融合蛋白IL-21-scFv的CART19组。
实施例5 构建表达Anti PD1-IL-21融合蛋白和CCR2b的MSLN CART慢病毒载体及其对照载体
本发明实施例构建的质粒的结构示意图如图3所示，基因合成图3所示结构的核苷酸序列，按照慢病毒载体酶切位点，将核苷酸序列构建到慢病毒载体上，设计引物，通过测序结果验证所构建质粒的正确性。
MSLN CAR氨基酸序列：如SEQ ID NO：1所示，核苷酸序列如SEQ ID NO：2所示；
其中，CCR2b趋化因子受体分子氨基酸序列：如SEQ ID NO：5所示，核苷酸序列：如SEQ ID NO：6所示。
其他元件序列同实施例1。
将所构建的3个质粒MSLN CAR、MSLN CAR+Anti PD1-IL-21、MSLN CAR+Anti PD1-IL-21+CCR2b依次分别命名为PCDHF-85、PCDHF-86、PCDHF-89；其中，Anti PD1-IL-21代表由PD1抗体和IL-21组成的融合蛋白。
实施例6 包装慢病毒
将实施例5构建得到的PCDHF-85、PCDHF-86、PCDHF-89质粒按照如表2所示的慢病毒包装体系包装慢病毒，步骤如下：
(1)接种293T细胞(细胞冻存密度为5×10 6 cells/mL)于10cm细胞培养皿中，加入10mL含10％FBS的DMEM培养基(DMEM Gibco，11995040-1L；FBS Gibco，10091-148)，5％CO 2，37℃条件下CO 2培养箱中培养24h；
(2)按表2进行慢病毒包装；包装后48h收细胞上清，25000rpm超速离心后检测慢病毒滴度，检测方法如下：
将收集的慢病毒原液梯度体积同样条件下感染293T细胞，48h后流式检测293T细胞MSLN抗体和CCR2b阳性率百分比(用标记FITC的MSLN抗原检测和CCR2抗体检测，ACRO，FITC-LabeLed Human MSLN(296-580)Protein，Fc Tag，Cat.No.MSN-HF253)，根据以下计算公式计算慢病毒原液滴度：
慢病毒原液滴度(TU/mL)＝1.5*(10×10 5)*293T细胞MSLN抗体和CCR2b阳性率百分比/慢病毒原液体积μL*1000。
表2慢病毒包装体系
Figure PCTCN2022117831-appb-000023
表3慢病毒滴度
质粒 慢病毒滴度
PCDHF-85 3.96×10 8 TU/mL
PCDHF-86 3.02×10 8 TU/mL
PCDHF-89 1.44×10 8 TU/mL
293T细胞MSLN抗体和CCR2b阳性率百分比结果如图4所示，各慢病毒滴度检测结果如表3所示，结果显示，PCDHF-85、PCDHF-86、PCDHF-89慢病毒的滴度依次分别为3.96×10 8TU/mL、3.02×10 8TU/mL、1.44×10 8TU/mL。
实施例7 CART细胞制备
利用Ficoll淋巴分离液(达科为，AS1114546)从50mL血液中分离PBMC细胞，利用 偶联CD3/CD28抗体的磁珠(Dynabeads，CD3/CD28 CTS，货号40203D，批号A2-011710E)阳选法分离获得T细胞，将实施例6制备得到的慢病毒按MOI＝5∶1感染T细胞以制备CART细胞，同时设置对照T细胞，CART细胞培养7天后，通过检测CART细胞的MSLN抗体表达和CCR2b表达来确定所制备89CART细胞的双阳率；其中，PCDHF-85慢病毒得到85CART细胞、PCDHF-86慢病毒得到86CART细胞、PCDHF-89慢病毒得到89CART细胞，89CART细胞的慢病毒载体结构示意图如图5所示，pCDHF空载体核苷酸如SEQ ID NO：8所示。
本发明制备的89CART细胞的双阳率结果如图6所示，结果显示，89CART细胞的双阳率为30.31％，说明本发明实施例成功制备得到了89CART细胞。
实施例8 CART细胞的趋化作用
趋化因子CCL2能趋化表达趋化因子受体CCR2b的细胞向高浓度CCL2处迁移，而肿瘤部位高表达CCL2。因此，为了验证实施例7制备得到的89CART细胞向肿瘤部位的迁移能力，购买人趋化因子CCL2(金斯瑞，产品编号：Z02829)，X-VIVO 15造血干细胞无血清培养基(LONZA，批号，8MB230)，配制成30ng/mL浓度，共计2.4mL。
在Transwell小室自带的24孔板(康宁，货号3421，6.5mm直径，5.0um孔径PC膜)下层加入X-VIVO溶液，600μL/孔；然后加入30ng/mL的CCL2溶液，同时设置不加CCL2溶液的对照组，重复3次；缓慢放入Transwell小室上层，然后向上层加入实施例7制备得到的86CART细胞、89CART细胞和对照T细胞，细胞浓度均为2×10 5cells/100μL，趋化实验方案如表4所示。
5％CO 2培养箱37℃培养2h，用镊子小心取出Transwell小室，镜下观察86CART细胞和89CART细胞的迁移情况，吹打混匀下层培养基，移至1.5mL EP管中，2500rpm离心5min，弃上清，40μL的DPBS(Hyclone，批号AE29431662)重悬并计数。
表4趋化实验方案
Figure PCTCN2022117831-appb-000024
本发明制备的89CART细胞向肿瘤部位的迁移情况结果如图7所示，结果显示，与对 照T细胞和86CART细胞相比，89CART细胞向肿瘤部位的迁移能力显著提高。
实施例9 CART细胞的体外杀伤肿瘤细胞性能
向E-Plate 16 PET(ACEA，批号，20190125)板各孔加入10000个卵巢癌细胞(OVCAR-3细胞)(ATCC HTB161)，培养基为RPMI1640(Gibco，批号2215748)+20％FBS(Gibco批号2152441P)，100μL/孔，放在xCELLigence RTCA S16仪器上，5％CO 2，37℃培养48h，分别取T细胞，85CART细胞，89CART细胞，计数之后用T细胞将85CART细胞稀释到阳性率为37.79％，按效应细胞(CAR+CART细胞)∶靶细胞＝10∶1的比例分别加入各孔，CART细胞的培养基为X-VIVO 15，100μL/孔，CART细胞与肿瘤细胞混合培养25h，测定89CART细胞体外杀伤肿瘤细胞的能力。
本发明实施例制备的89CART细胞体外杀伤肿瘤细胞的能力结果如图8所示，结果显示，与对照T细胞和85CART细胞相比，89CART细胞体外杀伤肿瘤细胞的能力显著提高。
另外，分别检测T细胞，85CART细胞和89CART细胞培养(细胞起始密度均为2×10 5cells/mL，培养72h后收集上清，IL-21 ELISA检测试剂盒，Invitrogen，批号220657)上清中IL-21的表达水平。
结果显示，本发明制备的89CART细胞正常分泌IL-21和PD1抗体。
实施例10 CART细胞在小鼠体内杀伤肿瘤细胞的性能
构建ASPC-1-CCL2细胞株：从NCBI数据库查找人CCL2基因序列(见SEQ ID NO：13)构建在pCDHF载体上，命名为pCDHF-91质粒，再包装慢病毒(包装过程参考实施例6)，用包装的慢病毒感染ASPC-1细胞(ATCC CRL1682 TM)，第2天将感染后的细胞铺单克隆，选择高表达CCL2的ASPC-1扩大培养建立ASPC-1-CCL2细胞株。
Figure PCTCN2022117831-appb-000025
第0天，ASPC-1-CCL2细胞皮下接种至NPG小鼠(百奥赛图)，5.0E+06 cells/100μl+100μl基质胶(公司BD，货号356234)/只，3组小鼠分别在第7天尾静脉回输T细胞、85CART细胞 和89CART细胞(各2.0E+06 CAR+cells)，定期测量小鼠肿瘤体积变化(如图9所示)并在第20天检测外周血和肿瘤中的CCL2浓度(如图10所示)和肿瘤部位CART细胞比例(如图11所示)。
结果显示，皮下接种ASPC-1-CCL2后在肿瘤部位和外周血都可以检测到CCL2的表达，并且肿瘤部位的CCL2浓度较外周血中CCL2浓度高，可以趋化更多的89CART细胞至肿瘤部位，并且能更快更有效地在小鼠体内杀伤肿瘤细胞。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (23)

  1. 一种转基因免疫效应细胞，其特征在于，所述转基因免疫效应细胞共表达嵌合抗原受体、融合蛋白和趋化因子受体分子；
    所述嵌合抗原受体包括依次串联的胞外区、跨膜区和胞内区；其中，所述胞外区特异性识别肿瘤抗原，所述跨膜区嵌入所述转基因免疫效应细胞的细胞膜中，所述胞内区包括免疫共刺激分子的胞内段；
    所述融合蛋白包括免疫检查点抗体和细胞因子；
    所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
  2. 根据权利要求1所述转基因免疫效应细胞，其特征在于，所述趋化因子受体为CXC趋化因子受体、CC趋化因子受体、CX3C趋化因子受体或XC趋化因子受体中的任一种或几种；
    优选地，所述CXC趋化因子受体为CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6或CXCR7中的任一种或几种；
    优选地，所述CC趋化因子受体为CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10或CCR11中的任一种或几种；
    优选地，所述CX3C趋化因子受体为CX3CR1；
    优选地，所述XC趋化因子受体为XCR1；
    优选地，所述趋化因子受体分子为CCR2；
    优选地，所述趋化因子受体分子的氨基酸序列如SEQ ID NO：5所示。
  3. 根据权利要求1-2任一项所述转基因免疫效应细胞，其特征在于，所述免疫共刺激分子选自4-1BB或其衍生物、CD28或其衍生物、CD3或其衍生物、OX-40或其衍生物、CD40L或其衍生物、CD27或其衍生物、以及CD30或其衍生物的任一种或几种。
  4. 根据权利要求1-3任一项所述转基因免疫效应细胞，其特征在于，所述免疫检查点选自PD1、PD-L1、TIGIT、LAG3、CTLA4、BTLA或TIM3中的任一种或几种。
  5. 根据权利要求1-4任一项所述转基因免疫效应细胞，其特征在于，所述细胞因子为白细胞介素；
    优选地，所述细胞因子选自IL-21、IL-23、IL-2、IL-7、IL-9、IL-12、IL-15或IL-18中的任一种或几种；
    优选地，所述细胞因子为IL-21。
  6. 根据权利要求1-5任一项所述转基因免疫效应细胞，其特征在于，所述融合蛋白还 包括连接肽；所述免疫检查点抗体通过连接肽与所述细胞因子相连；
    优选地，所述连接肽为含有G和S氨基酸的短肽；
    优选地，所述连接肽的氨基酸序列如SEQ ID NO：7所示。
  7. 根据权利要求1-6任一项所述转基因免疫效应细胞，其特征在于，所述免疫检查点抗体为PD1抗体，所述细胞因子为IL-21；
    优选地，所述PD1抗体的C端与所述连接肽的N端相连，所述IL-21的N端与所述连接肽的C端相连。
  8. 根据权利要求1-7任一项所述转基因免疫效应细胞，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO：3或SEQ ID NO：9所示。
  9. 根据权利要求1-8任一项所述转基因免疫效应细胞，其特征在于，所述胞外区包括特异性识别肿瘤抗原的抗体和CD8铰链区；
    所述跨膜区包括CD8的跨膜段；
    所述免疫共刺激分子为4-1BB，所述胞内区包括4-1-BB的胞内段和CD3 Zeta链；
    所述胞外区的CD8铰链区与所述跨膜区相连，所述跨膜区与所述胞内区的4-1-BB的胞内段相连。
  10. 根据权利要求1-9任一项所述转基因免疫效应细胞，其特征在于，所述肿瘤抗原为MSLN、GD2、GPC3、CD19、EGFR VIII、GUCY2C、HER2、MUC16或Claudin 18.2中的任一种或几种。
  11. 根据权利要求1-10任一项所述转基因免疫效应细胞，其特征在于，所述肿瘤抗原为MSLN或CD19；所述嵌合抗原受体的氨基酸序列如SEQ ID NO：1或SEQ ID NO：11所示。
  12. 根据权利要求1-11任一项所述转基因免疫效应细胞，其特征在于，所述嵌合抗原受体、融合蛋白和趋化因子受体分子通过2A肽相连；
    优选地，所述2A肽选自P2A、T2A、F2A或E2A中的任一种或几种；
    优选地，所述嵌合抗原受体通过P2A与所述融合蛋白相连，所述融合蛋白通过T2A与所述趋化因子受体分子相连。
  13. 根据权利要求1-12任一项所述转基因免疫效应细胞，其特征在于，所述转基因免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的任一种或几种。
  14. 一种慢病毒，其特征在于，所述慢病毒携带以下核酸：
    1)编码权利要求1-13任一项所述转基因免疫效应细胞中的嵌合抗原受体的核酸，所述 嵌合抗原受体的胞外区特异性识别肿瘤抗原；
    2)编码权利要求1-13任一项所述转基因免疫效应细胞中的融合蛋白的核酸，所述融合蛋白包括免疫检查点抗体和细胞因子；
    3)编码权利要求1-13任一项所述转基因免疫效应细胞中的趋化因子受体分子的核酸，所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
  15. 根据权利要求14所述慢病毒，其特征在于，所述肿瘤抗原为MSLN或CD19，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：1或SEQ ID NO：11所示，
    优选地，编码所述嵌合抗原受体的核酸具有如SEQ ID NO：2或SEQ ID NO：12所示的核苷酸序列；
    优选地，所述免疫检查点抗体为PD1抗体，所述细胞因子为IL-21，所述融合蛋白的氨基酸序列如SEQ ID NO：3或SEQ ID NO：9所示，优选地，编码所述融合蛋白的核酸具有如SEQ ID NO：4或SEQ ID NO：10所示的核苷酸序列；
    优选地，所述趋化因子受体分子为CCR2b，所述趋化因子受体分子的氨基酸序列如SEQ ID NO：5所示，优选地，编码所述趋化因子受体分子的核酸具有如SEQ ID NO：6所示的核苷酸序列。
  16. 一种构建体，其特征在于，所述构建体包括：
    第一核酸分子，所述第一核酸分子编码权利要求1-13任一项所述转基因免疫效应细胞中的嵌合抗原受体，所述嵌合抗原受体的胞外区特异性识别肿瘤抗原；
    第二核酸分子，所述第二核酸分子编码权利要求1-13任一项所述转基因免疫效应细胞中的融合蛋白，所述融合蛋白包括免疫检查点抗体和细胞因子；
    第三核酸分子，所述第三核酸分子编码权利要求1-13任一项所述转基因免疫效应细胞中的趋化因子受体分子，所述趋化因子受体分子为趋化因子受体或其趋化因子结合片段。
  17. 根据权利要求16所述构建体，其特征在于，所述第一核酸分子、第二核酸分子和第三核酸分子在免疫效应细胞中依次分别表达嵌合抗原受体、融合蛋白和趋化因子受体分子；
    所述嵌合抗原受体、融合蛋白和趋化因子受体分子呈非融合形式；
    所述转基因免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的任一种或几种；
    任选地，所述转基因免疫效应细胞为T细胞。
  18. 根据权利要求16-17任一项所述构建体，其特征在于，所述构建体还包括：第一启 动子，所述第一启动子与所述第一核酸分子可操作的连接；
    任选地，所述第一启动子选自U6、H1、CMV、EF-1、LTR或RSV启动子中的任一种或几种。
  19. 根据权利要求16-18任一项所述构建体，其特征在于，所述构建体的载体是非致病性病毒载体；
    任选地，所述非致病性病毒载体选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体中的任一种或几种；
    任选地，所述非致病性病毒载体为慢病毒载体；
    任选地，编码所述慢病毒载体的核酸具有SEQ ID NO：8所示的核苷酸序列。
  20. 一种制备权利要求1～13任一项所述转基因免疫效应细胞的方法，其特征在于，将权利要求14或15所述慢病毒、或权利要求16～19任一项所述构建体导入免疫效应细胞中；
    所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的任一种或几种；
    任选地，所述免疫效应细胞为T细胞。
  21. 权利要求1～13任一项所述的转基因免疫效应细胞、或权利要求14或15所述的慢病毒、或权利要求16～19任一项所述的构建体在制备癌症治疗药物中的应用；
    优选地，所述癌症为实体瘤。
  22. 一种癌症治疗药物，其特征在于，包括权利要求1～13任一项所述转基因免疫效应细胞、权利要求14或15所述慢病毒或权利要求16～19任一项所述构建体；
    优选地，所述癌症为实体瘤。
  23. 一种治疗癌症的方法，其特征在于，其包括：向需要治疗的主体施用治疗有效量的如权利要求22所述的癌症治疗药物；
    优选地，所述癌症为实体瘤。
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