WO2024120506A1 - 一种修饰的细胞及其用途 - Google Patents

一种修饰的细胞及其用途 Download PDF

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Publication number
WO2024120506A1
WO2024120506A1 PCT/CN2023/137333 CN2023137333W WO2024120506A1 WO 2024120506 A1 WO2024120506 A1 WO 2024120506A1 CN 2023137333 W CN2023137333 W CN 2023137333W WO 2024120506 A1 WO2024120506 A1 WO 2024120506A1
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Prior art keywords
cells
cell
present
expression
activity
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English (en)
French (fr)
Inventor
刘雅容
孙静玮
盛耀
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Shenzhen Grit Biotechnology Co Ltd
Shanghai Grit Biotechnology Co Ltd
Suzhou Grit Biotechnology Co Ltd
Zhuhai Tuoyu Biotechnology Co Ltd
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Shenzhen Grit Biotechnology Co Ltd
Shanghai Grit Biotechnology Co Ltd
Suzhou Grit Biotechnology Co Ltd
Zhuhai Tuoyu Biotechnology Co Ltd
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Application filed by Shenzhen Grit Biotechnology Co Ltd, Shanghai Grit Biotechnology Co Ltd, Suzhou Grit Biotechnology Co Ltd, Zhuhai Tuoyu Biotechnology Co Ltd filed Critical Shenzhen Grit Biotechnology Co Ltd
Priority to EP23900075.5A priority Critical patent/EP4632074A1/en
Priority to CN202380084095.7A priority patent/CN120344671A/zh
Publication of WO2024120506A1 publication Critical patent/WO2024120506A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/19—Omega peptidases (3.4.19)
    • C12Y304/19012—Ubiquitinyl hydrolase 1 (3.4.19.12)
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a modified cell and a use thereof.
  • immunotherapy is an effective method for treating patients with poor prognosis.
  • the immune cells used in immunotherapy have problems such as weak cell function or weak proliferation and survival after infusion. Therefore, how to provide a modified immune cell and a robust and reliable immune cell culture method are urgent issues to be solved.
  • the present invention provides a method for culturing cells, which has one or more of the following advantages: enhanced target cell killing ability, enhanced cell proliferation ability, enhanced cytokine release ability, increased proportion of activated cells, reduced proportion of regulatory cells, reduced proportion of exhausted cells, increased proportion of central memory cells and/or immature cells, reduced proportion of apoptotic cells and increased proportion of stem cell-like cells.
  • the present invention provides a method for culturing cells, the method comprising: reducing the expression and/or attenuating the activity of family members and/or functionally active fragments thereof selected from the peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family in the cells.
  • family members and/or functionally active fragments thereof selected from the peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family in the cells.
  • SSI STAT-induced STAT inhibitor
  • the present invention provides a cell obtained by the method of the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the cell of the present invention, and optionally a pharmaceutically acceptable carrier.
  • the present invention provides a method of influencing cell growth, comprising administering The cell of the invention and/or the pharmaceutical composition of the invention.
  • the present invention provides use of the cell of the present invention and/or the pharmaceutical composition of the present invention in the preparation of a medicament for preventing and/or treating a disease and/or symptom.
  • FIG1A shows the human TNFAIP3 gene editing targeting segment relative to the start codon provided by the present invention, for example, can be a continuous region with about 3 or more transcription factor binding numbers; and can be an exon region of the gene or an intron region about 20 bp away from the exon.
  • Figure 1B shows the human ZC3H12A gene editing targeting segment relative to the start codon provided by the present invention, for example, can be a continuous region with about 3 or more transcription factor binding numbers; and can be an exon region of the gene or an intron region about 20 bp away from the exon.
  • Figure 1C shows the human SOCS1 gene editing targeting segment relative to the start codon provided by the present invention, for example, can be a continuous region with about 3 or more transcription factor binding numbers; and can be an exon region of the gene or an intron region about 20 bp away from the exon.
  • FIG. 1D shows the human CBLB gene editing targeting segment relative to the start codon provided by the present invention, for example, can be a continuous region with about 3 or more transcription factor binding numbers; and can be an exon region of the gene or an intron region about 20 bp away from the exon. Sub-area.
  • Figure 2A shows the fold expansion of TNFAIP3 gene-edited TCR-T cells in the non-stimulation medium group.
  • Figures 2B-2C show the fold amplification of TNFAIP3 gene-edited TCR-T cells in the TransACT stimulation group.
  • Figures 2D-2G show the target cell killing ability of TCR-T cells from different donors after TNFAIP3 gene editing.
  • Figures 2D and 2F show the killing curves at each time point, and
  • Figures 2E and 2G show the killing conditions of each test group at the end of the test, which are all higher than the unedited NT group.
  • Figures 2H-2K show the various cytokine release capabilities of TNFAIP3 gene-edited TCR-T cells.
  • Figure 3A shows the expansion fold of ZC3H12A gene-edited TCR-T in the non-stimulation medium group.
  • Figure 3B shows the fold expansion of ZC3H12A gene-edited TCR-T cells in the TransACT stimulation group.
  • Figures 3C-3F show the target cell killing ability of ZC3H12A gene-edited TCR-T cells.
  • Figures 3C and 3E show the killing curves at each time point, and
  • Figures 3D and 3F show the killing conditions of each test group at the end of the test, which are all higher than the unedited NT group.
  • Figures 3G-3J show the various cytokine release capabilities of ZC3H12A gene-edited TCR-T cells.
  • Figure 4A shows the expansion fold of SOCS1 gene-edited TCR-T in the non-stimulation culture medium group.
  • Figure 4B shows the fold expansion of SOCS1 gene-edited TCR-T cells in the TransACT stimulation group.
  • Figure 4C shows the expansion fold of SOCS1 gene-edited TILs from different donors in the non-stimulation culture medium group.
  • FIG. 4D shows the expansion fold of SOCS1 gene-edited TILs from donor 306 in the TransACT stimulation group.
  • FIGS 4E-4H show the target cell killing ability of SOCS1 gene-edited TCR-T cells.
  • FIG4I shows the target cell killing ability of SOCS1 gene-edited TIL cells.
  • Figure 4J shows the cytokine expression of SOCS1 gene-edited TCR-T cells in the unstimulated group.
  • Figure 4K shows the cytokine expression of SOCS1 gene-edited TCR-T cells in the CD3 antibody stimulation group.
  • FIG4L shows the cytokine release capacity of SOCS1 gene-edited TCR-T cells co-cultured with A375 target cells.
  • Figure 4M shows the cytokine expression of SOCS1 gene-edited TIL cells in the unstimulated group.
  • Figure 4N shows the cytokine expression of SOCS1 gene-edited TIL cells in the TransACT stimulation group.
  • Figure 4O shows that TIL cells after SOCS1 gene editing have a higher proportion of stem cells.
  • Figure 4P shows that TIL cells after SOCS1 gene editing have a lower proportion of exhausted T cells.
  • Figures 5A-5B show the fold expansion of CBLB gene-edited TCR-T cells in the non-stimulation culture medium group.
  • Figures 5C-5D show the fold expansion of CBLB gene-edited TCR-T cells in the TransACT stimulation group.
  • Figures 5E-5H show the target cell killing ability of CBLB gene-edited TCR-T cells.
  • Figures 5E and 5G show the killing curves at each time point, and
  • Figures 5F and 5H show the killing conditions of each test group at the end of the test, which are all higher than the unedited NT group.
  • Figures 5I-5L show the various cytokine release capabilities of CBLB gene-edited TCR-T cells.
  • Figure 6A shows the expansion fold of TILs edited with the combination of CBLB and ZC3H12A in the non-stimulation medium group.
  • Figure 6B shows the expansion fold of TILs edited with the combination of SOCS1 and CBLB in the non-stimulation medium group.
  • Figure 6C shows the expansion fold of TILs edited with the combination of SOCS1 and TNFAIP3 genes in the non-stimulation medium group.
  • Figure 6D shows the expansion fold of TILs edited with the combination of SOCS1 and TNFAIP3 genes in the CD3 antibody stimulation group.
  • Figure 6E shows the expansion fold of TILs edited with the combination of SOCS1 and ZC3H12A in the non-stimulation medium group.
  • Figure 6F shows the expansion fold of TILs edited with the combination of SOCS1 and ZC3H12A in the CD3 antibody stimulation group.
  • Figure 6G shows the expansion fold of TILs edited with the combination of TNFAIP3 and CBLB in the non-stimulation medium group.
  • Figure 6H shows the expansion fold of TILs edited with the combination of TNFAIP3 and ZC3H12A in the non-stimulation medium group.
  • Figure 6I shows the expansion fold of TILs edited with the combination of TNFAIP3 and ZC3H12A in the CD3 antibody stimulation group.
  • Figure 6J shows the expansion fold of TILs edited with the combination of TNFAIP3 and SOCS1 in the non-stimulation medium group.
  • Figure 6K shows the expansion fold of TILs with combined gene editing of TNFAIP3 and SOCS1 in the TransACT antibody stimulation group.
  • FIG7A shows the target cell killing ability of TIL cells edited by the CBLB and ZC3H12A combination gene.
  • FIG7B shows the target cell killing ability of TIL cells edited by the CBLB and ZC3H12A combination gene.
  • FIG7C shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and CBLB.
  • FIG7D shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and CBLB.
  • FIG7E shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and CBLB.
  • FIG7F shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and TNFAIP3.
  • Figure 7G shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and TNFAIP3.
  • Figure 7H shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and TNFAIP3.
  • FIG7I shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and TNFAIP3.
  • Figure 7J shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and ZC3H12A.
  • Figure 7K shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and ZC3H12A.
  • Figure 7L shows the target cell killing ability of TIL cells edited by the combination gene of SOCS1 and ZC3H12A.
  • FIG7M shows the target cell killing ability of TIL cells edited by the TNFAIP3 and CBLB combination genes.
  • Figure 7N shows the target cell killing ability of TIL cells edited by the TNFAIP3 and CBLB combination genes.
  • FIG. 7O shows the target cell killing ability of TIL cells edited by the combination gene of TNFAIP3 and ZC3H12A.
  • Figure 7P shows the target cell killing ability of TIL cells edited by the TNFAIP3 and ZC3H12A combination gene.
  • Figure 7Q shows the target cell killing ability of TIL cells edited by the combination gene of TNFAIP3 and ZC3H12A.
  • Figure 7R shows the target cell killing ability of TIL cells edited by the combination gene of TNFAIP3 and ZC3H12A.
  • Figure 7S shows the target cell killing ability of TIL cells edited by the combination gene of TNFAIP3 and SOCS1.
  • Figure 7T shows the killing ability of TIL cells edited by the combination of TNFAIP3 and SOCS1 genes on autologous tumor organoids.
  • Figure 8A shows that TIL cells after combined gene editing of CBLB and ZC3H12A have a lower proportion of exhausted T cells.
  • Figure 8B shows that TIL cells after combined gene editing of SOCS1 and CBLB have a higher proportion of central memory T cells.
  • Figure 8C shows that TIL cells after combined gene editing of SOCS1 and CBLB have a lower proportion of exhausted T cells.
  • Figure 8D shows that TIL cells after combined gene editing of SOCS1 and TNFAIP3 have a higher proportion of central memory T cells.
  • Figure 8E shows that TIL cells after combined gene editing of SOCS1 and TNFAIP3 have a lower proportion of exhausted T cells.
  • Figure 8F shows that TIL cells after combined gene editing of SOCS1 and ZC3H12A have a higher proportion of central memory T cells.
  • FIGS. 8G-8H show that TIL cells after combined gene editing of SOCS1 and ZC3H12A had a lower proportion of exhausted T cells.
  • Figure 8I shows that TIL cells after TNFAIP3 and CBLB combination gene editing have a lower proportion of exhausted T cells.
  • Figure 8J shows that TIL cells after combined gene editing of TNFAIP3 and ZC3H12A have a higher proportion of naive T cells.
  • Figure 8K shows that TIL cells after combined gene editing of TNFAIP3 and ZC3H12A have a higher proportion of central memory T cells.
  • Figures 8L-8M show that TIL cells after combined gene editing of TNFAIP3 and ZC3H12A have a lower proportion of exhausted T cells.
  • Figure 8N shows that TIL cells after combined gene editing of TNFAIP3 and SOCS1 have a higher proportion of stem cells.
  • Figure 8O shows that TIL cells after combined gene editing of TNFAIP3 and SOCS1 have a lower proportion of exhausted T cells.
  • Figure 9A shows that TIL cells after gene editing of CBLB and ZC3H12A combination in the CD3 antibody stimulation group had a higher cytokine expression ratio.
  • Figure 9B shows that TIL cells after gene editing of SOCS1 and CBLB combination in the non-stimulated Medium group had a higher cytokine expression ratio.
  • Figure 9C shows that TIL cells after gene editing of SOCS1 and CBLB combination in the CD3 antibody stimulation group had a higher cytokine expression ratio.
  • Figure 9D shows that TIL cells after gene editing of SOCS1 and TNFAIP3 combination in the unstimulated Medium group had a higher cytokine expression ratio.
  • Figure 9E shows that TIL cells after SOCS1 and TNFAIP3 combination gene editing in the CD3 antibody stimulation group had a higher cytokine expression ratio.
  • Figure 9F shows that TIL cells after gene editing of SOCS1 and ZC3H12A combination in the unstimulated Medium group had a higher cytokine expression ratio.
  • Figure 9G shows that TIL cells after gene editing of SOCS1 and ZC3H12A combination in the CD3 antibody stimulation group had a higher cytokine expression ratio.
  • Figure 9H shows that TIL cells after gene editing of TNFAIP3 and CBLB combination in the unstimulated Medium group had a higher cytokine expression ratio.
  • Figure 9I shows that TIL cells after gene editing of TNFAIP3 and CBLB combination in the CD3 antibody stimulation group have a higher cytokine expression ratio.
  • Figures 9J-9K show that TIL cells after gene editing of TNFAIP3 and ZC3H12A combination in the unstimulated Medium group had a higher cytokine expression ratio.
  • Figure 9L shows that TIL cells after TNFAIP3 and ZC3H12A combination gene editing in the CD3 antibody stimulation group had a higher cytokine expression ratio.
  • Figure 9M shows that TIL cells after TNFAIP3 and SOCS1 combination gene editing co-cultured with autologous tumor organoids have higher cytokine release capacity.
  • FIG. 10 shows the results of apoptosis detection of TIL cells derived from donor 504.
  • CBL family member generally refers to a family member protein having an SH3 domain or a functionally active fragment thereof.
  • a CBL family member may include CBLB.
  • the UniProt number of a CBL family member may be Q13191.
  • the CBL family members of the present invention may also include functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing the functionally active fragments thereof produced after processing and/or modification in cells.
  • the CBL family members of the present invention may include functionally active fragments thereof and any other domains.
  • STAT-induced STAT inhibitor (SSI) family member generally refers to a family member protein having an SH2 domain or a functionally active fragment thereof.
  • a STAT-induced STAT inhibitor (SSI) family member may include SOCS1.
  • the UniProt number of the STAT-induced STAT inhibitor (SSI) family member can be O15524.
  • the STAT-induced STAT inhibitor (SSI) family member of the present invention can also cover its functionally active fragments, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, its active fragments, or substances containing the functionally active fragments produced after processing and/or modification thereof in cells.
  • the STAT-induced STAT inhibitor (SSI) family member of the present invention can include its functionally active fragments and other arbitrary domains.
  • the term "peptidase C64 family member” generally refers to a family member protein or a functionally active fragment thereof having a ubiquitin binding domain.
  • the peptidase C64 family member may include TNFAIP3.
  • the UniProt number of the peptidase C64 family member may be P21580.
  • the peptidase C64 family member of the present invention may also encompass its functionally active fragments, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, its active fragments, or substances containing the functionally active fragments produced after processing and/or modification thereof in cells.
  • the peptidase C64 family member of the present invention may include its functionally active fragments and any other domains.
  • the term "ZC3H12 family member” generally refers to a family member protein or a functionally active fragment thereof having a C3H1-type zinc finger domain.
  • the ZC3H12 family member may include ZC3H12A.
  • the UniProt number of the ZC3H12 family member may be Q5D1E8.
  • the ZC3H12 family members of the present invention may also include functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing the functionally active fragments thereof produced after processing and/or modification in cells.
  • the ZC3H12 family members of the present invention may include functionally active fragments thereof and any other domains.
  • the term "IKAROS zinc finger protein family member” generally refers to a family member protein having a zinc finger domain or a functionally active fragment thereof.
  • the IKAROS zinc finger protein family member may include IKZF1.
  • the UniProt number of the IKAROS zinc finger protein family member may be Q13422.
  • the IKAROS zinc finger protein family member of the present invention may also include It can include functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing the functionally active fragments produced after processing and/or modification thereof in cells.
  • the IKAROS zinc finger protein family members of the present invention can include functionally active fragments thereof and any other domains.
  • TNFAIP3 tumor necrosis factor alpha-induced protein 3
  • TNFAIP3 can ubiquitinate a signal transduction substance of the NF- ⁇ B pathway.
  • the UniProt accession number of TNFAIP3 can be P21580.
  • TNFAIP3 can include unprocessed TNFAIP3, any form of processed TNFAIP3, a variant of TNFAIP3, or a substance containing a functionally active fragment of TNFAIP3.
  • GTPase activating protein 1 family member generally refers to a family member protein having a GTPase activation domain or a functionally active fragment thereof.
  • a GTPase activating protein 1 family member may include RASA2.
  • the UniProt number of a GTPase activating protein 1 family member may be Q15283.
  • the GTPase activating protein 1 family member of the present invention may also encompass its functionally active fragments, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, its active fragments, or substances containing the functionally active fragments produced after processing and/or modification thereof in cells.
  • the GTPase activating protein 1 family member of the present invention may include its functionally active fragments and any other domains.
  • the term "FGF binding protein family member” generally refers to a family member protein having an FGF binding domain or a functionally active fragment thereof.
  • the FGF binding protein family member may include FIBP.
  • the UniProt number of the FGF binding protein family member may be O43427.
  • the FGF binding protein family member of the present invention may also encompass its functionally active fragments, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, its active fragments, or substances containing the functionally active fragments produced after processing and/or modification thereof in cells.
  • the FGF binding protein family member of the present invention may include its functionally active fragments and any other domains.
  • the term "Mediator (MED) family member” generally refers to a family member protein or a functionally active fragment thereof having a CDK8 binding domain.
  • the Mediator (MED) family member may include MED12.
  • the UniProt number of the Mediator (MED) family member may be Q93074.
  • the Mediator (MED) family members of the present invention may also include functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing the functionally active fragments thereof produced after processing and/or modification in cells.
  • the Mediator (MED) family members of the present invention may include functionally active fragments thereof and any other domains.
  • the term "immune cell” generally refers to cells involved in innate and adaptive immune responses.
  • lymphocytes such as T cells (including thymocytes) and B cells
  • natural killer (NK) cells such as T cells (including thymocytes) and B cells
  • NK natural killer
  • NKT cells NKT cells
  • macrophages monocytes
  • eosinophils basophils
  • neutrophils dendritic cells and mast cells
  • the modified immune effector cells are T cells, such as CD4+T cells, CD8+T cells (also referred to as cytotoxic T cells or CTL), regulatory T cells (Treg), Th1 cells, Th2 cells, Th17 cells ⁇ T cells and/or ⁇ T cells.
  • the immune cells of the present invention also include immune cells derived from stem cell differentiation.
  • the immune cells of the present invention also include immune cells derived from pluripotent stem cell differentiation.
  • obtaining the stem cells of the present invention can be produced by induction.
  • the above-mentioned stem cells of the present invention can include induced pluripotent stem cells (iPSC).
  • the term "chimeric antigen receptor" generally refers to an engineered antigen receptor.
  • CAR may include an extracellular antigen binding domain fused to a cytoplasmic domain comprising a signaling domain via a hinge and a transmembrane domain.
  • the CAR extracellular domain can bind to an antigen expressed by a target cell in an MHC-independent manner, resulting in cell activation and proliferation.
  • the extracellular domain of CAR can recognize a tag fused to an antibody or its antigen-binding fragment.
  • a single CAR construct can be made to target multiple different antigens by replacing one antibody with another.
  • the extracellular domain of CAR may include a protein derived from an antibody. Antigen binding fragments.
  • Antigen binding domains useful in the present disclosure may include, for example, scFv, antibodies, antigen binding regions of antibodies, variable regions of heavy/light chains, and/or single chain antibodies.
  • T cell receptor generally refers to an engineered antigen receptor.
  • TCR may include TCR alpha and/or TCR beta chains that have been isolated and cloned from a T cell population that recognizes a specific target antigen.
  • TCR alpha and/or TCR beta genes i.e., TRAC and TRBC
  • TRAC and TRBC may be cloned from a T cell population isolated from an individual with a specific malignancy or from a T cell population isolated from a humanized mouse immunized with a specific tumor antigen or tumor cell.
  • Engineered TCRs can recognize antigens (e.g., by recognizing their cognate antigens presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of target cells) by the same mechanism as their endogenous counterparts, thereby resulting in activation and proliferation of TCR engineered cells.
  • MHC major histocompatibility complex
  • a gene regulation system generally refers to a system that regulates the expression or activity of a target gene.
  • a gene regulation system may include gene regulation molecules.
  • a gene regulation system may regulate the expression or activity of a gene, such as rendering the gene in an inactive or activated state, increasing or decreasing the amount of the gene, rendering the gene in a state of increased or decreased transcription, and/or rendering the transcription product of the gene in an inactive or activated state; for example, a gene regulation system may regulate the expression or activity of a gene, such as increasing or decreasing the amount of the expression product of the gene in a single cell and/or increasing or decreasing the number of cells expressing the expression product of the gene.
  • the term "guide nucleic acid molecule” generally refers to a nucleic acid molecule that can be used for gene editing.
  • a guide nucleic acid molecule can provide information for nucleotide insertion or deletion to guide the editing process.
  • a guide nucleic acid molecule can be a guide RNA or a guide RNA (gRNA).
  • gRNA can refer to an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific position in a target DNA.
  • the term "enzyme protein” generally refers to a protein with enzymatic activity.
  • the enzyme protein may refer to a Cas protein.
  • the Cas protein may include at least one RNA recognition or binding domain, which may interact with the gRNA.
  • the Cas protein may also include a nuclease domain (e.g., a DNA enzyme or RNA enzyme domain), a DNA binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and/or other domains.
  • the nuclease domain may have catalytic activity for nucleic acid cleavage. The cleavage may include the breaking of covalent bonds of nucleic acid molecules.
  • the Cas protein may be a wild-type protein (i.e., a protein existing in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein.
  • the Cas protein may also be an active variant or fragment of a wild-type or modified Cas protein.
  • the Cas protein may include an unprocessed Cas protein, any form of processed Cas protein, a variant of the Cas protein, or a substance comprising a functionally active fragment of the Cas protein.
  • the term "ribonucleoprotein complex” generally refers to a complex formed by a protein and a nucleic acid.
  • the protein in the ribonucleoprotein complex can have nuclease activity.
  • the ribonucleoprotein complex can cut the target sequence under the guidance of the nucleic acid therein.
  • the ribonucleoprotein complex can be a complex formed by a Cas protein and a gRNA.
  • lipid nanoparticle generally refers to a lipid-nucleic acid particle or a nucleic acid-lipid particle.
  • LNP refers to a particle made of lipids (e.g., cationic lipids, non-cationic lipids, and conjugated lipids that prevent particle aggregation) and nucleic acids, wherein the nucleic acid (e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, self-amplifying RNA or plasmid, including plasmids from which interfering RNA or mRNA is transcribed) is encapsulated in lipids.
  • nucleic acid e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA
  • proteins can be encapsulated in LNPs, for example, Cas proteins known in the art can be encapsulated in LNPs.
  • the lipids in LNPs include (1) "simple lipids", which include fats and oils and waxes; (2) "complex lipids", which include phospholipids and glycolipids; and (3) "derived lipids” such as steroids.
  • the lipids in LNPs may also include lipid derivatives, such as lipids covalently or non-covalently bound to proteins or polypeptides.
  • the components in LNPs may also include polypeptide components, which The polypeptide component in the LNP can replace one or more lipid components in traditional LNP to maintain or improve the delivery ability of LNP.
  • exon generally refers to a portion of a gene that can be expressed as a protein.
  • an exon can refer to a portion of a gene that has the ability to be expressed as a protein during protein biosynthesis.
  • shearing the exon sequence of a target gene can reduce the activity or function of the target gene.
  • intron generally refers to a segment in DNA that does not encode part or all of the expressed protein. Usually under endogenous conditions, introns are transcribed into RNA molecules, but they are sheared off from endogenous RNA before being translated into proteins. For example, editing by targeting the position of introns can reduce the activity or function of the target gene. For example, editing by targeting the junction of introns and exons, such as editing intron regions about 0 bp to about 100 bp upstream or downstream of the exon, preferably about 0 bp to about 20 bp, can reduce the activity or function of the target gene.
  • start codon generally refers to a unit of adjacent nucleotides ('codon') on a gene that can define the start of protein synthesis (mRNA translation). For example, targeting the region 0 bp to 1500 bp upstream of the start codon, preferably 0 bp to 100 bp upstream of the start codon for editing, can reduce the activity or function of the target gene.
  • the term "protospacer adjacent motif (PAM)” generally refers to a short sequence after a target sequence.
  • PAM sequence can be used to determine the location of the cleavage. For example, by determining the region of the PAM, a person skilled in the art can easily determine the appropriate target sequence location, and can easily design a gRNA sequence for cleaving the target sequence.
  • the term "reduced expression” generally refers to a decrease in the amount of expression of a product or its gene and/or a decrease in the proportion of cells capable of expressing the product (e.g., at least about 5-100%). For example, it may be a decrease in the amount of the product expressed by the gene in the cell or a decrease in the amount of the product containing the gene expressed by the gene.
  • the proportion of cells expressing the product of the gene is reduced, or the proportion of cells secreting the product expressed by the gene is reduced.
  • the amount of knockout of the gene in the genome of the cell can be detected to indirectly indicate that the expression of the gene is reduced.
  • the proportion of cells in which the gene is knocked out in a cell population can be detected to indirectly indicate that the expression of the gene is reduced.
  • the term "activity" generally refers to the biological function of a substance.
  • the activity of a gene may refer to the transcription and/or translation state of the gene.
  • a reduction in the activity of a gene e.g., at least about 5-100% may refer to a reduction in the transcription function of the gene, the gene being unable to be normally transcribed, or the function of the transcription product of the gene being inhibited.
  • CD80 generally refers to a cell stimulatory molecule.
  • CD80 can be a ligand of CD28.
  • CD80 can be found in GenBank Accession No. P33681.
  • the CD80 protein of the present invention can also cover its functionally active fragments, not limited to substances containing functionally active fragments of CD80 produced after processing and/or modification in cells.
  • the CD80 of the present invention can include functionally active fragments of CD80 and other arbitrary domains.
  • CD86 generally refers to a cell stimulatory molecule.
  • CD86 can be a ligand of CD28.
  • CD86 can be found in GenBank Accession No. P42081.
  • the CD86 protein of the present invention can also cover its functionally active fragments, not limited to substances containing functionally active fragments of CD86 produced after processing and/or modification in cells.
  • the CD86 of the present invention can include functionally active fragments of CD86 and any other domains.
  • secreted generally refers to a substance that can be located outside of a cell.
  • a secreted substance can be transported to the extracellular space of a cell after being synthesized inside the cell.
  • whether a substance is a secreted substance can be detected by enzyme-linked immunosorbent assay or other detection methods.
  • T cell receptor generally refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigens.
  • TCRs may be responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • TCRs may be composed of alpha ( ⁇ ) and beta ( ⁇ ) chains.
  • a TCR may be a heterodimer, or composed of a gamma and delta ( ⁇ / ⁇ ) chain.
  • TCRs may exist in ⁇ / ⁇ and ⁇ / ⁇ forms, which are structurally similar but have unique anatomical locations and functions.
  • a TCR may be a modified TCR on any cell that expresses the TCR.
  • the type of TCR may be analyzed by a TCR subtyping reagent.
  • the term "clonal diversity” generally refers to a substance having multiple clonal types.
  • the clonal diversity of TCR may mean that TCR may have different sequence structures and/or antigen recognition capabilities.
  • the diversity of TCR is often distinguished by ⁇ chain subtypes, which may include V ⁇ 23, V ⁇ 7.2, V ⁇ 5.2, V ⁇ 11, V ⁇ 16, V ⁇ 3, etc. When a T cell population has more ⁇ chain subtypes, it can be considered that the T cell population has higher clonal diversity.
  • CD4 + cells generally refer to CD4-positive cells, such as T cells.
  • CD4 + cells and “CD4-positive cells” can be used synonymously.
  • These cells can be identified by methods known in the art, such as by staining the cells with fluorescently labeled antibodies against CD4 and using fluorescence-activated cell sorting.
  • existing data can show that an increase in the proportion of CD4 + cells can increase the ability of the cell population to secrete IFN and/or TNF, and can increase the effect of the T cell population in promoting tumor suppression.
  • Tay, RE, Richardson, EK et al. 2020. Cancer Gene Therapy, 1-13.
  • the art lacks a method for increasing the proportion of CD4 + cells, and the present invention can provide a method for affecting the proportion of CD4 + cells.
  • CD8 + cells generally refer to cells that are positive for CD8, such as T cells.
  • IC50 value or “IC50 value” generally refers to the concentration of the target substance required to obtain 50% inhibition of a biological process.
  • the IC50 value can be converted into an absolute inhibition constant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22: 3099).
  • KD value or “KD value” generally refers to the dissociation constant, which can be determined by surface plasmon resonance.
  • surface plasmon resonance analysis uses the BIAcore system (Pharmacia Biosensor, Piscataway, NJ) to measure the real-time binding interaction between the ligand (the substance immobilized on the biosensor matrix) and the analyte (the substance in the solution) by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • SPR surface plasmon resonance
  • SPR surface plasmon resonance
  • SPR surface plasmon resonance
  • the term "encoding” generally refers to the ability to directly or indirectly infer the structure or composition information of another type of molecule related to it from the structure or composition information of one molecule according to basically determined rules.
  • the nucleotide sequence can be inferred from the sequence of amino acids, such as the characteristics of complementary nucleic acids transcribed from deoxyribonucleic acid, including nucleic acids that can be translated into polypeptides.
  • deoxyribonucleic acid can encode RNA transcribed from deoxyribonucleic acid.
  • Deoxyribonucleic acid can similarly encode polypeptides translated from RNA transcribed from deoxyribonucleic acid.
  • small molecule compound generally refers to peptides, peptide mimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic substances with a molecular weight of less than about 10,000 g/mole (i.e., including heterologous organic substances and organometallic compounds), organic or inorganic substances with a molecular weight of less than about 5,000 g/mole, organic or inorganic substances with a molecular weight of less than about 1,000 g/mole, organic or inorganic substances with a molecular weight of less than about 500 g/mole, and salts, esters and other pharmaceutically acceptable forms of such drugs.
  • NK cell is also called “natural killer cell”, which generally refers to a cell with large granules in the cytoplasm.
  • NK cells develop from bone marrow lymphoid stem cells and can differentiate and develop depending on the bone marrow or thymus microenvironment.
  • the proportion of NK cells in TIL cells can be changed by the method of the present invention.
  • the term "antibody” generally refers to an immunoglobulin or its fragment or derivative, and covers any polypeptide comprising an antigen binding site, whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, Non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated and transplanted antibodies.
  • antibody also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specific binding to CD3). Generally, such fragments should include an antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies are composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contain 10 antigen binding sites, while IgA antibodies include 2-5 basic 4-chain units that can be combined with J chains to form a multivalent combination.
  • the 4-chain unit is generally about 150,000 Daltons.
  • Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for ⁇ and ⁇ chains, and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are considered to form an interface between the light chain and the heavy chain variable domain. VH and VL pair together to form a single antigen binding site.
  • L chains from any vertebrate species can be divided into one of two distinct types based on the amino acid sequence of their constant domains, called ⁇ and ⁇ .
  • immunoglobulins can be divided into different categories or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the term "antigen binding fragment” generally refers to one or more polypeptide fragments that have the ability to specifically bind to an antigen.
  • the antigen binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the term "expression” generally refers to the transcription and/or translation process of a gene encoding a target polypeptide in a cell.
  • the transcription level of a gene encoding a target polypeptide in a host cell can be determined by measuring the amount of the corresponding mRNA present in the cell.
  • the mRNA transcribed by the gene encoding the target polypeptide can be quantitatively measured by PCR or by RNA hybridization.
  • the translation level of the gene encoding the target polypeptide can be measured by a variety of methods, such as by ELISA, by a polypeptide biological activity test, or by Western blotting or radioimmunoassay.
  • the term "expression" can also refer to the transcription and/or translation process of the product generally.
  • the expression of a cytokine can be the process of cell transcription and/or translation of the cytokine.
  • the expression of a cytokine can be determined by detecting the amount of the corresponding mRNA present in the cell or detecting the amount of the cytokine produced by the cell, or both.
  • stage of in vitro expansion of a stage generally refers to a period of expansion process that TIL passes through in vitro.
  • each stage can be divided by the change in the number of TIL cells.
  • each stage can also be divided by the conditions of TIL cell culture.
  • T cell activators and/or T cell growth factors when added or supplemented to the cell culture medium, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In one embodiment, after the TIL cells are centrifuged and/or the cells are washed, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the number of days of TIL cell culture.
  • TIL cells after the TIL cells are cultured in vitro for about 1-100 days, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days or about 100 days. After 14 days, TIL cells can be considered to have entered the next stage of in vitro expansion.
  • the term "first stage in vitro expansion” generally refers to the stage of amplification using T cell growth factors after obtaining primary TIL from tissue.
  • the tissue of the present invention can be selected from the following groups: tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion, and the pleural effusion of the present invention can be pleural effusion of patients with metastatic cancer.
  • the amplification of the present invention can be in vivo amplification performed by autologous or allogeneic, or it can be in vitro amplification.
  • the first stage in vitro amplification of the present invention can also be referred to as the preREP (pre-rapid amplification) stage.
  • TIL derived from tumor tissue and not amplified in vitro can be referred to as the first TIL group.
  • the TIL obtained by the first stage in vitro amplification in the culture method of the present invention divided by the two-step method can be referred to as the second TIL group.
  • the term "second stage in vitro expansion" generally refers to the stage of amplification again after the tissue removed from the subject is amplified.
  • the number of TIL cells amplified in vitro in the second stage of the present invention increases, for example, it can increase by at least about 10 times (or at least about 20, 30, 40, 50, 60, 70, 80 or 90 times), or the number of cells in one embodiment can increase by at least about 100 times.
  • the second stage in vitro expansion can be different from the culture conditions of the first stage in vitro expansion, for example, the culture material added can be different.
  • the second stage in vitro expansion can also be referred to as the REP (rapid expansion) stage in the culture method of the present invention divided by the two-step method.
  • the TIL obtained by the second stage in vitro expansion in the culture method of the present invention divided by the two-step method can be referred to as the third TIL group.
  • in vivo generally refers to events occurring within the body of a subject.
  • in vitro generally refers to events that occur outside the body of a subject.
  • ex vivo generally refers to events involving treatment or surgery on cells, tissues and/or organs that have been removed from the subject's body.
  • the cells, tissues and/or organs may be returned to the subject's body by surgery or therapeutic means.
  • secretion capacity generally refers to the ability of a cell to express a polypeptide or protein and transfer the polypeptide or protein of the present invention to the extracellular environment.
  • irradiation generally refers to the treatment of a substance by radiation.
  • irradiation may refer to irradiating a substance by X-rays, ⁇ -rays, ⁇ -rays or ⁇ -rays.
  • the term "engineered cell” generally refers to a cell that has been genetically modified by adding additional genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • the engineered cell can be genetically modified to express a TIL of a T cell activator and/or a T cell growth factor of the present invention.
  • co-culture generally refers to culturing two or more different populations of cells with a certain degree of contact between them.
  • the "contact" of two or more different populations of cells of the present invention may be by direct contact in one embodiment, i.e., direct physical contact between cells of one population and cells of another population. Or in one embodiment, it may be indirect contact mediated by a shared culture medium.
  • the shared culture medium of the present invention may contain metabolites produced and released by at least one population of co-cultured cells, and be used to culture cells of another population.
  • the term "contact” generally refers to two or more different types of substances being in contact with each other in any order, in any manner, and for any duration.
  • direct contact may be used, for example, one or more feeder cells, T cell activators, and/or T cell growth factors may be added to the culture medium of TIL cells, for example, a culture medium containing one or more feeder cells, T cell activators, and/or T cell growth factors may be added to and/or replace the culture medium of TIL cells, for example, a culture medium containing one or more feeder cells, T cell activators, and/or T cell growth factors may be used for the culture of TIL cells; in one embodiment, indirect contact may be used, for example, metabolic products produced and released by feeder cells may be added to the culture medium of TIL cells. The product is used to culture TIL cells.
  • the terms “contacting simultaneously”, “contacting together”, “contacting at the same time as", “simultaneously” and “together” generally refer to the administration of two or more substances to a subject and/or a cell, so that the substances are present in the subject and/or the environment of the cell culture at the same time.
  • Concurrent contact may include simultaneous administration with different compositions, administration with different compositions at different times, or administration with a composition in which two or more active pharmaceutical ingredients are present.
  • “contacting simultaneously” in the present invention generally may refer to contacting substantially simultaneously.
  • the term “expanded” generally refers to an increase in the number of cells by several times over a period of time. In one embodiment, the number of cells can be increased by at least about 3 times (or 4, 5, 6, 7, 8 or 9 times), in one embodiment, the number of cells can be increased by at least about 10 times (or 20, 30, 40, 50, 60, 70, 80 or 90 times), or in one embodiment, the number of cells can be increased by at least about 100 times. In the term “expanded” generally refers to cells of the present invention undergoing one or more of the above-mentioned amplifications.
  • the term "polymer” generally refers to a molecule consisting of separate chemical parts connected together, and the polymer parts of the present invention can be the same or different.
  • the term “polymer” can refer to separate chemical parts that are connected end to end to form a linear molecule, as well as separate chemical parts that are connected together in the form of a branched (such as a "multi-arm” or "star") structure.
  • the polymer can include, for example, a polysaccharide, a dextran, a hydrogel, a polyethylene glycol, or a poloxamer.
  • Poloxamer is a nonionic triblock copolymer having a polyoxypropylene (poly (propylene oxide)) central hydrophobic chain and two polyoxyethylene (poly (ethylene oxide)) hydrophilic chains on the side.
  • the substances included in the present invention can be formulated with any polymer described herein or known in the art, or administered together with them.
  • the term "chimeric antibody” generally refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody.
  • a hybridoma that secretes mouse-specific monoclonal antibodies can be established, and then the variable region gene can be cloned from the mouse hybridoma cells, which can be used as needed.
  • the constant region gene of a human antibody the mouse variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into an expression vector.
  • the chimeric antibody molecule can be expressed in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody
  • CDR-grafted antibody generally refers to an antibody produced by transplanting mouse CDR sequences into human antibody variable region frameworks, that is, different types of human germline antibody framework sequences.
  • the heterologous response induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the antibody or ligand described in the present invention can be a fully human monoclonal antibody.
  • the relevant technologies for the preparation of fully human antibodies can be: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
  • CDR generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.
  • One of the most commonly used definitions of the six CDRs can be provided by Kabat E.A. et al., Chothia et al., and MacCallum et al.
  • the Kabat definition of CDR can be applied to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR1, CDR2, and CDR3 (CDR H1, CDR H2, CDR H3 or H1, H2, H3) of the heavy chain variable domain.
  • IL-2 or "IL2” generally refers to a T cell protein called interleukin 2.
  • Cell growth factors and include all forms of IL-2, may include in one embodiment human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, or active fragments thereof.
  • the GeneID encoding the IL-2 gene may be 3558.
  • the term "antigen presenting cell”, “antigen presenting cell”, or “APC” generally refers to an immune system cell, such as an auxiliary cell (e.g., B cell, dendritic cell, etc.), that displays an exogenous antigen complexed with a major histocompatibility complex (MHC) on its surface.
  • auxiliary cell e.g., B cell, dendritic cell, etc.
  • MHC major histocompatibility complex
  • T cells can recognize these complexes using their T cell receptors (TCR).
  • TCR T cell receptors
  • APC can process antigens and present them to T cells.
  • antigen presenting cells may include those selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • TIL characteristics generally refers to the characteristics of TIL cells obtained by the culture method of the present invention. Changes in TIL characteristics may include: increased TIL cell number, increased viable cell ratio, increased survival ability, improved T cell subset ratio, increased cytokine secretion ability, increased in vitro tumor cell killing ability, increased in vivo tumor killing ability, increased T cell receptor (TCR) clonal diversity and increased TIL cell number in tissue, or any combination thereof.
  • the changes of the present invention may be increased or decreased.
  • an increase in the persistence of TIL cells may refer to an increase in the time that TIL cells exist in vivo.
  • an increase in persistence may refer to an increase in the time that cells exist in a subject's tissue, such as a tumor, spleen, bone marrow, lung tissue, and blood.
  • an increase in persistence may be an increase in the persistence of TIL cells after IL-2 is removed from the culture medium.
  • the term "artificial antigen presenting cell” generally refers to an artificially constructed immune cell for presenting exogenous antigens.
  • the exogenous antigen can be presented by the surface of the artificial antigen presenting cell containing a complex of the exogenous antigen and the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • aAPC isolated artificial antigen presenting cell
  • aAPC isolated artificial antigen presenting cell
  • the gene encoding it can be GeneID 3105, 3106 or 3107
  • CD64 the gene encoding it can be GeneID 2209
  • CD80 the gene encoding it can be
  • the cells may contain 1 or more T cell activators, and may be modified to express one or more T cell activators.
  • the term "fusion protein” generally refers to a polypeptide or protein containing the amino acid sequence of a first polypeptide or protein or a fragment, analog or derivative thereof and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein or a fragment, analog or derivative thereof that is different from the first polypeptide or protein or a fragment, analog or derivative thereof, or that is not usually a part of the first polypeptide or protein or a fragment, analog or derivative thereof).
  • the fusion protein may include a preventive or therapeutic drug fused with a heterologous protein, polypeptide or peptide.
  • the heterologous protein, polypeptide or peptide of the present invention may or may not be a preventive or therapeutic drug of a different type.
  • two different proteins, polypeptides or peptides having immunomodulatory activity may be fused together to form a fusion protein.
  • the fusion protein may retain or increase the activity compared to the activity of the initial polypeptide or protein before the fusion of the heterologous protein, polypeptide or protein.
  • the term "killing ability” generally refers to killing target cells by contacting the cells of the present invention with an effective amount of a substance.
  • the substance of the present invention may be a TIL cell.
  • the killing of the present invention may include killing cells by itself or by promoting CDC, apoptosis, ADCC and/or phagocytosis of other cells or substances, or by a combination of two or more of these mechanisms.
  • administering generally refers to delivering a substance to a subject in need thereof by any route known in the art.
  • Pharmaceutical carriers and formulations or compositions are also well known in the art. Routes of administration may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral and/or mucosal.
  • the term "kit” generally refers to two or more components packaged together in a container, a receptacle or other container, one of which corresponds to the substance of the present invention.
  • a container a receptacle or other container, one of which corresponds to the substance of the present invention.
  • it contains the TIL cells of the present invention.
  • the term "subject” generally refers to a cell or an animal, which may be a mammal, such as a human, a non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs).
  • Human subjects include fetuses, newborns, infants, adolescents and adult subjects.
  • Subjects include animal disease models, such as tumor animal models, and other animal models known to those skilled in the art.
  • the term "feeder cell” generally refers to a cultured cell that can be used to support the growth of another target cell. For example, it can be grown in vitro and secrete at least one factor into the culture medium.
  • the feeder cell may include an antigen presenting cell.
  • the term "specific binding” generally refers to a binding substance that recognizes a specific target substance, but does not substantially recognize or bind to other molecules in the sample.
  • a binding substance can specifically bind to a specific target substance of the present invention from one species
  • the binding substance of the present invention can also specifically bind to a target substance of the present invention or a homologous target substance from one or more other species. This interspecies reactivity itself may not change the classification of the binding substance as specific.
  • a binding substance that specifically binds to a target substance can also bind to different allelic forms of the target substance.
  • the term "complete culture process” generally refers to the complete process starting from isolating cells from tumor tissue isolated from a patient, undergoing one or more amplifications, and finally obtaining cells that can be administered to a subject.
  • cell culture medium generally refers to a nutrient solution in which cells, such as mammalian cells, are grown.
  • the preparation of cell culture media is well known in the art.
  • cell culture media include buffers, salts, carbohydrates, amino acids, vitamins, and essential trace elements.
  • Cell culture media may or may not contain serum, peptone, and/or protein.
  • Cell culture media may be supplemented with additional components or increased concentrations of components, such as amino acids. Acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements etc., depending on the requirements of the cells to be cultured and/or the desired cell culture parameters.
  • the term "pharmaceutical composition” or “pharmaceutical preparation” generally refers to a preparation that allows the biological activity of the active ingredient to be effective and may not contain additional components that are unacceptably toxic to the subject to whom the preparation will be administered. Such preparations are sterile.
  • “Pharmaceutically acceptable” excipients carriers, additives are those excipients that can be reasonably administered to a subject mammal to provide an effective dose of the active ingredient used.
  • TIL tumor infiltrating lymphocytes
  • TIL generally refers to a cell population originally obtained as leukocytes, and the cells of the present invention have left the subject's bloodstream and migrated into the tumor.
  • TIL may include, but is not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages.
  • TIL may include primary TIL and secondary TIL.
  • Primary TIL may be those TIL cells obtained from a subject's tissue sample, and “secondary TIL” may be any TIL population that has been amplified or amplified in the present invention.
  • the tumor infiltrating lymphocytes of the present invention may be unisolated and purified, or may be mutually infiltrated with tumor cells.
  • the TIL of the present invention may refer to a TIL population.
  • central memory T cells generally refers to T cells that have long-term memory and can be re-stimulated by antigens.
  • Central memory T cells may have a phenotype of CD45RO + CD62L + , for example, central memory T cells may be identified by CD45RO + and CD62L + .
  • Central memory T cells may have a stronger ability to resist tumor growth than ordinary T cells.
  • regulatory T cells generally refers to a type of T cell subpopulation that controls autoimmune reactivity in vivo. Regulatory T cells may have a phenotype of CD4 + CD25 + Foxp3 + , for example, regulatory T cells may be identified by CD4 + , CD25 + and Foxp3 + . Regulatory T cells may have the ability to inhibit the anti-tumor growth of T cells.
  • activated T cells generally refers to T cells that have been activated to have the ability to resist tumor growth.
  • Activated T cells may have a phenotype of PD-1 + (PD1 + ), LAG-3 + (LAG3 + ) or CD28 + , for example, activated T cells may be identified by PD-1 + , LAG-3 + or CD28 + .
  • Activated T cells may have the ability to resist tumor growth.
  • tumor-specific T cells generally refers to T cells that can specifically resist tumor growth.
  • Tumor-specific T cells can have a phenotype of CD103 + CD39 + , for example, tumor-specific T cells can be identified by CD103 + and CD39 + .
  • Tumor-specific T cells can have a more specific ability to resist tumor growth than ordinary T cells.
  • stem cell-like T cells generally refers to a type of T cells that can have the potential for self-proliferation and/or differentiation.
  • stem cell-like cells For example, cells with differentiation potential and/or sustained proliferation ability in the present invention can be considered as stem cell-like cells.
  • naive T cells CD45RO - CD62L +
  • stem cell-like T cells can have a phenotype of CD45RO - CD62L + .
  • stem cell-like T cells can be identified by CD45RO - CD62L + .
  • stem cell-like T cells can be identified by CD39 - CD69- .
  • stem cell-like T cells can have a phenotype of TCF1 + , for example, stem cell-like T cells can be identified by TCF1 + .
  • Stem cell-like T cells can have a stronger and/or longer-term ability to resist tumor growth than ordinary T cells.
  • tumor fragments generally refers to tumor fragments formed by mechanical disruption, enzymatic hydrolysis and/or other disruption methods after tumor tissue is removed from a subject.
  • composition or “pharmaceutical composition” generally refers to a mixture of at least one cell and at least one and optionally more than one other pharmaceutically acceptable chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients.
  • the term "pharmaceutically acceptable carrier” generally refers to a carrier that does not interfere with the active ingredients.
  • the pharmaceutically acceptable carrier may not interfere with the biological activity of the active ingredient; for example, the pharmaceutically acceptable carrier may not interfere with the effectiveness of the biological activity possessed by the active ingredient.
  • Such preparations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans.
  • envisioned carriers, excipients and/or additives that can be used in the formulations described herein may include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), etc.
  • pharmaceutically acceptable carrier can be understood as a vector that does not contain a nucleic acid form used for genetic engineering.
  • the term "functionally active fragment” generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid.
  • a functionally active fragment can retain or partially retain the ability of the full-length protein to bind to another molecule.
  • T cell activator generally refers to a substance that binds to a corresponding binding receptor on a T cell and mediates a T cell co-stimulatory response.
  • a T cell activator can be a substance other than an antigen receptor required for a T cell to produce an effective immune response.
  • a T cell activator can refer to a T cell co-stimulatory molecule.
  • the T cell activator of the present invention can include any substance comprising a variant, a homolog, or a functionally active fragment thereof.
  • T cell activators may include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activation receptors, BTLA (the gene encoding it may be 151888), Toll ligand receptors, OX40 (the gene encoding it may be 7293), CD2 (the gene encoding it may be 914), CD7 (the gene encoding it may be 924), CD27 (the gene encoding it may be 939), CD28 (the gene encoding it may be 940), CD30 (the gene encoding it may be 943), CD40 (the gene encoding it may be 950), CD50 (the gene encoding it may be 951), CD60 (the gene encoding it may be 952), CD70 (the gene encoding it may be 953), CD80 (the gene encoding it may be 954), CD90 (the gene encoding it may
  • GeneID can be 3676), VLA1 (the gene encoding it can be 3672), CD49a (the gene encoding it can be 3672), IA4 (the gene encoding it can be 3732), CD49D (the gene encoding it can be 3676), ITGA6 (the gene encoding it can be 3655), VLA-6 (the gene encoding it can be 3655), CD49f (the gene encoding it can be 3655), ITGAD (the gene encoding it can be 3681), CD11d (the gene encoding it can be 3681), ITGAE (the gene encoding it can be 3682), CD103 (the gene encoding it can be 3682), ITGAL (the gene encoding it may be GeneID 3683), CD11a (the gene encoding it may be GeneID 3683), LFA-1 (the gene encoding it may be GeneID 3683), ITGAM (the gene encoding it may be GeneID 3684), CD11b (the gene encoding it
  • the co-stimulatory intracellular signaling domain may refer to the intracellular portion of a T cell activator.
  • the intracellular signaling domain may include a complete intracellular portion of a molecule derived therefrom or a complete native intracellular signaling domain or a functional fragment thereof.
  • T cell growth factor generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation.
  • the T cell growth factor of the present invention may include any substance comprising its variant, homologue or functionally active fragment.
  • the T cell growth factor may be selected from one or more of the following groups: IL-2 (encoding its The gene GeneID can be 3558), IL-4 (the gene encoding it GeneID can be 3565), IL-6 (the gene encoding it GeneID can be 3569), IL-7 (the gene encoding it GeneID can be 3574), IL-10 (the gene encoding it GeneID can be 3586), IL-12 (the gene encoding it GeneID can be 3592 or 3593), IL-15 (the gene encoding it GeneID can be 3600), IL-21 (the gene encoding it GeneID can be 59067), TNF- ⁇ (the gene encoding it GeneID can be 100137091), interferon ⁇ (the gene encoding it GeneID can be 3458), GZMB (the gene encoding it GeneID can be 3002), CD107a (the gene encoding it GeneID can be 6499), and the like.
  • IL-2 encoding its The gene GeneID can be 3558
  • substantially simultaneously generally refers to a period of time during the contact process in which the TIL can be in contact with two or more substances at the same time, but may not be limited to always being in contact with two or more substances at the same time during the entire contact process.
  • substantially simultaneously may mean that the TIL can be in contact with at least 10-95%, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of each of the two or more substances at the same time during a period of time.
  • the term "dendritic cell” generally refers to an antigen presenting cell present in vivo, in vitro, in vitro or in a host or subject or derived from a hematopoietic stem cell or a monocyte.
  • Dendritic cells and their precursors can be separated from various lymphoid organs such as spleen, lymph nodes, bone marrow and peripheral blood.
  • the dendritic cells of the present invention can have a characteristic morphology, such as a thin layer (lamellipodia) extending in multiple directions of the dendritic cell body.
  • dendritic cells can express high levels of MHC and costimulatory (such as B7-1 and B7-2) molecules. Dendritic cells can induce antigen-specific differentiation of T cells in vitro, and can trigger primary T cell responses in vitro and in vivo.
  • in vitro expansion generally refers to a change in the number of cells produced by culture.
  • the expanded cells may also produce a change in the number and/or ratio of cells, a change in secretion ability, a change in killing ability or a change in expression ability, or any combination thereof.
  • the change of the present invention may be an increase or decrease.
  • in vitro expansion may be for the purpose of expansion; for the purpose of detecting the function of TIL cells, such as detecting the release of cytokines by TIL cells;
  • the operation steps performed on TIL cells (such as adding one or more substances to the culture medium of TIL cells to detect the cytokine release ability of TIL cells) may not belong to the in vitro expansion of the present invention.
  • peripheral mononuclear cell or “peripheral blood mononuclear cell” generally refers to a cell having a single nucleus in peripheral blood.
  • the peripheral blood mononuclear cell of the present invention may include lymphocytes, monocytes and/or dendritic cells.
  • cytokine generally refers to a protein released by a cell population that acts as an intercellular regulator on another cell.
  • the cytokine of the present invention can be a lymphokine, a monokines, and a polypeptide hormone.
  • the cytokine of the present invention can include interleukins (ILs) such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-15, IL-21 and/or IL-12.
  • ILs interleukins
  • the term cytokine can include proteins from natural sources or from recombinant cell culture, biologically active equivalents of native sequence cytokines, and functionally active fragments thereof.
  • the term “diameter” generally refers to the diameter of the cross section of the substance of the present invention.
  • the term “diameter” generally refers to the maximum diameter and/or average diameter of the largest cross section of the substance of the present invention.
  • the method for determining the diameter of the substance can be a method commonly used in the art, such as transmission electron microscopy.
  • the term “tumor” generally refers to any new pathological tissue proliferation.
  • the tumor of the present invention may be benign or malignant.
  • the tumor of the present invention may be solid or blood.
  • the term “tumor” may be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.
  • tumor tissue generally refers to a sample from a tumor in a subject, including any solid tumor and/or any tissue of a non-solid tumor in the subject.
  • T cell subset ratio generally refers to the proportion of different T cell subsets in TIL cells or TIL groups.
  • different T cell subsets of the present invention have different immunological activities and/or differentiation abilities.
  • the T cell subsets of the present invention can be distinguished according to T cell surface markers.
  • central memory T cells can have a phenotype of CD45RO + CD62L + .
  • naive T cells can have a phenotype of CD45RO - CD62L + .
  • regulatory T cells can have a phenotype of CD4 + CD25 + Foxp3 + .
  • activated T cells can have a phenotype of CD25 + , CD28 + , PD-1 + or 41BB + .
  • tumor-specific T cells can have a phenotype of CD103 + CD39 + .
  • stem cell-like T cells can have a phenotype of TCF1 + .
  • the term "TIL cell number” generally refers to the number of cells in the TIL cells of the present invention.
  • the number of TIL cells may refer to the number of cells in the TIL population obtained at any stage of the present invention.
  • the number of TIL cells may refer to the number of cells of a first TIL population derived from a tumor tissue and not amplified in vitro.
  • the number of TIL cells may refer to the number of cells of a second TIL population amplified in vitro in the first stage.
  • the number of TIL cells may refer to the number of cells of a third TIL population amplified in vitro in the second stage.
  • the number of TIL cells may refer to the cells of the TIL finally obtained by any one of the culture methods of the present invention.
  • the number of TIL cells can be measured by methods commonly used in the art, such as, for example, manual cell counting with a cell counting plate and/or counting with an automatic cell counter may be included but is not limited to.
  • the terms "about” and “approximately” generally refer to a statistically significant numerical range. Such a range can be within an order of magnitude of a given value or range, can be included within 50%, preferably included within 20%, more preferably included within 10%, and most preferably included within 5%. The permissible variation contained in the term “about” or “approximately” may depend on the specific system under study, and can be easily understood by those of ordinary skill in the art.
  • the present invention provides a method for reducing the expression and/or weakening the activity of peptidase C64 family members and/or functionally active fragments thereof in the cell.
  • the present invention provides a method for culturing cells, so that the expression and/or activity of the peptidase C64 family member and/or its functionally active fragment is reduced.
  • the peptidase C64 family member may contain a ubiquitin binding domain.
  • the peptidase C64 family member may contain TNFAIP3.
  • the target gene of the present invention can be a gene encoding a peptidase C64 family member and/or a functionally active fragment thereof.
  • the cells obtained by reducing the expression and/or weakening the activity of at least one target gene of the cell can show improved cell characteristics.
  • the cells whose expression and/or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and/or weakened the activity of at least one target gene of the cell.
  • the cells whose expression and/or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and/or weakened the activity of other genes (e.g., knocking out the other genes, which have basically no effect on cell function) other than the target gene of the cell.
  • the cell comprises an immune cell.
  • the cell comprises an immune effector cell.
  • the cell comprises an immune effector T cell, an immune effector NK cell, an immune effector NKT cell.
  • the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and/or a basophil.
  • the cells comprise monocytes, macrophages and/or dendritic cells.
  • the cells of the present invention also include cells derived from differentiation of stem cells.
  • the cells of the present invention also include cells derived from differentiation of pluripotent stem cells.
  • obtaining the stem cells of the present invention can be produced by induction.
  • the above-mentioned stem cells of the present invention can include induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells, and the like. cells and/or peripheral blood stem cells.
  • stem cells of the present invention also include pluripotent cells, multipotent cells, precursor cells and progenitor cells.
  • stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, placental stem cells obtained from placental tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from reproductive tissue of a fetus.
  • pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs.”
  • the cell comprises B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • “unmodified cells” or “unmodified cells” may refer to cells or cell colonies in which the genome is not modified and does not comprise a gene regulatory system or comprises a control gene regulatory system (e.g., an empty vector control, a non-targeted gRNA, an interfering siRNA, etc.).
  • the cell comprises ⁇ T cells and/or ⁇ T cells.
  • the cell comprises tumor infiltrating lymphocytes (TIL).
  • the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion TIL and/or TIL revived after cryopreservation.
  • the TIL of the present invention can be TIL derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and/or TIL derived from cryopreservation and resuscitation.
  • the TIL of the present invention can be obtained by processing tumor tissue into tumor fragments.
  • the volume of the tumor fragment of the present invention is about 1-27 cubic millimeters.
  • the volume of the tumor fragments of the present invention is about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 14 cubic millimeters, about 15 cubic millimeters, about 16 cubic millimeters, about 17 cubic millimeters, about 18 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or about 27 cubic millimeters. About 27 cubic millimeters.
  • the cell comprises an engineered immune receptor displayed on the cell surface.
  • the engineered immune receptor specifically binds to an antigen expressed on a target cell.
  • the cell comprises a chimeric antigen receptor and/or a T cell receptor.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: reducing the expression and/or activity of peptidase C64 family members and/or functionally active fragments thereof in the TIL.
  • TIL tumor infiltrating lymphocytes
  • TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and/or activity of peptidase C64 family members and/or functionally active fragments thereof in the TILs can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage of in vitro amplification and a second stage of in vitro amplification, and in the second stage of in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification and a second stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present invention and not expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced. The expression and/or activity of the segment is reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage of in vitro amplification, a second stage of in vitro amplification, and a third stage of in vitro amplification, and in the first stage of in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the second stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the third stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced, and in the second stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced, and in the third stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the second stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced, and in the third stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced, and in the second stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced, and in the third stage in vitro amplification of the present invention, the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the TIL can be reduced.
  • cells obtained by reducing the expression and/or attenuating the activity of a peptidase C64 family member exhibit improved cell properties compared to cells in which the expression and/or activity of the peptidase C64 family member is not altered.
  • the improved cell number of the present invention means that the cell number of the cells of the present invention in which the expression and/or activity of the peptidase C64 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to cells in which the expression and/or activity of the peptidase C64 family member is not altered.
  • the increased proportion of live cells can be expressed as an increase in cell viability.
  • the increased proportion of live cells of the present invention can mean that the proportion of live cells of the cells of the present invention in which the expression and/or activity of the peptidase C64 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least
  • the improved cytokine secretion capacity of the present invention may refer to the improved cytokine secretion capacity of the cell selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention may refer to the expression and/or activity of the peptidase C64 family member in the cells of the present invention that reduce the expression and/or activity of the peptidase C64 family member in at least one in vitro expansion stage.
  • the proportion of cells that secrete cytokines can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the improved cytokine secretion capacity of the present invention may mean that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and/or activity of the peptidase C64 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%
  • the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of the peptidase C64 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared with cells in which the expression and/or activity of the peptidase C64 family member is not changed.
  • the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of the peptidase C64 family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, compared to cells in which the expression and/or activity of the peptidase C64 family member is not changed.
  • the tumor cell killing rate of the cells of the present invention can be measured by the IncuCyte system or CFSE and DAPI staining.
  • the tumor cell killing of the cells of the present invention can refer to the ability of the cells to kill solid tumor cells.
  • the improved cell subpopulation ratio of the present invention may include one or more selected from the following groups: an increased CD8 + cell ratio, an increased central memory cell and/or naive cell ratio, a decreased regulatory cell ratio, an increased activated cell ratio, an increased tumor-specific cell ratio, and an increased stem-like cell ratio.
  • the proportion of cells, tumor-specific cells and/or stem-like cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the reduced ratio of exhausted cells of the present invention may be an increase in the ratio of PD-1 + , LAG-3 + , TIM-3 + , and/or CD39 + cells in cells.
  • the reduced ratio of regulatory cells of the present invention may be a decrease in the ratio of CD4 + CD25 + Foxp3 + cells in cells.
  • the reduced ratio of apoptotic cells of the present invention may be a decrease in the ratio of CD95 + caspass3 + cells and/or CD95 + DR5 + cells in cells.
  • the proportion of exhausted cells, regulatory cells and/or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 19%, at least about 18 ...
  • the amount of protein in the composition may be about 4% less, at least about 3% less, at least about 2% less, at least about 1% less, at least about 0.5% less, at least about 0.4% less, at least about 0.3% less, at least about 0.2% less, or at least about 0.1% less, or may be at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold less.
  • the culture method of the present invention may include a gene editing step for the cells.
  • it includes: subjecting the cells to at least one stage of in vitro expansion, wherein at least one stage of in vitro expansion is carried out.
  • a gene regulatory system may be introduced into the cells.
  • the gene regulation system can destroy the target gene at the DNA level.
  • the gene regulation system can destroy the region or fragment of the target gene in the genome of the cell.
  • the DNA region or fragment where the target gene in the cell is located is sheared and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and continuous.
  • the genomic region of the present invention is determined according to the human reference genome hg38 version.
  • the gene regulation system may include a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have a nucleic acid shearing enzyme activity, and the guide nucleic acid molecule may guide the enzyme protein to specifically shear the region or fragment thereof where the target gene is located.
  • the guide nucleic acid molecule and the enzyme protein may exist in the form of a ribonucleoprotein complex (RNP) or exist independently of each other.
  • the enzyme protein may include a Cas protein.
  • a polynucleotide encoding a gRNA and a Cas protein may be introduced or independently introduced into a target cell.
  • the present invention reduces the expression and/or weakens the activity of at least one target gene of a cell and may include: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell.
  • the enzyme protein may include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.
  • the guide nucleic acid molecule may include a guide RNA (gRNA).
  • the guide nucleic acid molecule may include a guide RNA (gRNA).
  • gRNA guide nucleic acid molecule
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • a complex comprising a polynucleotide encoding a gRNA and a Cas protein may be introduced into the cell.
  • a complex comprising a gRNA and a Cas protein may be introduced into the cell.
  • the gRNA can be used to bind to the sequence of the target gene.
  • the binding of the gRNA to the sequence of the target gene can be completely complementary, partially complementary, or hybridized to the sequence of the target gene under moderate or stringent conditions.
  • the binding of the gRNA to the sequence of the target gene can make the gRNA The CRISPR system specifically cuts the target gene.
  • the editing target region of the present invention may be a region before the promoter.
  • the editing target region of the present invention may be a region with high binding affinity of transcription factors.
  • the editing target region of the present invention may be a region with a specific number of transcription factor binding numbers.
  • the editing target region of the present invention may be a continuous region with about 3 or more transcription factor binding numbers.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) downstream, and the protospacer adjacent motif (PAM) may be AGG, TGG, GGG or CGG.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene is determined, a person skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides upstream of the 5' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the following group: AGG, TGG, GGG and CGG.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of the protospacer adjacent motif (PAM) selected from the group consisting of NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: NTTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV), wherein N may be A, T, C or G, Y may be T or C, V may be A, C or G, and R may be A or G.
  • TTYN TTTN / TTCN
  • VTTV ATTV / CTTV / GTTV
  • TRTV TRTV
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV (TTTA, TTTC, or TTTG), wherein V may be A, C or G.
  • TTTV TTTA, TTTC, or TTTG
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV, TATV, or TYCV, wherein V may be A, C or G, and Y may be T or C.
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TNN, or NTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can comprise a target sequence consisting of about 10 to about 30 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG in the DNA where the gene encoding the peptidase C64 family member and/or its functionally active fragment is located.
  • the guide nucleic acid molecule can comprise a target sequence that can bind to a gene encoding a peptidase C64 family member and/or a functionally active fragment thereof, and that consists of about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG.
  • the target sequence can be selected from a region defined by the genomic coordinates shown in Table 1A, or a fragment thereof.
  • the target sequence of the present invention can be the OUT structural functional domain of TNFAIP3.
  • the target sequence of the present invention can be the zinc finger structural functional domain of TNFAIP3.
  • the target sequence of the present invention can be chr6:137871529-137871637, chr6:137874734-137874807, chr6:137874895-137874943, chr6:137875008-137875040, chr6:137875046-137875149, chr6:137875614-137875650, chr6:137875666-137875724, chr6:1 37875789-137875816 ⁇ chr6:137875844-137876075 ⁇ chr6:137879031-137879264 ⁇ chr6:137879337-137879460 ⁇ chr6:137879958-137880090 ⁇ chr6:137880155-137880268 ⁇ chr6:137880275-13788
  • the guiding nucleic acid molecule may include a targeting domain that is complementary to a target sequence selected from the following group: SEQ ID NO: 107-212, 1562-2532.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 1-106, 591-1561, 7267-7324, 7419, 7420.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 7267-7324, 7419, 7420.
  • the proportion of cells expressing the product of the target gene in the cells obtained by reducing the expression and/or attenuating the activity of the target gene can be reduced and/or the expression level of the target gene in a single cell can be decreased.
  • the proportion of cells expressing the product of the target gene in the cell obtained by reducing the expression and/or weakening the activity of the target gene is reduced by at least about 5%.
  • the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and/or its functionally active fragment is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and/or its functionally active fragment can be from the proportion of cells that can be observed to 0%.
  • the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and/or its functionally active fragment can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the proportion of cells expressing the product of the gene encoding the peptidase C64 can be from the
  • the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and/or its functionally active fragment in the cells obtained by reducing the expression and/or weakening the activity of at least one target gene of the cells in the method of the present invention can be up to about 95%.
  • the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and/or its functionally active fragment can be up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to about 14%, up to about 13%, up to about 12%, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, or up to about 5%.
  • expression of the peptidase C64 family member and/or its functional activity The cellular proportion of the product of the gene fragment can be detected by cell flow cytometry.
  • the expression of at least one target gene of the cell is reduced and/or the activity is weakened, and the expression of the target gene in a single cell can be reduced by at least about 5%.
  • the expression of the target gene in a single cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the expression of the target gene in a single cell can be from an observable amount to 0%.
  • the expression level of the target gene in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression level of the target gene in a single cell obtained by reducing the expression and/or weakening the activity of at least one target gene in the cell can be up to about 95% of the cell in which the expression and/or activity of the target gene is not changed.
  • the expression level of the gene encoding the peptidase C64 family member and/or its functionally active fragment (e.g., the gene encoding TNFAIP3) in a single cell can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5% of the cells in which the expression and/or activity of the gene encoding the peptidase C64 family member and/or its functionally active fragment is not altered.
  • the method of the present invention comprises: subjecting the cells to at least one stage of in vitro expansion, wherein, during at least one stage of in vitro expansion, the expression and/or activity of a member of the peptidase C64 family of the cells is reduced.
  • the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion, the expression of peptidase C64 family members of the TILs expanded in vitro in the first stage is reduced and/or the activity is attenuated.
  • the first stage in vitro expansion is performed for at least about 7 days.
  • the second stage in vitro expansion is performed for at least about 7 days.
  • the cell in a single stage of in vitro expansion of the present invention, the cell is contacted with the one or more cell activators and the expression and/or activity of the peptidase C64 family members and/or their functionally active fragments in the cell is reduced.
  • the cell activator may include an agonist of one or more targets selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the expression and/or activity of the peptidase C64 family members of the cell of the present invention are reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the TIL of the present invention in the first stage of in vitro expansion of the present invention, can be reduced in expression and/or the activity of the peptidase C64 family members of the present invention and contacted with one or more cell activators of the present invention.
  • the expression and/or activity of the peptidase C64 family members of the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the expression and/or activity of the peptidase C64 family members of the TIL of the present invention can be reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the cells of the present invention can be Substantially simultaneously, the expression of the peptidase C64 family member is reduced and/or the activity is weakened, and one or more cell activators of the present invention are contacted.
  • the cells of the present invention can first reduce the expression of the peptidase C64 family member and/or weaken the activity, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell activators of the present invention.
  • the cells of the present invention can first contact with one or more cell activators of the present invention, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce the expression of the peptidase C64 family member and/or weaken the activity.
  • one or more cell activators of the present invention for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc.
  • the TIL of the present invention in the first stage of in vitro amplification of the present invention, can be made to substantially simultaneously reduce the expression and/or weaken the activity of the peptidase C64 family member and contact one or more cell activators of the present invention.
  • the TIL of the present invention in the second stage of in vitro amplification of the present invention, can be made to substantially simultaneously reduce the expression and/or weaken the activity of the peptidase C64 family member and contact one or more cell activators of the present invention.
  • the TIL of the present invention can be made to substantially simultaneously reduce the expression and/or weaken the activity of the peptidase C64 family member and contact one or more cell activators of the present invention.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and/or weakening the activity of peptidase C64 family members of the second TIL population; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the first stage in vitro amplification of the present invention can be used interchangeably with step (A) in the above method.
  • the second stage in vitro amplification of the present invention can be used interchangeably with step (B) in the above method.
  • the first stage in vitro amplification of the present invention can be used interchangeably with step (B) in the above method.
  • the TIL amplified in vitro can be arbitrarily replaced with the second TIL group obtained in step (A) of the method in the above aspect.
  • the TIL amplified in vitro in the second stage of the present invention can be arbitrarily replaced with the third TIL group obtained in step (B) of the method in the above aspect.
  • the third stage in vitro amplification of the present invention can be arbitrarily replaced with any additional step (C) in the method in the above aspect.
  • the TIL amplified in vitro in the third stage of the present invention can be arbitrarily replaced with the fourth TIL group obtained by any additional step (C) in the method in the above aspect.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may include: (A) contacting a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with multiple cell growth factors; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with multiple cell growth factors, multiple cell activators, reducing the expression and/or weakening the activity of peptidase C64 family members, and co-culturing the TIL with feeder cells; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro may be contacted with a cell growth factor; wherein a second TIL population is obtained through step (A); (B) the second TIL population may be contacted with a cell growth factor, with a cell activator, the expression and/or activity of a peptidase C64 family member may be reduced and the TIL may be co-cultured with feeder cells, and the peptidase C64 family member may include TNFAIP3; wherein a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the method for obtaining TIL cells from a tissue sample of a subject can be to obtain an in situ tumor sample or a metastatic tumor sample from a patient during surgery, and the weight can be at least about 1g, or multiple pieces of tissue can be combined.
  • Tumor tissue, pleural effusion and/or peritoneal effusion are placed in a sample transport fluid, such as a commercially available tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, with a volume of about 2-8 degrees transport, processing within 48 hours.
  • Tissue blocks can be mechanically broken into pieces of about 1-27 cubic millimeters in size, transferred into a breathable culture bag or Grex, added with cell serum-free culture medium and IL-2 at a concentration of 300-9000 IU/mL (for example, 1000-9000 IU/mL, for example, 6000 IU/mL) and cultured for about 3-14 days.
  • Cells in the culture medium are collected and transferred into a breathable culture bag, or Grex, or Xuri device, and the cell serum-free culture medium can be added with CD28 antibodies, CD3 antibodies and CD28 antibodies of the present invention, magnetic beads (such as Dynabeads) containing CD3 antibodies and CD28 antibodies and/or nanomatrices (such as transACT) containing CD3 antibodies and CD28 antibodies, IL-2 at a concentration of 300-9000 IU/mL (for example, 1000-9000 IU/mL, for example, 6000 IU/mL) L-2 and reduce the expression and/or activity of peptidase C64 family members (peptidase C64 family members may include TNFAIP3, for example, by transducing with a ribonucleoprotein complex (RNP) containing the gRNA of the present invention and the Cas protein to make the proportion of cells encoding genes of peptidase C64 family members in the TIL about 95% or less), activate the TIL of the present invention for a certain
  • Cells in the culture medium can be collected using a cell processing system, washed, frozen, and tested.
  • the final product CD3 ratio can be greater than 80%, the cell viability can be greater than 50%, and cells greater than 80% can be memory effector cells and effector cells.
  • IFN- ⁇ can be secreted after stimulation, and/or it can have the characteristic of an increased proportion of activated cells.
  • a method for culturing cells comprising: reducing the expression and/or weakening the activity of a ZC3H12 family member and/or a functionally active fragment thereof in the cells.
  • the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and/or basophils.
  • immune cells comprise monocytes, macrophages and/or dendritic cells.
  • stem cells comprise induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • immune cells comprise B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • TILs tumor infiltrating lymphocytes
  • TIL is derived from tumor tissue fragments, TIL from pleural effusion and/or peritoneal effusion and/or TIL derived from cryopreservation and resuscitation.
  • volume of the fragments is from about 1 cubic millimeter to about 27 cubic millimeters.
  • improved cell characteristics include one or more selected from the following groups: improved cell proliferation ability, increased proportion of living cells, improved proportion of cell subpopulations, increased cytokine secretion ability and improved tumor cell killing ability.
  • the improved proportion of cell subpopulations comprises one or more selected from the following groups: an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and/or immature cells, a decreased proportion of apoptotic cells and an increased proportion of stem-like cells.
  • reducing the expression and/or attenuating the activity of the ZC3H12 family member comprises: introducing into the cell a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and a Cas protein.
  • RNP ribonucleoprotein complex
  • guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • PAM protospacer adjacent motif
  • the guiding nucleic acid molecule includes a targeting domain
  • the targeting domain comprises a sequence as shown in any one of SEQ ID NOs: 213-280, 2533-3115, 7325-7345, 7416, 7417.
  • composition comprising the cell of embodiment 34.
  • a pharmaceutical composition comprising the cell of embodiment 34 and/or the composition of embodiment 35, and optionally a pharmaceutically acceptable carrier.
  • a method of influencing cell growth comprising administering the cell of embodiment 34, the composition of embodiment 35 and/or the pharmaceutical composition of embodiment 36.
  • disease and/or symptoms comprise one or more selected from the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.
  • the present invention provides a method for reducing the expression and/or weakening the activity of ZC3H12 family members and/or functionally active fragments thereof in the cell.
  • the present invention provides a method for culturing cells, so that the expression and/or activity of a ZC3H12 family member and/or a functionally active fragment thereof is reduced in the cell.
  • the ZC3H12 family member may comprise a C3H1-type zinc finger domain.
  • the ZC3H12 family member may comprise ZC3H12A.
  • the target gene of the present invention can be a gene encoding a ZC3H12 family member and/or a functionally active fragment thereof.
  • the cells obtained by reducing the expression and/or weakening the activity of at least one target gene of the cell can show improved cell characteristics.
  • the cells whose expression and/or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and/or weakened the activity of at least one target gene of the cell.
  • the cells whose expression and/or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and/or weakened the activity of other genes (e.g., knocking out the other genes, having substantially no effect on cell function) other than the target gene of the cell.
  • the cells include immune cells.
  • the cells include immune effector cells.
  • the cells include immune effector T cells, immune effector NK cells, immune effector NKT cells.
  • the cells include phagocytes, lymphocytes, neutrophils, cells, eosinophils and/or basophils.
  • the cells comprise monocytes, macrophages and/or dendritic cells.
  • the cells of the present invention also include cells derived from stem cell differentiation.
  • the cells of the present invention also include cells derived from pluripotent stem cell differentiation.
  • obtaining the stem cells of the present invention can be produced by induction.
  • the above-mentioned stem cells of the present invention can include induced pluripotent stem cells (iPSC), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and/or peripheral blood stem cells.
  • stem cells of the present invention also include pluripotent cells, multipotent cells, precursor cells and progenitor cells.
  • stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, placental stem cells obtained from placental tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from reproductive tissue of a fetus.
  • pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs.”
  • the cell comprises B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • “unmodified cells” or “unmodified cells” may refer to cells or cell colonies in which the genome is not modified and does not comprise a gene regulatory system or comprises a control gene regulatory system (e.g., an empty vector control, a non-targeted gRNA, an interfering siRNA, etc.).
  • the cell comprises ⁇ T cells and/or ⁇ T cells.
  • the cell comprises tumor infiltrating lymphocytes (TIL).
  • the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion TIL and/or TIL revived after cryopreservation.
  • the TILs of the present invention may be TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion, and/or TILs derived from cryopreservation and resuscitation.
  • Tumor tissue is processed into tumor fragments to obtain TILs of the present invention.
  • the volume of the tumor fragments of the present invention is about 1-27 cubic millimeters.
  • the volume of the tumor fragments of the present invention is about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 14 cubic millimeters, about 15 cubic millimeters, about 16 cubic millimeters, about 17 cubic millimeters, about 18 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters or about 27 cubic millimeters.
  • the cell comprises an engineered immune receptor displayed on the cell surface.
  • the engineered immune receptor specifically binds to an antigen expressed on a target cell.
  • the cell comprises a chimeric antigen receptor and/or a T cell receptor.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: reducing the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL.
  • TIL tumor infiltrating lymphocytes
  • TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TILs can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of ZC3H12 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TILs of the present invention derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and In the first stage of in vitro expansion of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification and a second stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced, and in the second stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage in vitro expansion, a second stage in vitro expansion and a third stage in vitro expansion, and in the second stage in vitro expansion of the present invention, the expression and/or activity of ZC3H12 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the third stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present invention and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of the ZC3H12 family members and/or their functionally active fragments in the TIL can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of the ZC3H12 family members in the TIL can be increased. and/or its functionally active fragments.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the second stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage in vitro amplification, a second stage in vitro amplification and a third stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced, and in the second stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage in vitro amplification of the present invention, the expression and/or activity of ZC3H12 family members and/or functionally active fragments thereof in the TIL can be reduced.
  • cells obtained by reducing the expression and/or attenuating the activity of a ZC3H12 family member exhibit improved cellular properties compared to cells in which the expression and/or activity of the ZC3H12 family member is not altered.
  • the improved cell number of the present invention refers to the expression of ZC3H12 family members
  • the number of cells of the invention having reduced expression and/or attenuated activity of the ZC3H12 family member in at least one in vitro expansion stage may be increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold compared to cells with unchanged expression and/or activity.
  • the increased proportion of live cells can be expressed as an increase in cell viability.
  • the increased proportion of live cells of the present invention can mean that the proportion of live cells of the cells of the present invention in which the expression and/or activity of the ZC3H12 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 25
  • the improved cytokine secretion capacity of the present invention may refer to an improved cytokine secretion capacity of a cell selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention may refer to an increase in the ...
  • proportion of cells secreting cytokines in the cells of the present invention in which the expression and/or activity of the ZC3H12 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage compared to cells in which the expression and/or activity of the ZC3H12 family member is not changed which can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the improved cytokine secretion capacity of the present invention may refer to an increase in the proportion of cells secreting cytokines in the cells of the present invention in which the expression and/or activity of the ZC3H12 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage.
  • the secretion capacity may refer to that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and/or activity of the ZC3H12 family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 30%, at least about 40%, at least about 5 ...
  • the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of the ZC3H12 family member is reduced in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared with cells in which the expression and/or activity of the ZC3H12 family member is not changed.
  • the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of the ZC3H12 family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 30%, at least about 40%, at least about 5 ...
  • the tumor cell killing rate of the cells of the present invention can be measured by the IncuCyte system or CFSE and DAPI staining.
  • the cells of the present invention Tumor cell killing can refer to the ability of a cell to kill solid tumor cells.
  • the improved cell subpopulation ratio of the present invention may include one or more selected from the following groups: an increased CD8 + cell ratio, an increased central memory cell and/or naive cell ratio, a decreased regulatory cell ratio, an increased activated cell ratio, an increased tumor-specific cell ratio, and an increased stem-like cell ratio.
  • the proportion of CD8 + cells, central memory cells and/or naive cells, activated cells, tumor-specific cells and/or stem-like cells in cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the reduced ratio of exhausted cells of the present invention may be an increase in the ratio of PD-1 + , LAG-3 + , TIM-3 + , and/or CD39 + cells in cells.
  • the reduced ratio of regulatory cells of the present invention may be a decrease in the ratio of CD4 + CD25 + Foxp3 + cells in cells.
  • the reduced ratio of apoptotic cells of the present invention may be a decrease in the ratio of CD95 + caspass3 + cells and/or CD95 + DR5 + cells in cells.
  • the proportion of exhausted cells, regulatory cells and/or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 1.5%, at least about 2.5%, at least about 3.5%, at least about 4.5%, at least about 5.5%, at least about 6.5%, at least about 7.5%, at least about 8.5%, at least about 9.5%, at least about
  • %, at least about 0.3%, at least about 0.2%, or at least about 0.1% can be reduced by at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the culture method of the present invention may include a gene editing step for cells.
  • a gene editing step for cells includes: subjecting the cells to at least one stage of in vitro amplification, wherein, during at least one stage of in vitro amplification, a gene regulatory system may be introduced into the cells.
  • the gene regulation system can destroy the target gene at the DNA level.
  • the gene regulation system can destroy the region or fragment of the target gene in the genome of the cell.
  • the DNA region or fragment where the target gene in the cell is located is sheared and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and continuous.
  • the genomic region of the present invention is determined according to the human reference genome hg38 version.
  • the gene regulation system may include a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have a nucleic acid shearing enzyme activity, and the guide nucleic acid molecule may guide the enzyme protein to specifically shear the region or fragment thereof where the target gene is located.
  • the guide nucleic acid molecule and the enzyme protein may exist in the form of a ribonucleoprotein complex (RNP) or exist independently of each other.
  • the enzyme protein may include a Cas protein.
  • a polynucleotide encoding a gRNA and a Cas protein may be introduced or independently introduced into a target cell.
  • the present invention can reduce the expression and/or weaken the activity of at least one target gene of a cell by: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell.
  • the enzyme protein can include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.
  • the guide nucleic acid molecule can include a guide RNA (gRNA).
  • the guide nucleic acid molecule can include a guide RNA (gRNA).
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • a complex comprising a polynucleotide encoding a gRNA and a Cas protein can be introduced into the cell.
  • a complex comprising a gRNA and a Cas protein can be introduced into the cell.
  • the gRNA can be used to bind to the sequence of the target gene.
  • the binding of the gRNA to the sequence of the target gene can be completely complementary, partially complementary, or hybridized to the sequence of the target gene under moderate stringency or stringent conditions.
  • the binding of the gRNA to the sequence of the target gene can enable the CRISPR system of the gRNA to specifically cleave the target gene.
  • the editing target region of the present invention may be a region before the promoter.
  • the editing target region of the present invention may be a region with high binding affinity of transcription factors.
  • the editing target region of the present invention may be a region with a specific number of transcription factor binding numbers.
  • the editing target region of the present invention may be a continuous region with about 3 or more transcription factor binding numbers.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) downstream, and the protospacer adjacent motif (PAM) may be AGG, TGG, GGG or CGG.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene is determined, a person skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides upstream of the 5' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the following group: AGG, TGG, GGG and CGG.
  • PAM protospacer adjacent motif
  • the gene editing system comprises CRISPR/Cas12
  • the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of the protospacer adjacent motif (PAM) selected from the following group: NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: NTTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV), wherein N may be A, T, C or G, Y may be T or C, V may be A, C or G, and R may be A or G.
  • TTYN TTTN / TTCN
  • VTTV ATTV / CTTV / GTTV
  • TRTV TRTV
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV (TTTA, TTTC, or TTTG), wherein V may be A, C or G.
  • TTTV TTTA, TTTC, or TTTG
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV, TATV, or TYCV, wherein V may be A, C or G, and Y may be T or C.
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TNN, or NTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule may comprise a target sequence that can bind to the target sequence consisting of about 10 to about 30 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG in the DNA encoding the ZC3H12 family member and/or its functionally active fragment.
  • the guide nucleic acid molecule can comprise a target sequence that is capable of binding to about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG in the DNA of the gene encoding the ZC3H12 family member and/or the functionally active fragment thereof.
  • the target sequence can be selected from a region defined by the genomic coordinates shown in Table 1B, or a fragment thereof.
  • the target sequence of the present invention can be the C3H1 type zinc finger structure functional domain of ZC3H12A.
  • the target sequence of the present invention can be R_22_chr1:37482635-37482714.
  • the guiding nucleic acid molecule may include a targeting domain that is complementary to a target sequence selected from the following group: SEQ ID NO: 281-348, 3116-3698.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 213-280, 2533-3115, 7325-7345, 7416, 7417.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 7325-7345, 7416, 7417.
  • the proportion of cells expressing the product of the target gene in the cells obtained by reducing the expression and/or attenuating the activity of the target gene can be reduced and/or the expression level of the target gene in a single cell can be decreased.
  • the proportion of cells expressing the product of the target gene is reduced by at least about 5% compared to cells in which the expression and/or activity of the target gene is not altered.
  • the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and/or its functionally active fragment is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and/or its functionally active fragment can be reduced from the observed proportion of cells to 0%.
  • the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and/or its functionally active fragment can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression and/or activity of at least one target gene of the cells obtained by reducing the expression and/or attenuating the activity of the cells can be at most about 95%.
  • the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and/or its functionally active fragment can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%.
  • expression of the protein encoding the ZC3H12 family member and/or its functional activity The cellular proportion of the product of the gene fragment can be detected by cell flow cytometry.
  • the expression of at least one target gene of the cell is reduced and/or the activity is weakened, and the expression of the target gene in a single cell can be reduced by at least about 5%.
  • the expression of the target gene in a single cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the expression of the target gene in a single cell can be from an observable amount to 0%.
  • the expression level of the target gene in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression level of the target gene in a single cell obtained by reducing the expression and/or weakening the activity of at least one target gene in the cell can be up to about 95% of the cell in which the expression and/or activity of the target gene is not changed.
  • the expression level of the gene encoding the ZC3H12 family member and/or the functionally active fragment thereof (e.g., the gene encoding ZC3H12A) in a single cell may be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5% of the expression and/or activity of the gene encoding the ZC3H12 family member and/or the functionally active fragment thereof in a single cell.
  • the method of the present invention comprises: subjecting the cell to at least one stage of in vitro expansion, wherein, during at least one stage of in vitro expansion, the expression and/or activity of a ZC3H12 family member of the cell is reduced.
  • the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion, the expression of ZC3H12 family members of the TILs expanded in vitro in the first stage is reduced and/or the activity is attenuated.
  • the first stage in vitro expansion is performed for at least about 7 days.
  • the second stage in vitro expansion is performed for at least about 7 days.
  • the cell in a single stage of in vitro expansion of the present invention, the cell is contacted with the one or more cell activators and the expression and/or activity of the ZC3H12 family members and/or their functionally active fragments in the cell is reduced.
  • the cell activator may include an agonist of one or more targets selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the expression of the ZC3H12 family members of the cells of the present invention is reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the TIL of the present invention in the first stage of in vitro expansion of the present invention, can be reduced in expression and/or the activity of the ZC3H12 family members of the present invention and contacted with one or more cell activators of the present invention.
  • the expression of the ZC3H12 family members of the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the expression of the ZC3H12 family members of the TIL of the present invention in the third stage of in vitro expansion of the present invention, can be reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the cells of the present invention can be Substantially simultaneously, the expression of the ZC3H12 family member is reduced and/or the activity is attenuated and one or more cell activators of the present invention are contacted.
  • the cells of the present invention may first be subjected to the expression of the ZC3H12 family member is reduced and/or the activity is attenuated, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contacted with one or more cell activators of the present invention.
  • the cells of the present invention may first be subjected to the one or more cell activators of the present invention, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then the expression of the ZC3H12 family member is reduced and/or the activity is attenuated.
  • the one or more cell activators of the present invention for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc.
  • the TIL of the present invention can be made to substantially simultaneously reduce the expression and/or weaken the activity of ZC3H12 family members and contact one or more cell activators of the present invention.
  • the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be made to substantially simultaneously reduce the expression and/or weaken the activity of ZC3H12 family members and contact one or more cell activators of the present invention.
  • the TIL of the present invention in the third stage of in vitro expansion of the present invention, can be made to substantially simultaneously reduce the expression and/or weaken the activity of ZC3H12 family members and contact one or more cell activators of the present invention.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and/or weakening the activity of ZC3H12 family members in the second TIL population; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the first stage in vitro amplification of the present invention can be used interchangeably with step (A) in the above method.
  • the second stage in vitro amplification of the present invention can be used interchangeably with step (B) in the above method.
  • the first stage in vitro amplification of the present invention can be used interchangeably with step (B) in the above method.
  • the TIL amplified in vitro can be arbitrarily replaced with the second TIL group obtained in step (A) of the method in the above aspect.
  • the TIL amplified in vitro in the second stage of the present invention can be arbitrarily replaced with the third TIL group obtained in step (B) of the method in the above aspect.
  • the third stage in vitro amplification of the present invention can be arbitrarily replaced with any additional step (C) in the method in the above aspect.
  • the TIL amplified in vitro in the third stage of the present invention can be arbitrarily replaced with the fourth TIL group obtained by any additional step (C) in the method in the above aspect.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with a plurality of cell growth factors; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with a plurality of cell growth factors, with a plurality of cell activators, reducing the expression and/or weakening the activity of ZC3H12 family members, and co-culturing the TIL with feeder cells; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro may be contacted with a cell growth factor; wherein a second TIL population is obtained through step (A); (B) the second TIL population may be contacted with a cell growth factor, with a cell activator, the expression and/or activity of a ZC3H12 family member may be reduced and the TIL may be co-cultured with a feeder cell, and the ZC3H12 family member may include ZC3H12A; wherein a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the method for obtaining TIL cells from a tissue sample of a subject can be to obtain an in situ tumor sample or a metastatic tumor sample from a patient during surgery, and the weight can be at least about 1g, or multiple pieces of tissue can be combined.
  • Tumor tissue, pleural effusion and/or peritoneal effusion are placed in a sample transport fluid, such as a commercially available tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, with a volume of about 2-8 degrees transport, processing within 48 hours.
  • Tissue blocks can be mechanically broken into pieces of about 1-27 cubic millimeters in size, transferred into a breathable culture bag or Grex, added with cell serum-free culture medium and IL-2 at a concentration of 300-9000 IU/mL (for example, 1000-9000 IU/mL, for example, 6000 IU/mL) and cultured for about 3-14 days.
  • Cells in the culture medium are collected and transferred into a breathable culture bag, or Grex, or Xuri device, and the cell serum-free culture medium can be added with CD28 antibodies, CD3 antibodies and CD28 antibodies of the present invention, magnetic beads (such as Dynabeads) containing CD3 antibodies and CD28 antibodies and/or nanomatrices (such as transACT) containing CD3 antibodies and CD28 antibodies, IL-2 at a concentration of 300-9000 IU/mL (for example, 1000-9000 IU/mL, for example, 6000 IU/mL) and/or nanomatrices (such as transACT) containing CD3 antibodies and CD28 antibodies.
  • magnetic beads such as Dynabeads
  • nanomatrices such as transACT
  • ZC3H12 family members may include ZC3H12A, for example, the proportion of cells encoding genes of ZC3H12 family members in the TIL may be about 95% or less by transduction with a ribonucleoprotein complex (RNP) containing the gRNA of the present invention and the Cas protein), after activating the TIL of the present invention for a certain period of time, adding irradiated PBMC (TIL and PBMC at a ratio of about 1:40-about 1:400), and amplifying and culturing for about 3-14 days.
  • Cells in the culture medium may be collected using a cell processing system, washed, frozen, and tested.
  • the final product CD3 ratio may be greater than 80%, the cell viability may be greater than 50%, and cells greater than 80% may be memory effector cells and effector cells.
  • IFN- ⁇ may be secreted after stimulation, and/or may have the characteristic of an increased proportion of activated cells.
  • a method for culturing cells comprising: reducing the expression and/or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member and/or a functionally active fragment thereof in the cells.
  • SSI STAT-induced STAT inhibitor
  • the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and/or basophils.
  • the immune cell comprises Contains monocytes, macrophages and/or dendritic cells.
  • stem cells comprise induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • immune cells comprise B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • TILs tumor infiltrating lymphocytes
  • TIL is derived from tumor tissue fragments, TIL from pleural effusion and/or peritoneal effusion and/or TIL derived from cryopreservation and resuscitation.
  • volume of the fragments is from about 1 cubic millimeter to about 27 cubic millimeters.
  • the immune cell comprises a chimeric antigen receptor and/or a T cell receptor.
  • reducing the expression and/or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member of the cell comprises inhibiting the function of negative regulation of cytokine signaling.
  • SSI STAT-induced STAT inhibitor
  • improved cell characteristics include one or more selected from the following groups: improved cell proliferation ability, increased proportion of living cells, improved proportion of cell subpopulations, increased cytokine secretion ability and improved tumor cell killing ability.
  • the improved proportion of cell subpopulations comprises one or more selected from the following groups: an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and/or immature cells, a decreased proportion of apoptotic cells and an increased proportion of stem-like cells.
  • STAT-induced STAT inhibitor (SSI) family member comprises an SH2 domain.
  • STAT-induced STAT inhibitor (SSI) family member comprises SOCS1.
  • SSI STAT-induced STAT inhibitor
  • a method according to embodiment 23, wherein reducing the expression and/or weakening the activity of the STAT-induced STAT inhibitor (SSI) family member comprises: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and a Cas protein into the cell.
  • RNP ribonucleoprotein complex
  • guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • PAM protospacer adjacent motif
  • guiding nucleic acid molecule includes a targeting domain
  • the targeting domain comprises a sequence as shown in any one of SEQ ID NOs: 349-398, 3699-4392, 7346-7375, 7418.
  • SSI STAT-induced STAT inhibitor
  • SSI STAT-induced STAT inhibitor
  • composition comprising the cell of embodiment 34.
  • a pharmaceutical composition comprising the cell of embodiment 34 and/or the composition of embodiment 35, and optionally a pharmaceutically acceptable carrier.
  • a method of influencing cell growth comprising administering the cell of embodiment 34, the composition of embodiment 35 and/or the pharmaceutical composition of embodiment 36.
  • disease and/or symptoms comprise one or more selected from the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.
  • the present invention provides a method for reducing the expression and/or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member and/or a functionally active fragment thereof in the cell.
  • SSI STAT-induced STAT inhibitor
  • the present invention provides a method for culturing cells, so that the expression and/or activity of a STAT-induced STAT inhibitor (SSI) family member and/or its functionally active fragment is reduced in the cell.
  • SSI STAT-induced STAT inhibitor
  • the STAT-induced STAT inhibitor (SSI) family member may comprise an SH2 domain.
  • the STAT-induced STAT inhibitor (SSI) family member may comprise SOCS1.
  • the target gene of the present invention may be a gene encoding a STAT-induced STAT inhibitor (SSI) family member and/or a functionally active fragment thereof.
  • SSI STAT-induced STAT inhibitor
  • cells obtained by reducing the expression and/or weakening the activity of at least one target gene of the cell may show improved cell characteristics.
  • cells in which the expression and/or activity of the target gene is not changed may refer to cells derived from the same donor and in which the expression and/or activity of at least one target gene of the cell has not been reduced.
  • cells in which the expression and/or activity of the target gene is not changed may refer to cells derived from the same donor and in which the expression and/or activity of other genes other than the target gene of the cell have not been reduced (e.g., knocking out the other gene has substantially no effect on the cell function).
  • the cell comprises an immune cell.
  • the cell comprises an immune effector cell.
  • the cell comprises an immune effector T cell, an immune effector NK cell, an immune effector NKT cell.
  • the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and/or a basophil.
  • the cells comprise monocytes, macrophages and/or dendritic cells.
  • the cells of the present invention also include cells derived from stem cell differentiation.
  • the cells of the present invention also include cells derived from pluripotent stem cell differentiation.
  • obtaining the stem cells of the present invention can be produced by induction.
  • the above-mentioned stem cells of the present invention can include induced pluripotent stem cells (iPSC), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and/or peripheral blood stem cells.
  • stem cells of the present invention also include pluripotent cells, multipotent cells, precursor cells and progenitor cells.
  • stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, placental stem cells obtained from placental tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from reproductive tissue of a fetus.
  • pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs.”
  • the cells include B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • “unmodified cells” or “unmodified cells” may refer to cells or cell populations in which the genome is not modified and does not include a gene regulatory system or includes a control gene regulatory system (e.g., an empty vector control, a non-targeting gRNA, an interfering siRNA, etc.).
  • the cells include ⁇ T cells and/or ⁇ T cells.
  • the cells include tumor infiltrating lymphocytes (TILs).
  • the TILs are derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusions and/or peritoneal effusions and/or TILs revived after cryopreservation.
  • the TIL of the present invention can be TIL derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and/or TIL derived from cryopreservation and resuscitation.
  • the TIL of the present invention can be obtained by processing tumor tissue into tumor fragments.
  • the volume of the tumor fragment of the present invention is about 1-27 cubic millimeters.
  • the volume of a tumor fragment of the present invention is about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 14 cubic millimeters, about 15 cubic millimeters, about 16 cubic millimeters, about 17 cubic millimeters, about 18 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters or about 27 cubic millimeters.
  • the cell comprises an engineered immune receptor displayed on the cell surface.
  • the engineered immune receptor specifically binds to an antigen expressed on a target cell.
  • the cell comprises a chimeric antigen receptor and/or a T cell receptor.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: reducing the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL.
  • TIL tumor infiltrating lymphocytes
  • SSI STAT-induced STAT inhibitor
  • TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or functionally active fragments thereof in the TILs can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs of the present invention that are derived from tumor tissue, pleural effusion and/or peritoneal effusion and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TILs can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs of the present invention that are derived from tumor tissue, pleural effusion and/or peritoneal effusion and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TILs can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage in vitro expansion and a second stage in vitro expansion, and in the first stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced, and in the second stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs of the present invention that are derived from tumor tissue, pleural effusion and/or peritoneal effusion and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TILs can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present invention and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the TILs can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion.
  • the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments are reduced in the human body.
  • SSI STAT-induced STAT inhibitor
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage in vitro expansion, a second stage in vitro expansion and a third stage in vitro expansion, and in the third stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage in vitro expansion, a second stage in vitro expansion and a third stage in vitro expansion, and in the first stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced, and in the second stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage in vitro expansion, a second stage in vitro expansion and a third stage in vitro expansion, and in the first stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced, and in the third stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present invention and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments can be reduced.
  • the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in TILs is reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been expanded in vitro can be subjected to a first stage in vitro expansion, a second stage in vitro expansion and a third stage in vitro expansion, and in the first stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced, and in the second stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced, and in the third stage in vitro expansion of the present invention, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments in the TIL can be reduced.
  • SSI STAT-induced STAT inhibitor
  • cells obtained by reducing the expression and/or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member exhibit improved cell properties compared to cells in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family member is not altered.
  • SSI STAT-induced STAT inhibitor
  • the improved cell number of the present invention means that the cell number of the cells of the present invention in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to cells in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family member is not altered.
  • SSI STAT-induced STAT inhibitor
  • the increased proportion of live cells can be expressed as an increase in cell viability.
  • the increased proportion of live cells of the present invention can refer to a decrease in the expression and/or activity of a STAT-induced STAT inhibitor (SSI) family member in at least one in vitro expansion stage compared to cells in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family member is not changed.
  • SSI STAT-induced STAT inhibitor
  • the proportion of live cells of the cells of the present invention with reduced activity or viability can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the improved cytokine secretion capacity of the present invention can refer to the improved cytokine secretion capacity of the cell selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention can refer to the expression and/or activity of the STAT inhibitor (SSI) family members of STAT induction.
  • SSI STAT inhibitor
  • the proportion of cells secreting cytokines in the cells of the present invention that reduce the expression and/or activity of the STAT inhibitor (SSI) family members induced by the STAT in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the improved cytokine secretion capacity of the present invention may mean that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about
  • the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family members is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to cells in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family members are not changed.
  • SSI STAT-induced STAT inhibitor
  • the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, compared to cells in which the expression and/or activity of the STAT-induced STAT inhibitor (SSI) family member is not changed.
  • SSI STAT-induced STAT inhibitor
  • the tumor cell killing rate of the cells of the present invention can be measured by the IncuCyte system or CFSE and DAPI staining.
  • the tumor cell killing of the cells of the present invention can refer to the ability of the cells to kill solid tumor cells.
  • the improved cell subpopulation ratio of the present invention may include one or more selected from the following groups: an increased CD8 + cell ratio, an increased central memory cell and/or naive cell ratio, a decreased regulatory cell ratio, an increased activated cell ratio, an increased tumor-specific cell ratio, and an increased stem-like cell ratio.
  • the proportion of cells, tumor-specific cells and/or stem-like cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the reduced ratio of exhausted cells of the present invention may be an increase in the ratio of PD-1 + , LAG-3 + , TIM-3 + , and/or CD39 + cells in cells.
  • the reduced ratio of regulatory cells of the present invention may be a decrease in the ratio of CD4 + CD25 + Foxp3 + cells in cells.
  • the reduced ratio of apoptotic cells of the present invention may be a decrease in the ratio of CD95 + caspass3 + cells and/or CD95 + DR5 + cells in cells.
  • the proportion of exhausted cells, regulatory cells and/or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 19%, at least about 18 ...
  • the amount of protein in the composition may be about 4% less, at least about 3% less, at least about 2% less, at least about 1% less, at least about 0.5% less, at least about 0.4% less, at least about 0.3% less, at least about 0.2% less, or at least about 0.1% less, or may be at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold less.
  • the culture method of the present invention may include a gene editing step for the cells.
  • it includes: subjecting the cells to at least one stage of in vitro expansion, wherein at least one stage of in vitro expansion is carried out.
  • a gene regulatory system may be introduced into the cells.
  • the gene regulation system can destroy the target gene at the DNA level.
  • the gene regulation system can destroy the region or fragment of the target gene in the genome of the cell.
  • the DNA region or fragment thereof where the target gene in the cell is located is sheared and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and continuous.
  • the genomic region of the present invention is determined according to the human reference genome hg38 version.
  • the gene regulation system may include a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have a nucleic acid shearing enzyme activity, and the guide nucleic acid molecule may guide the enzyme protein to specifically shear the region or fragment thereof where the target gene is located.
  • the guide nucleic acid molecule and the enzyme protein may exist in the form of a ribonucleoprotein complex (RNP) or exist independently of each other.
  • the enzyme protein may include a Cas protein.
  • a polynucleotide encoding a gRNA and a Cas protein may be introduced or introduced independently of each other into a target cell.
  • the present invention reduces the expression and/or weakens the activity of at least one target gene of a cell and may include: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell.
  • the enzyme protein may include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.
  • the guide nucleic acid molecule may include a guide RNA (gRNA).
  • the guide nucleic acid molecule may include a guide RNA (gRNA).
  • gRNA guide nucleic acid molecule
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • a complex comprising a polynucleotide encoding a gRNA and a Cas protein may be introduced into the cell.
  • a complex comprising a gRNA and a Cas protein may be introduced into the cell.
  • the gRNA can be used to bind to the sequence of the target gene.
  • the binding of the gRNA to the sequence of the target gene can be completely complementary, partially complementary, or hybridized to the sequence of the target gene under moderate or stringent conditions.
  • the binding of the gRNA to the sequence of the target gene can make the gRNA The CRISPR system specifically cuts the target gene.
  • the editing target region of the present invention may be a region before the promoter.
  • the editing target region of the present invention may be a region with high binding affinity of transcription factors.
  • the editing target region of the present invention may be a region with a specific number of transcription factor binding numbers.
  • the editing target region of the present invention may be a continuous region with about 3 or more transcription factor binding numbers.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) downstream, and the protospacer adjacent motif (PAM) may be AGG, TGG, GGG or CGG.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene is determined, a person skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides upstream of the 5' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the following group: AGG, TGG, GGG and CGG.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of the protospacer adjacent motif (PAM) selected from the group consisting of NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: NTTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV), wherein N may be A, T, C or G, Y may be T or C, V may be A, C or G, and R may be A or G.
  • TTYN TTTN / TTCN
  • VTTV ATTV / CTTV / GTTV
  • TRTV TRTV
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV (TTTA, TTTC, or TTTG), wherein V may be A, C or G.
  • TTTV TTTA, TTTC, or TTTG
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV, TATV, or TYCV, wherein V may be A, C or G, and Y may be T or C.
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TNN, or NTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can comprise a target sequence consisting of about 10 to about 30 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG in the DNA encoding a STAT-induced STAT inhibitor (SSI) family member and/or a functionally active fragment thereof.
  • SSI STAT-induced STAT inhibitor
  • the guide nucleic acid molecule can comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 21, about 15 to about 19, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 20 A target sequence consisting of about 19, or about 15 to about 17 nucleotides.
  • the target sequence can be selected from a region defined by the genomic coordinates shown in Table 1C, or a fragment thereof.
  • the guiding nucleic acid molecule may include a targeting domain that is complementary to a target sequence selected from the following group: SEQ ID NO: 399-448, 4393-5086.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 349-398, 3699-4392, 7346-7375, 7418.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 7346-7375, 7418.
  • the proportion of cells expressing the product of the target gene in the cells obtained by reducing the expression and/or attenuating the activity of the target gene can be reduced and/or the expression level of the target gene in a single cell can be decreased.
  • the proportion of cells expressing the product of the target gene is reduced by at least about 5% in cells obtained by reducing the expression and/or attenuating the activity of at least one target gene in the cells as compared to cells in which the expression and/or activity of the target gene is not altered.
  • the proportion of cells expressing the product of the gene encoding the STAT-induced STAT inhibitor (SSI) family member and/or its functionally active fragment is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • SSI STAT-induced STAT inhibitor
  • the proportion of cells expressing the product of the gene encoding the STAT-inducible STAT inhibitor (SSI) family member and/or its functionally active fragment can be reduced from the proportion of cells that can be observed to 0%.
  • the proportion of cells expressing the product of the gene encoding the STAT-inducible STAT inhibitor (SSI) family member and/or its functionally active fragment can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the proportion of cells expressing the product of the gene encoding the STAT-induced STAT inhibitor (SSI) family member and/or its functionally active fragment obtained by the method of the present invention in which the expression of at least one target gene of the cell is reduced and/or the activity is attenuated can be up to about 95%.
  • the proportion of cells expressing the product of the gene encoding the STAT-induced STAT inhibitor (SSI) family member and/or its functionally active fragment can be up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to about 14%, up to about 13%, up to about 12%, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, or up to about 5%.
  • the proportion of cells expressing the product of the gene encoding the STAT-induced STAT inhibitor (SSI) family member and/or its functionally active fragment can be detected by flow cytometry.
  • the expression level of the target gene in a single cell can be reduced by at least about 5%.
  • the expression level of the target gene in a single cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%,
  • the expression of the target gene in a single cell can be from an observable amount to 0%.
  • the expression level of the target gene in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression level of the target gene in a single cell obtained by reducing the expression and/or weakening the activity of at least one target gene in the cell can be up to about 95% of the cell in which the expression and/or activity of the target gene is not changed.
  • the expression level of the gene encoding a STAT-induced STAT inhibitor (SSI) family member and/or a functionally active fragment thereof (e.g., a gene encoding SOCS1) in a single cell can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5% of the cells in which the expression and/or activity of the gene encoding a STAT-induced STAT inhibitor (SSI) family member and/or a functionally active fragment thereof is not altered.
  • SSI STAT-induced STAT inhibitor
  • the method of the present invention comprises: subjecting the cells to at least one stage of in vitro expansion, wherein, during at least one stage of the in vitro expansion, the expression and/or activity of a STAT-induced STAT inhibitor (SSI) family member of the cells is reduced.
  • SSI STAT-induced STAT inhibitor
  • the tumor tissue, tumor-associated lymph nodes with or without tumor Metastases, tumor metastatic lesions, fragments of adjacent cancer tissues, pleural effusions and/or peritoneal effusions that have not been expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members of the TILs that have been expanded in vitro in the first stage are reduced.
  • SSI STAT-induced STAT inhibitor
  • the first stage in vitro expansion is performed for at least about 7 days.
  • the second stage in vitro expansion is performed for at least about 7 days.
  • the cell can be contacted with the one or more cell activators and the expression and/or activity of the STAT inhibitor (SSI) family members induced by STAT and/or their functionally active fragments contained in the cell can be reduced in a single stage of in vitro expansion of the present invention.
  • the cell activator can include an agonist of one or more targets selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the expression and/or activity of the STAT inhibitor (SSI) family members induced by STAT of the cell of the present invention are reduced and/or weakened and contacted with one or more cell activators of the present invention.
  • the expression and/or activity of the STAT inhibitor (SSI) family members induced by STAT of the TIL of the present invention can be reduced and/or weakened and contacted with one or more cell activators of the present invention.
  • the expression and/or activity of the STAT inhibitor (SSI) family members induced by STAT of the TIL of the present invention can be reduced and/or weakened and contacted with one or more cell activators of the present invention.
  • the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members of the TILs of the present invention can be reduced and/or attenuated and contacted with one or more cell activators of the present invention.
  • SSI STAT-induced STAT inhibitor
  • the cells of the present invention can be made to substantially simultaneously reduce the expression and/or weaken the activity of STAT-induced STAT inhibitor (SSI) family members and contact one or more cell activators of the present invention.
  • the cells of the present invention can first reduce the expression and/or weaken the activity of STAT-induced STAT inhibitor (SSI) family members, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 3 hours in advance.
  • the cells of the present invention may be first contacted with one or more cell activators of the present invention, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, and then the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members may be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TIL of the present invention can be made to substantially simultaneously reduce the expression and/or weaken the activity of STAT-induced STAT inhibitor (SSI) family members and contact one or more cell activators of the present invention.
  • the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be made to substantially simultaneously reduce the expression and/or weaken the activity of STAT-induced STAT inhibitor (SSI) family members and contact one or more cell activators of the present invention.
  • the TIL of the present invention can be made to substantially simultaneously reduce the expression and/or weaken the activity of STAT-induced STAT inhibitor (SSI) family members and contact one or more cell activators of the present invention.
  • SSI STAT-induced STAT inhibitor
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and/or weakening the activity of STAT-induced STAT inhibitor (SSI) family members in the second TIL population; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the first stage of in vitro expansion of the present invention can be used interchangeably with step (A) in the above method.
  • the second stage of in vitro expansion of the present invention can be used interchangeably with step (B) in the above method.
  • the first stage of in vitro expansion of the present invention can be used interchangeably with the second TIL population obtained in step (A) of the above method.
  • the second stage of in vitro expansion of the present invention can be used interchangeably with step (B) in the above method.
  • the TILs amplified in vitro can be arbitrarily replaced with the third TIL group obtained in step (B) of the method in the above aspects.
  • the third stage in vitro amplification of the present invention can be arbitrarily replaced with the step (C) arbitrarily added in the method in the above aspects.
  • the TILs amplified in vitro in the third stage of the present invention can be arbitrarily replaced with the fourth TIL group obtained by the step (C) arbitrarily added in the method in the above aspects.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may include: (A) a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro may be contacted with multiple cell growth factors; wherein, a second TIL population is obtained through step (A); (B) the second TIL population may be contacted with multiple cell growth factors, multiple cell activators, the expression and/or activity of STAT-induced STAT inhibitor (SSI) family members may be reduced, and the TIL may be co-cultured with feeder cells; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may include: (A) a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro may be contacted with a cell growth factor; wherein, a second TIL population is obtained through step (A); (B) the second TIL population may be contacted with a cell growth factor, with a cell activator, the expression and/or activity of a STAT-induced STAT inhibitor (SSI) family member may be reduced and the TIL may be co-cultured with feeder cells, and the STAT-induced STAT inhibitor (SSI) family member may include SOCS1; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the method for obtaining TIL cells from a tissue sample of a subject can be to obtain an in situ tumor sample or a metastatic tumor sample from a patient during surgery, the weight of which can be at least about 1 g, or multiple pieces of tissue can be combined.
  • Tumor tissue, pleural effusion and/or peritoneal effusion are transported in a sample transport fluid, such as a commercially available tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, at about 2-8 degrees and processed within 48 hours.
  • the tissue blocks can be mechanically broken into about 1-27 cubic meters per block.
  • the cells are collected from the culture medium and transferred into a breathable culture bag or Grex, and the cells are cultured for about 3-14 days with a serum-free culture medium and IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, e.g., 6000 IU/mL).
  • a serum-free culture medium and IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, e.g., 6000 IU/mL).
  • the cells are transferred into a breathable culture bag, or Grex, or Xuri device, and the serum-free culture medium may be supplemented with the CD28 antibody, CD3 antibody, and CD28 antibody of the present invention, magnetic beads (e.g., Dynabeads) containing CD3 antibody and CD28 antibody, and/or nanomatrix (e.g., transACT) containing CD3 antibody and CD28 antibody, IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, e.g., 6000 IU/mL), and a STAT inhibitor that induces STAT.
  • magnetic beads e.g., Dynabeads
  • nanomatrix e.g., transACT
  • STAT-induced STAT inhibitor (SSI) family members may include SOCS1, for example, the proportion of cells encoding genes of STAT-induced STAT inhibitor (SSI) family members in the TIL can be about 95% or less by transduction with a ribonucleoprotein complex (RNP) containing the gRNA of the present invention and the Cas protein).
  • RNP ribonucleoprotein complex
  • TIL and PBMC are at a ratio of about 1:40-about 1:400
  • the cells in the culture medium can be collected using a cell processing system, washed, frozen, and tested.
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 50%, and cells greater than 80% can be memory effector cells and effector cells.
  • IFN- ⁇ can be secreted after stimulation, and/or it can have the characteristic of an increased proportion of activated cells.
  • a method for culturing cells comprising: reducing the expression and/or weakening the activity of CBL family members and/or functionally active fragments thereof in the cells.
  • the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and/or basophils.
  • immune cells comprise monocytes, macrophages and/or dendritic cells.
  • stem cells comprise induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • immune cells comprise B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • TILs tumor infiltrating lymphocytes
  • TIL is derived from tumor tissue fragments, TIL from pleural effusion and/or peritoneal effusion and/or TIL derived from cryopreservation and resuscitation.
  • volume of the fragments is from about 1 cubic millimeter to about 27 cubic millimeters.
  • improved cell characteristics include one or more selected from the following groups: improved cell proliferation ability, increased proportion of living cells, improved proportion of cell subpopulations, increased cytokine secretion ability and improved tumor cell killing ability.
  • the improved proportion of cell subpopulations comprises one or more selected from the following groups: an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and/or immature cells, a decreased proportion of apoptotic cells and an increased proportion of stem-like cells.
  • a method according to embodiment 23, wherein reducing the expression and/or weakening the activity of the CBL family member comprises: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and a Cas protein into the cell.
  • RNP ribonucleoprotein complex
  • guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • PAM protospacer adjacent motif
  • the guiding nucleic acid molecule includes a targeting domain
  • the targeting domain comprises a sequence as shown in any one of SEQ ID NOs: 449-519, 5087-6176, 7376-7413, 7414, and 7415.
  • composition comprising the cell of embodiment 34.
  • a pharmaceutical composition comprising the cell of embodiment 34 and/or the composition of embodiment 35, and optionally a pharmaceutically acceptable carrier.
  • a method of influencing cell growth comprising administering the cell of embodiment 34, the composition of embodiment 35 and/or the pharmaceutical composition of embodiment 36.
  • disease and/or symptoms comprise one or more selected from the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.
  • the present invention provides a method for reducing the expression and/or attenuating the activity of CBL family members and/or functionally active fragments thereof in the cell.
  • the present invention provides a method for culturing cells, so that the expression and/or activity of CBL family members and/or functionally active fragments thereof in the cells are reduced.
  • the CBL family members may include an SH3 domain.
  • the CBL family members may include CBLB.
  • the target gene of the present invention may be a gene encoding a CBL family member and/or a functionally active fragment thereof.
  • a cell obtained by reducing the expression and/or weakening the activity of at least one target gene of the cell may show improved cell characteristics.
  • a cell in which the expression and/or activity of the target gene is not changed may refer to a cell derived from the same donor and in which the expression and/or activity of at least one target gene of the cell is not reduced and/or weakened.
  • a cell in which the expression and/or activity of the target gene is not changed may refer to a cell derived from the same donor and in which the expression and/or activity of other genes other than the target gene of the cell (e.g., knocking out the other gene has substantially no effect on the cell function) is not reduced and/or weakened.
  • the cells include immune cells.
  • the cells include immune effector cells.
  • the cells include immune effector T cells, immune effector NK cells, immune Effector NKT cells.
  • the cells include phagocytes, lymphocytes, neutrophils, eosinophils and/or basophils.
  • the cells comprise monocytes, macrophages and/or dendritic cells.
  • the cells of the present invention also include cells derived from stem cell differentiation.
  • the cells of the present invention also include cells derived from pluripotent stem cell differentiation.
  • obtaining the stem cells of the present invention can be produced by induction.
  • the above-mentioned stem cells of the present invention can include induced pluripotent stem cells (iPSC), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and/or peripheral blood stem cells.
  • stem cells of the present invention also include pluripotent cells, multipotent cells, precursor cells and progenitor cells.
  • stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, placental stem cells obtained from placental tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from reproductive tissue of a fetus.
  • pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs.”
  • the cell comprises B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • “unmodified cells” or “unmodified cells” may refer to cells or cell colonies in which the genome is not modified and does not comprise a gene regulatory system or comprises a control gene regulatory system (e.g., an empty vector control, a non-targeted gRNA, an interfering siRNA, etc.).
  • the cell comprises ⁇ T cells and/or ⁇ T cells.
  • the cell comprises tumor infiltrating lymphocytes (TIL).
  • the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion TIL and/or TIL revived after cryopreservation.
  • the TILs of the present invention may be derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or The TIL of peritoneal effusion and/or the TIL resuscitated after being derived from cryopreservation.
  • TIL of the present invention can be obtained by processing tumor tissue into tumor fragments.
  • the volume of tumor fragments of the present invention is about 1-27 cubic millimeters.
  • the volume of tumor fragments of the present invention is about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about
  • the cell comprises an engineered immune receptor displayed on the cell surface.
  • the engineered immune receptor specifically binds to an antigen expressed on a target cell.
  • the cell comprises a chimeric antigen receptor and/or a T cell receptor.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: reducing the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL.
  • TIL tumor infiltrating lymphocytes
  • TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TILs can be reduced.
  • the TILs of the present invention that are derived from tumor tissue, pleural effusion and/or peritoneal effusion and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TILs can be reduced.
  • the invention can be derived from tumor tissue, pleural effusion and/or peritoneal effusion and
  • the TIL that has not been expanded in vitro undergoes a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage of in vitro amplification and a second stage of in vitro amplification, and in the first stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced, and in the second stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TILs of the present invention that are derived from tumor tissue, pleural effusion and/or peritoneal effusion and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TILs can be reduced.
  • the TILs of the present invention that are derived from tumor tissue, pleural effusion and/or peritoneal effusion and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the third stage of in vitro expansion of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TILs can be reduced.
  • the TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present invention and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TILs can be reduced, and in the present invention, In the second stage of in vitro expansion of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage of in vitro amplification, a second stage of in vitro amplification and a third stage of in vitro amplification, and in the first stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage of in vitro amplification, a second stage of in vitro amplification and a third stage of in vitro amplification, and in the second stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced.
  • the TIL of the present invention that is derived from tumor tissue, pleural effusion and/or peritoneal effusion and has not been amplified in vitro can be subjected to a first stage of in vitro amplification, a second stage of in vitro amplification and a third stage of in vitro amplification, and in the first stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced, and in the second stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage of in vitro amplification of the present invention, the expression and/or activity of CBL family members and/or functionally active fragments thereof in the TIL can be reduced.
  • cells obtained by reducing the expression and/or attenuating the activity of a CBL family member exhibit improved cellular properties compared to cells in which the expression and/or activity of the CBL family member is not altered.
  • the improved cell number of the present invention means that the cell number of the cells of the present invention in which the expression and/or activity of the CBL family member is reduced in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to cells in which the expression and/or activity of the CBL family member is not altered.
  • an increased proportion of live cells can be expressed as an increase in cell viability.
  • the increased proportion of live cells of the present invention may mean that the proportion of live cells of the cells of the present invention in which the expression and/or activity of the CBL family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage may be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.
  • the improved cytokine secretion capacity of the present invention may refer to an improved cytokine secretion capacity of a cell selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention may refer to an increase in the ...
  • the improved cytokine secretion capacity of the present invention may refer to an increase in the proportion of cells secreting cytokines in the cells of the present invention in which the expression and/or activity of the CBL family member is reduced and/or the activity is attenuated in at least one in vitro expansion stage.
  • the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of the CBL family member is reduced in at least one in vitro expansion stage is increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to cells in which the expression and/or activity of the CBL family member is not changed.
  • the tumor cell killing rate of the cells of the invention can be measured by the IncuCyte system or CFSE and DAPI staining.
  • the tumor cell killing rate of the cells of the invention can be measured by the IncuCyte system or CFSE and DAPI staining method. Killing can refer to the ability of a cell to kill solid tumor cells.
  • the improved cell subpopulation ratio of the present invention may include one or more selected from the following groups: an increased CD8 + cell ratio, an increased central memory cell and/or naive cell ratio, a decreased regulatory cell ratio, an increased activated cell ratio, an increased tumor-specific cell ratio, and an increased stem-like cell ratio.
  • the proportion of CD8 + cells, central memory cells and/or naive cells, activated cells, tumor-specific cells and/or stem-like cells in cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the reduced ratio of exhausted cells of the present invention may be an increase in the ratio of PD-1 + , LAG-3 + , TIM-3 + , and/or CD39 + cells in cells.
  • the reduced ratio of regulatory cells of the present invention may be a decrease in the ratio of CD4 + CD25 + Foxp3 + cells in cells.
  • the reduced ratio of apoptotic cells of the present invention may be a decrease in the ratio of CD95 + caspass3 + cells and/or CD95 + DR5 + cells in cells.
  • the proportion of exhausted cells, regulatory cells and/or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 1.5%, at least about 2.5%, at least about 3.5%, at least about 4.5%, at least about 5.5%, at least about 6.5%, at least about 7.5%, at least about 8.5%, at least about 9.5%, at least about
  • %, at least about 0.3%, at least about 0.2%, or at least about 0.1% can be reduced by at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the culture method of the present invention may include a gene editing step for cells.
  • a gene editing step for cells includes: subjecting the cells to at least one stage of in vitro amplification, wherein, during at least one stage of in vitro amplification, a gene regulatory system may be introduced into the cells.
  • the gene regulation system can destroy the target gene at the DNA level.
  • the gene regulation system can destroy the region or fragment of the target gene in the genome of the cell.
  • the DNA region or fragment thereof where the target gene in the cell is located is sheared and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and continuous.
  • the genomic region of the present invention is determined according to the human reference genome hg38 version.
  • the gene regulation system may include a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have a nucleic acid shearing enzyme activity, and the guide nucleic acid molecule may guide the enzyme protein to specifically shear the region or fragment thereof where the target gene is located.
  • the guide nucleic acid molecule and the enzyme protein may exist in the form of a ribonucleoprotein complex (RNP) or exist independently of each other.
  • the enzyme protein may include a Cas protein.
  • a polynucleotide encoding a gRNA and a Cas protein may be introduced or independently introduced into a target cell.
  • the present invention can reduce the expression and/or weaken the activity of at least one target gene of a cell by: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell.
  • the enzyme protein can include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.
  • the guide nucleic acid molecule can include a guide RNA (gRNA).
  • the guide nucleic acid molecule can include a guide RNA (gRNA).
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • a complex comprising a polynucleotide encoding a gRNA and a Cas protein can be introduced into the cell.
  • a complex comprising a gRNA and a Cas protein can be introduced into the cell.
  • the gRNA can be used to bind to the sequence of the target gene.
  • the binding of the gRNA to the sequence of the target gene can be completely complementary, partially complementary, or hybridized to the sequence of the target gene under moderate stringency or stringent conditions.
  • the binding of the gRNA to the sequence of the target gene can enable the CRISPR system of the gRNA to specifically cleave the target gene.
  • the editing target region of the present invention may be a region before the promoter.
  • the editing target region of the present invention may be a region with high binding affinity of transcription factors.
  • the editing target region of the present invention may be a region with a specific number of transcription factor binding numbers.
  • the editing target region of the present invention may be a continuous region with about 3 or more transcription factor binding numbers.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) downstream, and the protospacer adjacent motif (PAM) may be AGG, TGG, GGG or CGG.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene is determined, a person skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides upstream of the 5' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the following group: AGG, TGG, GGG and CGG.
  • PAM protospacer adjacent motif
  • the gene editing system comprises CRISPR/Cas12
  • the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of the protospacer adjacent motif (PAM) selected from the following group: NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: NTTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV), wherein N may be A, T, C or G, Y may be T or C, V may be A, C or G, and R may be A or G.
  • TTYN TTTN / TTCN
  • VTTV ATTV / CTTV / GTTV
  • TRTV TRTV
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV (TTTA, TTTC, or TTTG), wherein V may be A, C or G.
  • TTTV TTTA, TTTC, or TTTG
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV, TATV, or TYCV, wherein V may be A, C or G, and Y may be T or C.
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TNN, or NTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule may comprise a target sequence that can bind to a gene encoding a CBL family member and/or a functionally active fragment thereof, which is located in the DNA containing the gene encoding the CBL family member and/or a functionally active fragment thereof and which consists of about 10 to about 30 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG.
  • the nucleic acid molecule may comprise a target sequence that is capable of binding to a gene encoding a CBL family member and/or a functionally active fragment thereof and that consists of about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG.
  • the target sequence can be a region defined by the genomic coordinates shown in Table 1D or a fragment thereof.
  • the target sequence of the present invention can be the PTB structural functional domain of CBLB.
  • the target sequence of the present invention can be the zinc finger structural functional domain of CBLB.
  • the target sequence of the present invention can be the UBA structural functional domain of CBLB.
  • the target sequence of the present invention can be chr3:105658893-105659215, chr3:105720184-105720231, chr3:105720234-105720278, chr3:105720300-105720327, chr3:105720333-105720375, chr3:105723918-105723956, chr3:105724025-105724052, chr3:105724100-105724131, chr3:105720300-105720327, chr3:105720333-105720375, r3:105724134-105724219 ⁇ chr3:105724232-105724310 ⁇ chr3:105733918-105733970 ⁇ chr3:105733988-105734073 ⁇ chr3:105734082-105734109 ⁇ chr3:105737162-105737268 ⁇ chr3:105740404-105740449 ⁇ chr3:105740470-105740551
  • the guiding nucleic acid molecule may include a targeting domain that is complementary to a target sequence selected from the following group: SEQ ID NO: 520-590, 6177-7266.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 449-519, 5087-6176, 7376-7413, 7414, 7415.
  • the guiding nucleic acid molecule may include a targeting domain, which may contain a sequence as shown in SEQ ID NO: 7376-7413, 7414, 7415.
  • the proportion of cells expressing the product of the target gene in the cells obtained by reducing the expression and/or attenuating the activity of the target gene can be reduced and/or the expression level of the target gene in a single cell can be decreased.
  • the proportion of cells expressing the product of the target gene is reduced by at least about 5% in the cells obtained by reducing the expression and/or attenuating the activity of at least one target gene in the cells, compared to cells in which the expression and/or activity of the target gene is not altered.
  • the proportion of cells expressing the product of the gene encoding the CBL family member and/or its functionally active fragment is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the proportion of cells expressing the product of the gene encoding the CBL family member and/or its functionally active fragment can be reduced from the observed proportion of cells to 0%.
  • the proportion of cells expressing the product of the gene encoding the CBL family member and/or its functionally active fragment can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the proportion of cells expressing the product of the gene encoding the CBL family member and/or its functionally active fragment can be detected by flow
  • the proportion of cells expressing the product of the gene encoding the CBL family member and/or its functionally active fragment obtained by reducing the expression and/or attenuating the activity of at least one target gene of the cells can be up to about 95%.
  • the proportion of cells expressing the product of the gene encoding the CBL family member and/or its functionally active fragment can be up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to about 14%, up to about 13%, up to about 12%, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, or up to about 5%.
  • the proportion of cells expressing the product of the gene encoding the CBL family member and/or the functionally active fragment thereof can be detected by flow cytometry.
  • the expression level of the target gene in a single cell can be reduced by at least about 5%.
  • the expression level of the target gene in a single cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 18 ...
  • the expression of the target gene in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression of the target gene in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression level of the target gene in a single cell obtained by reducing the expression and/or weakening the activity of at least one target gene in the cell can be up to about 95% of the cell in which the expression and/or activity of the target gene is not changed.
  • the expression level of the gene encoding the CBL family member and/or the functionally active fragment thereof (e.g., the gene encoding CBLB) in a single cell may be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5% of the expression and/or activity of the gene encoding the CBL family member and/or the functionally active fragment thereof in a single cell.
  • the method of the present invention comprises: subjecting the cell to at least one stage of in vitro expansion, wherein, during at least one stage of in vitro expansion, the expression and/or activity of a CBL family member of the cell is reduced.
  • the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion, the CBLs of the TILs expanded in vitro in the first stage are expanded. The expression and/or activity of family members are reduced.
  • the first stage in vitro expansion is performed for at least about 7 days.
  • the second stage in vitro expansion is performed for at least about 7 days.
  • the cell can be contacted with the one or more cell activators and the expression and/or activity of the CBL family members and/or their functionally active fragments contained in the cell can be reduced in a single stage of in vitro expansion of the present invention.
  • the cell activator can include an agonist of one or more targets selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the expression of the CBL family members of the cells of the present invention is reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the TIL of the present invention in the first stage of in vitro expansion of the present invention, can be reduced in expression and/or the activity of the CBL family members of the present invention and contacted with one or more cell activators of the present invention.
  • the expression of the CBL family members of the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the expression of the CBL family members of the TIL of the present invention in the third stage of in vitro expansion of the present invention can be reduced and/or the activity is weakened and contacted with one or more cell activators of the present invention.
  • the cells of the present invention can be made to reduce the expression and/or weaken the activity of CBL family members and contact one or more cell activators of the present invention at substantially the same time.
  • the cells of the present invention can be made to reduce the expression and/or weaken the activity of CBL family members first, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell activators of the present invention.
  • the cells of the present invention can be made to contact with one or more cell activators of the present invention first, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce the expression and/or weaken the activity of CBL family members.
  • the TIL of the present invention can be made to substantially simultaneously reduce the expression and/or weaken the activity of CBL family members and contact one or more cell activators of the present invention.
  • the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be made to substantially simultaneously reduce the expression and/or weaken the activity of CBL family members and contact one or more cell activators of the present invention.
  • the TIL of the present invention in the third stage of in vitro expansion of the present invention, can be made to substantially simultaneously reduce the expression and/or weaken the activity of CBL family members and contact one or more cell activators of the present invention.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and/or weakening the activity of CBL family members in the second TIL population; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the first stage in vitro expansion of the present invention can be arbitrarily replaced with step (A) in the method of the above aspect.
  • the second stage in vitro expansion of the present invention can be arbitrarily replaced with step (B) in the method of the above aspect.
  • the TIL of the present invention after the first stage in vitro expansion can be arbitrarily replaced with the second TIL group obtained by step (A) in the method of the above aspect.
  • the TIL of the present invention after the second stage in vitro expansion can be arbitrarily replaced with the third TIL group obtained by step (B) in the method of the above aspect.
  • the third stage in vitro expansion of the present invention can be arbitrarily replaced with any additional step (C) in the method of the above aspect.
  • the TIL of the present invention after the third stage in vitro expansion can be arbitrarily replaced with the fourth TIL group obtained by any additional step (C) in the method of the above aspect.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) culturing cells derived from tumor tissue, pleural effusion and/or peritoneal effusion; The method further comprises contacting a first TIL population that has been cultured and not expanded in vitro with a plurality of cell growth factors; wherein the second TIL population is obtained through step (A); (B) the second TIL population is contacted with a plurality of cell growth factors, with a plurality of cell activators, the expression and/or activity of CBL family members are reduced, and the TIL is co-cultured with feeder cells; wherein the third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with a cell growth factor; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with a cell growth factor, with a cell activator, reducing the expression and/or weakening the activity of a CBL family member, and co-culturing the TIL with feeder cells, wherein the CBL family member may include CBLB; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the method for obtaining TIL cells from a subject's tissue sample can be that the patient obtains an in situ tumor sample or a metastatic tumor sample by surgery, and the weight can be at least about 1g, or multiple tissues can be combined.
  • Tumor tissue, pleural effusion and/or peritoneal effusion are transported in a sample transport fluid, such as a commercially commonly used tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, at about 2-8 degrees and processed within 48 hours.
  • the tissue blocks can be mechanically broken into a size of about 1-27 cubic millimeters per piece, transferred into a breathable culture bag or Grex, and cell serum-free culture medium and IL-2 at a concentration of 300-9000IU/mL (for example, 1000-9000IU/mL, for example, 6000IU/mL) are added and cultured for about 3-14 days.
  • the cells in the culture medium are collected and transferred into a breathable culture bag, or a Grex, or a Xuri device.
  • the serum-free culture medium of the cells can be supplemented with the CD28 antibody, CD3 antibody and CD28 antibody of the present invention, magnetic beads (e.g., Dynabeads) comprising CD3 antibody and CD28 antibody and/or a nanomatrix (e.g., transACT) comprising CD3 antibody and CD28 antibody, IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, e.g., 6000 IU/mL), and IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, e.g., 6000 IU/mL), and IL-2 at a concentration of 300-9000 IU/mL, and IL-2 at a concentration of 6000 IU/mL, and IL-2 at a concentration of 10 ...
  • magnetic beads e.g., Dynabeads
  • Transduction makes the proportion of cells encoding genes of CBL family members in the TIL to be about 95% or less), after activating the TIL of the present invention for a certain period of time, irradiated PBMCs are added (TIL and PBMCs are at a ratio of about 1:40-about 1:400), and the culture is expanded for about 3-14 days.
  • the cells in the culture medium can be collected using a cell processing system, washed, frozen, and tested.
  • the final product CD3 ratio can be greater than 80%, the cell viability can be greater than 50%, and greater than 80% of the cells can be memory effector cells and effector cells.
  • IFN- ⁇ can be secreted after stimulation, and/or can have the characteristics of an increased proportion of activated cells.
  • a method for culturing cells comprising: reducing the expression and/or attenuating the activity of at least two family members selected from the group consisting of peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments in the cells.
  • family members selected from the group consisting of peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments in the cells.
  • the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and/or basophils.
  • immune cells comprise monocytes, macrophages and/or dendritic cells.
  • stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and/or peripheral blood stem cells.
  • iPSCs induced pluripotent stem cells
  • embryonic stem cells embryonic stem cells
  • bone marrow stem cells bone marrow stem cells
  • umbilical cord blood stem cells and/or peripheral blood stem cells.
  • the immune cell comprises Contains B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • TILs tumor infiltrating lymphocytes
  • TIL is TIL derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of peritumoral tissue, pleural effusion and/or peritoneal effusion and/or TIL derived from cryopreservation and resuscitation.
  • volume of the fragments is from about 1 cubic millimeter to about 27 cubic millimeters.
  • the immune cell comprises a chimeric antigen receptor and/or a T cell receptor.
  • the reduced expression and/or weakened activity of at least two family members selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the cell comprises at least two effects selected from the following groups: inhibiting the function of deubiquitinating enzymes and/or zinc finger nucleases, inhibiting the function of nucleases, inhibiting the function of negative regulation of cytokine signaling and inhibiting the function of E3 ubiquitin protein ligases.
  • improved cell characteristics include one or more selected from the following groups: improved cell proliferation ability, increased proportion of live cells, improved proportion of cell subpopulations, increased cytokine secretion ability, improved in vitro tumor cell killing ability and improved in vivo tumor killing ability.
  • the improved proportion of cell subpopulations comprises one or more selected from the following groups: an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and/or immature cells, a decreased proportion of apoptotic cells and an increased proportion of stem-like cells.
  • a method according to any one of embodiments 1-18, wherein the family member selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family comprises a ubiquitin binding domain, a C3H1-type zinc finger domain, an SH2 domain and an SH3 domain, respectively.
  • the family member selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family comprises TNFAIP3, ZC3H12A, SOCS1, and CBLB, respectively.
  • the gene regulation system destroys the family members selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family at the DNA level; and optionally, the cells have reduced expression and/or weakened activity selected from BRD4, FAS, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6 and TNIP1.
  • the gene regulation system destroys the family members selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family at the DNA level; and optionally, the cells have reduced expression and/or weakened activity selected from BRD4, FAS, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6 and TNIP1.
  • a method according to embodiment 23, wherein reducing the expression and/or weakening the activity of the family member selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family comprises: introducing into the cell a complex comprising the guide nucleic acid molecule and the enzyme protein, or a complex comprising the guide nucleic acid molecule and the nucleic acid encoding the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and an encoding Cas protein.
  • guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • a method according to any one of embodiments 23-27 wherein the guide nucleic acid molecule binds to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the following group: AGG, TGG, CGG and GGG, or binds to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of the protospacer adjacent motif (PAM) selected from the following group: NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, and NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the guiding nucleic acid molecule includes a targeting domain
  • the targeting domain comprises a sequence as shown in at least 2 of SEQ ID NOs: 1-106, 591-1561, 7267-7324, 7419, 7420, SEQ ID NOs: 213-280, 2533-3115, 7325-7345, 7416, 7417, SEQ ID NOs: 349-398, 3699-4392, 7346-7375, 7418, and SEQ ID NOs: 449-519, 5087-6176, 7376-7413, 7414, 7415.
  • family members selected from the peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family.
  • a pharmaceutical composition comprising the cell of embodiment 34, and optionally a pharmaceutically acceptable carrier.
  • a method of influencing cell growth comprising administering the cell of embodiment 34 and/or the pharmaceutical composition of embodiment 35.
  • a medicament for preventing and/or treating a disease and/or symptom comprising the cell of embodiment 34 and/or the pharmaceutical composition of embodiment 35 as an active ingredient.
  • a method for preventing and/or treating a disease and/or a symptom comprising administering the cell of embodiment 34 and/or the pharmaceutical composition of embodiment 35 to a subject in need thereof.
  • the disease and/or symptom comprises one or more selected from the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.
  • the present invention provides a method for reducing the expression and/or attenuating the activity of members of at least two families selected from the group consisting of peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments in the cell.
  • families selected from the group consisting of peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments in the cell.
  • the present invention provides a method for culturing cells, which reduces the expression and/or attenuates the activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, and/or their functionally active fragments.
  • families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, and/or their functionally active fragments.
  • the cell may further comprise reduced expression and/or reduced activity of a gene optionally selected from the group consisting of BRD4, FAS, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6, and TNIP1.
  • a gene optionally selected from the group consisting of BRD4, FAS, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6, and TNIP1.
  • the peptidase C64 family member may comprise a ubiquitin binding domain.
  • the peptidase C64 family member may comprise TNFAIP3.
  • the ZC3H12 family member may comprise a C3H1-type zinc finger domain.
  • the ZC3H12 family member may comprise ZC3H12A.
  • the STAT-induced STAT inhibitor (SSI) family member may comprise an SH2 domain.
  • the STAT-induced STAT inhibitor (SSI) family member may comprise SOCS1.
  • the CBL family member may comprise an SH3 domain.
  • the CBL family member may comprise a CBLB.
  • the expression of peptidase C64 family and ZC3H12 family members and/or their functionally active fragments is reduced and/or the activity is attenuated.
  • the expression of TNFAIP3 and ZC3H12A is reduced and/or the activity is attenuated.
  • the expression and/or activity of peptidase C64 family and STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments are reduced.
  • the expression and/or activity of TNFAIP3 and SOCS1 are reduced.
  • the expression of peptidase C64 family and CBL family members and/or their functionally active fragments is reduced and/or the activity is attenuated.
  • the expression of TNFAIP3 and CBLB is reduced and/or the activity is attenuated.
  • the expression and/or activity of ZC3H12 family and STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments are reduced.
  • the expression and/or activity of ZC3H12A and SOCS1 are reduced.
  • the expression of ZC3H12 family and CBL family members and/or their functionally active fragments are reduced and/or their activity is attenuated.
  • the expression of ZC3H12A and CBLB is reduced and/or their activity is attenuated.
  • the expression and/or activity of STAT-induced STAT inhibitor (SSI) family and CBL family members and/or their functionally active fragments are reduced.
  • the expression and/or activity of SOCS1 and CBLB are reduced.
  • the expression and/or activity of peptidase C64 family, ZC3H12 family and STAT-induced STAT inhibitor (SSI) family members and/or their functionally active fragments are reduced.
  • the expression and/or activity of TNFAIP3, ZC3H12A and SOCS1 are reduced.
  • the expression of peptidase C64 family, ZC3H12 family and CBL family members and/or their functionally active fragments are reduced and/or their activity is attenuated.
  • the expression of TNFAIP3, ZC3H12A and CBLB is reduced and/or their activity is attenuated.
  • the expression and/or activity of peptidase C64 family, STAT-induced STAT inhibitor (SSI) family and CBL family members and/or their functionally active fragments are reduced.
  • the expression and/or activity of TNFAIP3, SOCS1 and CBLB are reduced.
  • the expression and/or activity of members of the ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments are reduced.
  • the expression and/or activity of ZC3H12A, SOCS1 and CBLB are reduced.
  • the expression and/or activity of peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family members and/or their functionally active fragments are reduced.
  • the expression and/or activity of TNFAIP3, ZC3H12A, SOCS1 and CBLB are reduced.
  • the target gene of the present invention may be a gene encoding a member of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, and/or a functionally active fragment thereof.
  • a cell obtained by reducing the expression and/or weakening the activity of at least two target genes of the cell may show improved cell characteristics.
  • the cell whose expression and/or activity of the target gene is unchanged may refer to a cell derived from the same donor and whose expression and/or activity of at least two target genes of the cell have not been reduced and/or whose activity has not been weakened. In one embodiment, the cell whose expression and/or activity of the target gene is unchanged may refer to a cell derived from the same donor and whose expression and/or activity of other genes other than the target gene of the cell (e.g., knocking out the other gene has substantially no effect on cell function) have not been reduced and/or whose activity has been weakened.
  • the corresponding cells that have not reduced the expression and/or weakened the activity of at least two target genes of the cell may refer to cells isolated in the same manner from the same donor and have not reduced the expression and/or weakened the activity of at least two target genes of the cell. In one embodiment, the corresponding cells that have not reduced the expression and/or weakened the activity of at least two target genes of the cell may refer to cells from the same tumor source of the same donor and have not reduced the expression and/or weakened the activity of at least two target genes of the cell.
  • the corresponding cells that have not reduced the expression and/or weakened the activity of at least two target genes of the cell may refer to cells from the same tumor source of the same donor divided into two groups, wherein one group of cells that have not reduced the expression and/or weakened the activity of at least two target genes of the cell may be corresponding cells that have not reduced the expression and/or weakened the activity of at least two target genes of the cell.
  • the reduced expression and/or weakened activity of at least two target genes may mean that the target genes in natural cells are in a certain degree of expression state, and after the treatment of the present invention, the expression level of the target genes in the cell can be reduced, that is, the reduction in the expression level of the target genes can be a change in the natural cells from expressing the target genes to basically not expressing the target genes or reducing the amount of expression of the target genes.
  • the cell comprises an immune cell.
  • the cell comprises an immune effector cell.
  • the cell comprises an immune effector T cell, an immune effector NK cell, an immune effector NKT cell.
  • the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and/or a basophil.
  • the cells comprise monocytes, macrophages and/or dendritic cells.
  • the cells of the present invention also include cells derived from stem cell differentiation.
  • the cells of the present invention also include cells derived from pluripotent stem cell differentiation.
  • obtaining the stem cells of the present invention can be produced by induction.
  • the above-mentioned stem cells of the present invention can include induced pluripotent stem cells (iPSC), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and/or peripheral blood stem cells.
  • stem cells of the present invention also include pluripotent cells, multipotent cells, precursor cells and progenitor cells.
  • stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, placental stem cells obtained from placental tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from reproductive tissue of a fetus.
  • pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs.”
  • the cell comprises B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • “unmodified cells” or “unmodified cells” may refer to cells or cell colonies in which the genome is not modified and does not comprise a gene regulatory system or comprises a control gene regulatory system (e.g., an empty vector control, a non-targeted gRNA, an interfering siRNA, etc.).
  • the cell comprises ⁇ T cells and/or ⁇ T cells.
  • the cell comprises tumor infiltrating lymphocytes (TIL).
  • the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion TIL and/or TIL revived after cryopreservation.
  • the TILs of the present invention may be TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusions and/or peritoneal effusions, and/or TILs derived from cryopreservation and resuscitation.
  • the TILs of the present invention may be obtained by processing tumor tissue into tumor fragments.
  • the volume of the tumor fragments of the present invention is about 1-27 cubic millimeters.
  • the volume of the tumor fragments of the present invention is about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 14 cubic millimeters, about 15 cubic millimeters, about 16 cubic millimeters, about 17 cubic millimeters, about 18 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters, about 22 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, about 27 cubic millimeters, about 28 cubic millimeters, about 29 cubic millimeters, about 30 cubic millimeters, about 31 cubic millimeters, about 31 cubic millimeters, about 31 cubic
  • the cell comprises an engineered immune receptor displayed on the cell surface.
  • the engineered immune receptor specifically binds to an antigen expressed on a target cell.
  • the cell comprises a chimeric antigen receptor and/or a T cell receptor.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: reducing the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the TIL.
  • TIL tumor infiltrating lymphocytes
  • TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments can be reduced in the TILs.
  • SSI STAT-induced STAT inhibitor
  • the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue fragments, pleural effusion and/or peritoneal effusion of the present invention and not amplified in vitro can be subjected to a first stage of in vitro amplification and a second stage of in vitro amplification, and in the second stage of in vitro amplification of the present invention, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family in the TILs and/or their functionally active fragments can be reduced.
  • SSI STAT-induced STAT inhibitor
  • TILs that are not accompanied by tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and have not been expanded in vitro undergo a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family in the TILs and/or their functionally active fragments can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not amplified in vitro can be subjected to a first stage in vitro amplification and a second stage in vitro amplification, and in the first stage in vitro amplification of the present invention, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the TILs can be reduced, and in the second stage in vitro amplification of the present invention, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family in the TILs and/or their functionally active fragments can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion of the present invention and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention,
  • the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the TIL are reduced.
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the third stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family in the TILs and/or their functionally active fragments can be reduced.
  • SSI STAT-induced STAT inhibitor
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the TILs can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, The expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the TILs can be reduced, and In the second stage in vitro expansion of the present invention, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the C
  • each stage of in vitro expansion can be determined by changes in the number of TIL cells.
  • the number of TIL cells increases at least about 1 times, it can be considered that TIL cells have entered the next stage of in vitro expansion.
  • the number of TIL cells increases at least about 1-1000 times, such as at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times or at least about 1000 times, it can be considered that TIL cells have entered the next stage of in vitro expansion.
  • each stage of in vitro expansion can also be divided by the change of the conditions of TIL cell culture.
  • TIL cell culture medium when cell activators and/or cell growth factors are added or supplemented to the cell culture medium, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • IL-2 when added or supplemented to the cell culture medium, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • one or more gene regulatory systems are added or supplemented to the cell culture medium, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • feeder cells are added or supplemented to the cell culture medium, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • each stage can also be divided by the number of days of TIL cell culture.
  • TIL cells are cultured in vitro for about 1-100 days, such as about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days or about 100 days, the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • the reducing the expression and/or weakening the activity of the peptidase C64 family member in the cell comprises inhibiting the function of deubiquitinase and/or zinc finger nuclease.
  • the reducing the expression and/or weakening the activity of the ZC3H12 family member in the cell comprises inhibiting the function of a nuclease.
  • the reducing the expression and/or weakening the activity of a STAT-induced STAT inhibitor (SSI) family member in the cell comprises inhibiting the function of negative regulation of cytokine signaling.
  • SSI STAT-induced STAT inhibitor
  • the reducing the expression and/or weakening the activity of the CBL family member in the cell comprises inhibiting the function of E3 ubiquitin protein ligase.
  • cells obtained by reducing the expression and/or attenuating the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family show improved cell properties.
  • the improved cell properties include one or more selected from the following groups: improved cell proliferation ability (i.e., cell number), increased proportion of living cells, improved cell subpopulation proportions, enhanced cytokine secretion ability, enhanced in vitro tumor cell killing ability, and enhanced in vivo tumor killing ability.
  • the improved cell subpopulation ratio comprises one or more selected from the following groups: an increased ratio of activated cells, a decreased ratio of regulatory cells, a decreased ratio of exhausted cells, an increased ratio of central memory cells and/or immature cells, a decreased ratio of apoptotic cells, and an increased ratio of stem-like cells.
  • the improved cell number of the present invention means that the cell number of the cells of the present invention in which the expression and/or activity of at least two members of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family is reduced in at least one in vitro expansion stage can be increased by at least about 1-50 times, for example, to 100 times, compared to cells in which the expression and/or activity of at least two members of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family are not changed.
  • SSI STAT-induced STAT inhibitor
  • the increased proportion of live cells can be expressed as an increase in cell survival rate.
  • the increased proportion of live cells in the present invention can mean that the proportion of live cells of the present invention in which the expression and/or activity of at least two members of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about
  • the improved cytokine secretion capacity of the present invention may refer to an improved cytokine secretion capacity of the cell selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention may refer to a cell having an improved cytokine secretion capacity ... cell having an improved cytokine secretion capacity selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the increased cytokine secretion capacity of the present invention may refer to that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and/or activity of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family is reduced and/or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%.
  • At least about 70% at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the cytokine secretion capacity of the cells of the present invention is determined by flow cytometry or CBA (Cytometric Bead Array).
  • the improved in vitro tumor cell killing ability and/or improved in vivo tumor killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family are reduced in at least one in vitro expansion stage can be increased by at least about 1-50 times, for example, at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 21 times, at least about 22 times, at least about 23 times, at least about 24 times, at least about 25 times, at least about 26 times, at least about
  • the tumor cell killing rate of the cells of the present invention can be measured by the IncuCyte system or CFSE and DAPI staining.
  • tumor cell killing by a cell of the invention may refer to the ability of a cell to kill solid tumor cells.
  • the improved cell subpopulation ratio of the present invention may include one or more selected from the following groups: an increased CD8 + cell ratio, an increased central memory cell and/or naive cell ratio, a decreased regulatory cell ratio, an increased activated cell ratio, an increased tumor-specific cell ratio (having a CD103 + CD39 + phenotype), an increased stem-like cell ratio, a decreased exhausted cell ratio, and a decreased apoptotic cell ratio.
  • the increased CD8 + cell ratio of the present invention can be an increase in the ratio of CD8 positive cells in cells.
  • the CD8 + cell ratio in cells can be increased by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, to at least about 16%.
  • the increased proportion of activated cells of the present invention can be an increase in the proportion of CD28 + , CD25 + and/or 41BB + cells in the cells.
  • the proportion of activated cells in the cells can be increased by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%
  • the reduced proportion of exhausted cells of the present invention can be an increase in the proportion of PD-1 + , LAG-3 + , TIM-3 + , CD39 + , CD38 + and/or CD101 + cells in the cells.
  • the proportion of exhausted cells in the cells can be reduced by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 5%
  • the proportion of regulatory cells decreased in the present invention may be a decrease in the proportion of CD4 + CD25 + Foxp3 + cells in cells.
  • the proportion of regulatory cells in a cell can be reduced by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%, at least about 0.4%
  • the ratio of apoptotic cells decreased in the present invention may be a reduction in the ratio of Annexin V + 7-AAD + cells and/or Annexin V + 7-AAD ⁇ cells in cells.
  • the proportion of apoptotic cells in a cell can be reduced by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%,
  • the increased proportion of cells having stemness in the present invention may be an increase in the proportion of CD69 - CD39 - cells and/or TCF1 + cells among cells.
  • the proportion of cells having stemness in the cells can be increased by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about at least about
  • the increased proportion of central memory cells in the present invention may be an increase in the proportion of CD45RA - CCR7 + or CD45RO + CD62L + cells among cells.
  • the proportion of central memory cells in the cells can be increased by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about at least about
  • the increased proportion of naive T cells in the present invention can be an increase in the proportion of CD45RO - CD62L + cells in cells.
  • the proportion of naive cells in cells can be increased by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about
  • the culture method of the present invention may include a gene editing step for cells.
  • a gene editing step for cells includes: subjecting the cells to at least one stage of in vitro amplification, wherein, during at least one stage of in vitro amplification, a gene regulatory system may be introduced into the cells.
  • the gene regulation system can destroy the target gene at the DNA level.
  • the gene regulation system can destroy the region or fragment of the target gene in the genome of the cell.
  • the DNA region or fragment where the target gene is located in the cell is sheared and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and continuous.
  • the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family is inhibited.
  • the genomic region described in the present invention is determined based on the human reference genome version hg38.
  • the activity of TNFAIP3 and ZC3H12A in the cells of the present invention is inhibited.
  • the preferred subregions of TNFAIP3 and ZC3H12A in the cells shown in Tables 1A and 1B of the present invention are knocked out and/or inhibited.
  • the preferred targeted subregions shown in TNFAIP3 R_1 to 30 in Table 1A of the present invention and ZC3H12A R_1 to 9 in Table 1B, the combination of combination numbers 1 to 270 are knocked out and/or inhibited.
  • the activities of TNFAIP3 and SOCS1 in the cells of the present invention are inhibited.
  • the preferred subunits of TNFAIP3 and SOCS1 in the cells shown in Tables 1A and 1C of the present invention are
  • the preferred targeted subregions shown in Table 1A of the present invention are combined with TNFAIP3 R_1 to 30 and SOCS1 R_1 to 10 in Table 1C, and the combination of combination numbers 271 to 570 is knocked out and/or inhibited.
  • the activity of TNFAIP3 and CBLB in the cells of the present invention is inhibited.
  • the preferred subregions of TNFAIP3 and CBLB in the cells shown in Tables 1A and 1D of the present invention are knocked out and/or inhibited.
  • the combination of the preferred targeted subregions shown in TNFAIP3 R_1 to 30 in Table 1A of the present invention and CBLB R_1 to 44 in Table 1D, the combination of combination numbers 571 to 1890 is knocked out and/or inhibited.
  • the activity of ZC3H12A and SOCS1 in the cell of the present invention is inhibited.
  • the preferred subregions of ZC3H12A and SOCS1 in the cell shown in Tables 1B and 1C of the present invention are knocked out and/or inhibited.
  • the combination of the preferred targeted subregions shown in ZC3H12A R_1 to 9 in Table 1B of the present invention and SOCS1 R_1 to 10 in Table 1C, the combination of combination numbers 1891 to 1980 is knocked out and/or inhibited.
  • the activity of ZC3H12A and CBLB in the cell of the present invention is inhibited.
  • the preferred subregions of ZC3H12A and CBLB in the cell shown in Tables 1B and 1D of the present invention are knocked out and/or inhibited.
  • the combination of the preferred targeted subregions shown in ZC3H12A R_1 to 9 in Table 1B of the present invention and CBLB R_1 to 44 in Table 1D, the combination of combination numbers 1981 to 2376 is knocked out and/or inhibited.
  • the activities of SOCS1 and CBLB in the cells of the present invention are inhibited.
  • the preferred subregions of SOCS1 and CBLB in the cells shown in Tables 1C and 1D of the present invention are knocked out and/or inhibited.
  • the combination of the preferred targeted subregions shown in SOCS1 R_1 to 10 in Table 1C of the present invention and CBLB R_1 to 44 in Table 1D, the combination of combination numbers 2377 to 2816 is knocked out and/or inhibited.
  • the gene regulation system may include a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have a nucleic acid cleavage enzyme activity, and the guide nucleic acid molecule may guide the The enzyme protein specifically shears the region or fragment thereof where the target gene is located.
  • the guide nucleic acid molecule and the enzyme protein can exist in the form of a ribonucleoprotein complex (RNP) or exist independently of each other.
  • the enzyme protein can include a Cas protein.
  • a polynucleotide encoding a gRNA and a Cas protein can be introduced or introduced independently of each other into a target cell.
  • the present invention reduces the expression and/or weakens the activity of at least two target genes of a cell, and may include: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell.
  • RNP ribonucleoprotein complex
  • the enzyme protein may include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.
  • the guide nucleic acid molecule may include a guide RNA (gRNA).
  • gRNA guide RNA
  • gRNA guide RNA
  • a complex comprising a polynucleotide encoding a gRNA and a Cas protein may be introduced into the cell.
  • a complex comprising a gRNA and a Cas protein may be introduced into the cell.
  • the gRNA can be used to bind to the sequence of the target gene.
  • the binding of the gRNA to the sequence of the target gene can be completely complementary, partially complementary, or hybridized to the sequence of the target gene under moderate stringency or stringent conditions.
  • the binding of the gRNA to the sequence of the target gene can enable the CRISPR system of the gRNA to specifically cleave the target gene.
  • the editing target region of the present invention may be a region before the start codon.
  • the editing target region of the present invention may be a region with high transcription factor binding ability.
  • the editing target region of the present invention may be a region with a specific number of transcription factor binding numbers.
  • the editing target region of the present invention may be a continuous region with about 3 or more transcription factor binding numbers.
  • the genomic coordinates of the editing target region of the present invention may be selected from the preferred targeting sub-regions shown in Tables 1A to 1D.
  • the guide nucleic acid molecule targeting TNFAIP3 of the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 107-212, 1562-2532.
  • the guide nucleic acid molecule targeting ZC3H12A of the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 281-348, 3116-3698.
  • the present invention The guide nucleic acid molecule targeting SOCS1 can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 399-448, 4393-5086.
  • the guide nucleic acid molecule targeting CBLB of the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 520-590, 6177-7266.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) downstream, and the protospacer adjacent motif (PAM) may be AGG, TGG, GGG or CGG.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene is determined, a person skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides upstream of the 5' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the following group: AGG, TGG, GGG and CGG.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of the protospacer adjacent motif (PAM) selected from the following group: NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: NTTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV), wherein N may be A, T, C or G, Y may be T or C, V may be A, C or G, and R may be A or G.
  • TTYN TTTN / TTCN
  • VTTV ATTV / CTTV / GTTV
  • TRTV TRTV
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: TTTV (TTTA, TTTC, or TTTG), wherein V may be A, C or G.
  • TTTV TTTA, TTTC, or TTTG
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guiding nucleic acid molecule of the present invention targets
  • the upstream region may have a PAM sequence selected from the following: TTTV, TATV, or TYCV, wherein V may be A, C or G, and Y may be T or C.
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.
  • the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TNN, or NTN, wherein N may be A, T, C or G.
  • a person skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and a suitable gRNA can be designed for the target sequence.
  • the guide nucleic acid molecule may comprise a target sequence consisting of about 15 to about 25 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG in the DNA encoding a member of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or its functionally active fragments.
  • SSI STAT-induced STAT inhibitor
  • the guide nucleic acid molecule may comprise a target sequence consisting of about 15 to about 25 nucleotides before the PAM region represented by AGG, TGG, GGG and/or CGG in the DNA encoding a member of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or its functionally active fragments.
  • SSI STAT-induced STAT inhibitor
  • a target sequence consisting of about 15 to about 20 nucleotides, about 17 to about 23 nucleotides, about 19 to about 23 nucleotides, about 20 to about 23 nucleotides, about 21 to about 23 nucleotides, about 22 to about 23 nucleotides, about 15 to about 21 nucleotides, about 17 to about 21 nucleotides, about 19 to about 21 nucleotides, about 20 to about 21 nucleotides, about 15 to about 20 nucleotides, about 17 to about 20 nucleotides, about 19 to about 20 nucleotides, about 15 to about 19 nucleotides, about 17 to about 19 nucleotides, or about 15 to about 17 nucleotides.
  • the sequence can be a region or a fragment thereof defined by the genomic coordinates shown in Tables 2A-2D.
  • some target sequences are displayed in 2A-2D as target sequences for double knockout; however, the target sequences of two or more targets selected in Tables 3A-3D of the present invention are combined with each other, which can also be used for double knockout or multiple knockout of the target.
  • the guiding nucleic acid molecule can comprise a targeting domain of an sgRNA targeting TNFAIP3 as shown in any one of SEQ ID NOs: 1-106, 591-1561, 7267-7324, 7419, 7420, a targeting domain of an sgRNA targeting ZC3H12A as shown in any one of SEQ ID NOs: 213-280, 2533-3115, 7325-7345, 7416, 7417, a targeting domain of an sgRNA targeting SOCS1 as shown in any one of SEQ ID NOs: 349-398, 3699-4392, 7346-7375, 7418, or a targeting domain of an sgRNA targeting CBLB as shown in any one of SEQ ID NOs: 449-519, 5087-6176, 7376-7413, 7414, 7415.
  • the activity of TNFAIP3 and ZC3H12A in the cells of the present invention is inhibited.
  • the guide sequences shown in SEQ ID NO: 1-106 and SEQ ID NO: 213-280 of the present invention are combined in pairs to form editing combination numbers 1 to 7208, which are knocked out and/or inhibited.
  • the activity of TNFAIP3 and SOCS1 in the cells of the present invention is inhibited.
  • the guide sequences shown in SEQ ID NO: 1-106 and SEQ ID NO: 349-398 of the present invention are combined in pairs to form a combination of editing combination numbers 7209 to 12508, which is knocked out and/or inhibited.
  • the activity of TNFAIP3 and CBLB in the cells of the present invention is inhibited.
  • the guide sequences shown in SEQ ID NO: 1-106 and SEQ ID NO: 449-519 of the present invention are combined in pairs to form a combination of editing combination numbers 12509 to 20034, which is knocked out and/or inhibited.
  • the activities of ZC3H12A and SOCS1 in the cells of the present invention are inhibited.
  • the guide sequences shown in SEQ ID NOs: 213-280 and 349-398 of the present invention The sequences were combined pairwise to form edited combinations, and the combinations of sequence numbers 20035 to 23434 were knocked out and/or suppressed.
  • the activity of ZC3H12A and CBLB in the cells of the present invention is inhibited.
  • the combination of the guide sequences shown in SEQ ID NO: 213-280 and SEQ ID NO: 449-519 of the present invention is combined in pairs to form the combination of editing combination numbers 23435 to 28262, which is knocked out and/or inhibited.
  • the activities of SOCS1 and CBLB in the cells of the present invention are inhibited.
  • the guide sequences shown in SEQ ID NO: 349-398 and SEQ ID NO: 449-519 of the present invention are combined in pairs to form a combination of editing combination numbers 28263 to 31812, which is knocked out and/or inhibited.
  • the proportion of cells expressing the products of the target genes in the cells obtained by reducing the expression and/or attenuating the activity of at least two target genes in the cells can be reduced and/or the expression level of the target genes in a single cell can be decreased.
  • the proportion of cells expressing the products of the target genes in the cells obtained by reducing the expression and/or attenuating the activity of at least two target genes in the cells is reduced by at least about 5% compared to cells in which the expression and/or activity of the target genes are not changed.
  • the proportion of cells expressing the product of a gene encoding a member of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or a functionally active fragment thereof is reduced by at least about 100-5%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • SSI STAT-induced STAT inhibitor
  • the proportion of cells expressing the product of a gene encoding a member of at least two of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or a functionally active fragment thereof can be reduced from the proportion of cells that can be observed to 1%.
  • the proportion of cells expressing the product of a gene encoding a member of at least two of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or a functionally active fragment thereof can be reduced to at least about 100-1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • SSI STAT-induced STAT inhibitor
  • the proportion of cells expressing the product of the gene encoding members of at least two families selected from the peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments can be detected by flow cytometry.
  • the expression of at least two target genes in the cells is reduced and/or the activity is attenuated, and the proportion of cells expressing the products of the genes encoding members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments can be up to about 95%.
  • the proportion of cells expressing the products of the genes encoding members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments can be up to about 95%.
  • the proportion of cells expressing the product of the gene encoding members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments can be at most about 95-5%, such as at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%.
  • SSI STAT-induced STAT inhibitor
  • the proportion of cells expressing the product of the gene encoding members of at least two families selected from the peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments can be detected by flow cytometry.
  • the expression of at least two target genes of the cell is reduced and/or the activity is weakened, and the expression of the target gene in a single cell can be reduced by at least about 5%.
  • the expression of the target gene in a single cell can be reduced by at least about 100-5%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the expression of the target gene in a single cell can be from the observable expression to 1%.
  • the expression level of the target gene in a single cell can be reduced to at least about 100-1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1%.
  • the expression level of the target gene in a single cell obtained by reducing the expression and/or weakening the activity of at least two target genes in the cell can be at most about 95% of the cell in which the expression and/or activity of the target gene is not changed.
  • the expression level of the gene encoding members of at least two families selected from the peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family and/or their functionally active fragments (e.g., genes encoding TNFAIP3, ZC3H12A, SOCS1, CBLB) in a single cell can be at most about 95-5%, such as at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 50%, at most about 60%, at most about 70%, at most about 8 ...
  • the method of the present invention comprises: subjecting the cell to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family in the cell are reduced.
  • the expression and/or activity of members of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family in the cell are reduced.
  • the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion, the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family of the TILs expanded in vitro in the first stage are reduced.
  • SSI STAT-induced STAT inhibitor
  • the first stage in vitro expansion is performed for at least about 7 days.
  • the second stage in vitro expansion is performed for at least about 7 days.
  • the cell in a single stage of in vitro expansion of the present invention, can be contacted with the one or more cell activators and the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family and/or their functionally active fragments in the cell can be reduced.
  • the cell activator can include an agonist of one or more targets selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family of the cell of the present invention are reduced and/or the activity is weakened and the cell is contacted with one or more cell activators of the present invention.
  • the TIL of the present invention can be selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family.
  • the expression of members of at least 2 families is reduced and/or the activity is weakened, and the TIL is contacted with one or more cell activators of the present invention.
  • the expression of members of at least 2 families selected from the peptidase C64 family, the ZC3H12 family, the STAT inhibitor (SSI) family of STAT induction and the CBL family of the TIL of the present invention can be reduced and/or the activity is weakened, and the TIL is contacted with one or more cell activators of the present invention.
  • the expression of members of at least 2 families selected from the peptidase C64 family, the ZC3H12 family, the STAT inhibitor (SSI) family of STAT induction and the CBL family of the TIL of the present invention can be reduced and/or the activity is weakened, and the TIL is contacted with one or more cell activators of the present invention.
  • the cells of the present invention substantially simultaneously reduce the expression and/or weaken the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, and contact with one or more cell activators of the present invention.
  • families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, and contact with one or more cell activators of the present invention.
  • the cells of the present invention first reduce the expression and/or weaken the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, for example, 2-48 hours in advance, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, and then contact with one or more cell activators of the present invention.
  • families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family
  • the cells of the present invention are first contacted with one or more cell activators of the present invention, for example, 2-48 hours in advance, such as 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then the expression and/or activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family are reduced.
  • 2-48 hours in advance such as 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc.
  • the TIL of the present invention substantially simultaneously reduces the expression and/or weakens the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, and contacts with one or more cell activators of the present invention.
  • the TIL of the present invention substantially simultaneously reduces the expression and/or weakens the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family.
  • the expression of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family is reduced and/or the activity is weakened, and contacted with one or more cell activators of the present invention.
  • the TILs of the present invention substantially simultaneously reduce the expression and/or the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family, and contacted with one or more cell activators of the present invention.
  • the second stage in vitro expansion of the present invention is carried out for at least about 7 days.
  • the second stage in vitro expansion of the present invention can be carried out for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the second stage in vitro expansion of the present invention can be carried out for about 9 days to about 14 days
  • the second stage in vitro expansion of the present invention can be carried out for about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the second stage in vitro expansion of the present invention can be considered as the REP (rapid expansion protocol) stage.
  • the first stage in vitro expansion of the present invention can be considered as the preREP stage.
  • the first stage in vitro amplification of the present invention is carried out for at least about 7 days.
  • the second stage in vitro amplification of the present invention can be carried out for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the second stage in vitro amplification of the present invention can be carried out for about 9 days to about 14 days
  • the second stage in vitro amplification of the present invention can be carried out for about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the number of days that the second stage in vitro amplification of the present invention is performed can be calculated from the start time of the second stage in vitro amplification.
  • the second stage in vitro amplification starts, it can be considered that the second stage in vitro amplification has been performed for about 0 hours.
  • the second stage in vitro amplification has been performed for about 1 day.
  • the day when the second stage in vitro amplification starts can be considered that the second stage in vitro amplification has been performed for about 0 days.
  • the number of days that the second stage in vitro amplification of the present invention is performed can be calculated by the number of days that the second stage in vitro amplification is performed. For example, the day after the start of the second stage in vitro amplification, it can be considered that the second stage in vitro amplification has been performed for about 1 day.
  • the cell activator of the present invention may include one or more selected from the following groups: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and their functionally active fragments.
  • the cell activator of the present invention may include an agonist of one or more targets selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the cell activator of the present invention may include an antibody selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB and their antigen-binding fragments.
  • the cell activator of the present invention may include a CD3 agonist.
  • the cell activator of the present invention may include an anti-CD3 antibody and/or an antigen-binding fragment thereof, such as OKT3 of Miltenyi Biotech, and SP34 of BD.
  • the cell activator of the present invention may include a CD28 agonist.
  • the cell activator of the present invention may include an anti-CD28 antibody and/or an antigen-binding fragment thereof, such as 15E8 of Merck.
  • the cell activator of the present invention may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, it may comprise the light chain VL and heavy chain VH of OKT3 of Miltenyi Biotech, or it may comprise the light chain VL and heavy chain VH of SP34 of BD.
  • the cell activator of the present invention may comprise a CD28 agonist.
  • the cell activator of the present invention may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example, it may comprise the light chain VL and heavy chain VH of 15E8 of Merck.
  • the cell activator of the present invention may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, it may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of OKT3 of Miltenyi Biotech, or it may comprise the light chain VL and heavy chain VH of SP34 of BD.
  • LCDR1-3 and heavy chain HCDR1-3, the anti-CD3 antibody and/or its antigen-binding fragment of the present invention may have CD3 binding ability.
  • the cell activator of the present invention may include a CD28 agonist.
  • the cell activator of the present invention may include an anti-CD28 antibody and/or its antigen-binding fragment, such as the light chain LCDR1-3 and heavy chain HCDR1-3 of Merck's 15E8, and the anti-CD28 antibody and/or its antigen-binding fragment of the present invention may have CD28 binding ability.
  • the antibody or its antigen-binding protein of the present invention comprises at least one CDR in the heavy chain variable region VH of the antibody and/or at least one CDR in the light chain variable region VL of the antibody.
  • the CDR of the present invention may be defined according to the IMGT nomenclature, the CDR of the present invention may be defined according to Chothia, or the CDR of the present invention may be defined according to Kabat.
  • contacting the cells of the present invention with one or more cell activators of the present invention may include one or more methods selected from the following groups: (1) adding the cell activators of the present invention to the cell culture medium of the cells of the present invention; (2) adding engineered cells expressing the cell activators of the present invention to the cell culture medium of the cells of the present invention; (3) adding a solid phase medium containing the cell activators of the present invention to the cell culture medium of the cells of the present invention.
  • contacting the cells of the present invention with one or more cell activators of the present invention may include adding a solid phase medium containing the cell activators of the present invention to the cell culture medium of the cells of the present invention.
  • contacting the cells of the present invention with one or more cell activators of the present invention may include adding a solid phase medium containing the CD28 antibody and the CD3 antibody of the present invention to the cell culture medium of the cells of the present invention.
  • the initial concentration of the cell activator in the cell culture medium of the cells of the present invention can be at least about 30 ng/mL.
  • the initial concentration of the CD28 antibody of the present invention in the cell culture medium of the cells of the present invention can be at least about 30 ng/mL;
  • the initial concentration of the CD3 antibody of the present invention in the cell culture medium of the cells of the present invention can be at least about 30 ng/mL.
  • the selection of the initial concentration of the CD28 antibody of the present invention can be independent of the selection of the initial concentration of the CD3 antibody of the present invention; for example, the initial concentrations of the CD28 antibody of the present invention and the CD3 antibody of the present invention in the cell culture medium of the cells of the present invention can be arbitrarily combined.
  • the initial concentration of the CD28 antibody of the present invention in the cell culture medium of the cells of the present invention can be arbitrarily combined. Meaning is selected from about 30ng/mL to about 300ng/mL.
  • the initial concentration of the CD3 antibody of the present invention in the cell culture medium of the cells of the present invention can be arbitrarily selected from about 30ng/mL to about 300ng/mL.
  • the initial concentration of the CD28 antibody of the present invention in the cell culture medium of the cells of the present invention can be arbitrarily selected from about 30ng/mL to about 300ng/mL
  • the initial concentration of the CD3 antibody of the present invention in the cell culture medium of the cells of the present invention can be arbitrarily selected from about 30ng/mL to about 300ng/mL
  • the selection of the initial concentration of the CD28 antibody of the present invention can be independent of the selection of the initial concentration of the CD3 antibody of the present invention.
  • the diameter of the solid phase medium of the present invention can be about 500 nanometers to about 10 microns.
  • the diameter of the solid phase medium of the present invention can be measured by transmission electron microscopy.
  • the diameter of the solid phase medium of the present invention can be about 1 nanometer to about 500 nanometers.
  • the diameter of the solid phase medium of the present invention can be about 100 nanometers to about 500 nanometers.
  • the diameter of the solid phase medium of the present invention can be about 200 nanometers to about 500 nanometers.
  • the diameter of the solid phase medium of the present invention can be measured by transmission electron microscopy.
  • the solid phase medium of the present invention may comprise a polymer.
  • the solid phase medium of the present invention may comprise dextran.
  • the solid phase medium of the present invention contains at least about 25 ⁇ g of the cell activating agent of the present invention per mg.
  • the solid phase medium containing the cell activator of the present invention is added to the cell culture medium of the cells of the present invention at a ratio of about 100:1 to about 1:2000, preferably about 1:100 to about 1:2000.
  • the solid phase medium containing the cell activator of the present invention is added to the cell culture medium of the cells of the present invention at a ratio of about 2:1 to about 1:2.
  • the solid phase medium containing the cell activator of the present invention can be added to the cell culture medium of the cells of the present invention at a ratio of about 2:1 to about 1:2.
  • the solid phase medium comprising the cell activating agent of the present invention such as a CD3 agonist and/or a CD28 agonist, is added to the cell culture medium of the cells of the present invention at a ratio of the solid phase medium of the present invention to the cells of the present invention of between about 2:1 and about 1:2, at a ratio of about 2:1 to about 1:1, or at a ratio of about 1:1 to about 1:2.
  • the solid phase medium containing the cell activator of the present invention can be added to the cell culture medium of the cells of the present invention at a ratio of about 1:100 to about 1:2000 of the solid phase medium of the present invention to the cells of the present invention.
  • the solid phase medium containing the cell activator of the present invention can be added to the cell culture medium of the cells of the present invention at a ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:300 to about 1:2000, about 1:400 to about 1:2000, about 1:500 to about 1:2000, about 1:600 to about 1:2000, about 1:700 to about 1:2000, about 1:800 to about 1:2000, about 1:900 to about 1:1000, about 1:1100 to about 1:12000, about 1:1200 to about 1:1300, about 1:1400 to about 1:1500, about 1:1600 to about 1:1600, about 1:1700 to about 1:1800, about 1:1900 to about 1:1900
  • a solid phase medium comprising a CD28 agonist and a CD3 agonist of the present invention can be added to the cell culture medium of the cells of the present invention at a ratio of about 1:900 to about 1:2000, about 1:1000 to about 1:2000, about 1:1
  • the method of the present invention may further comprise: contacting the cells of the present invention with one or more cell growth factors during at least one stage of the in vitro expansion of the present invention.
  • the cells of the present invention can be contacted with the cell activator of the present invention and with one or more cell growth factors of the present invention.
  • the TIL of the present invention in the first stage of in vitro expansion of the present invention, can be contacted with the cell activator of the present invention and with one or more cell growth factors of the present invention.
  • the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be contacted with the cell activator of the present invention and with one or more cell growth factors of the present invention.
  • the TIL of the present invention in the third stage of in vitro expansion of the present invention, can be contacted with the cell activator of the present invention and with one or more cell growth factors of the present invention.
  • the cells of the present invention are contacted with the cell activating agent of the present invention and one or more cell growth factors of the present invention substantially simultaneously.
  • the cells of the present invention can be contacted with one or more cell growth factors of the present invention and one or more cell activators of the present invention substantially at the same time.
  • the cells of the present invention can be contacted with one or more cell growth factors of the present invention first, for example, 2-48 hours in advance, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contacted with one or more cell activators of the present invention.
  • one or more cell growth factors of the present invention first, for example, 2-48 hours in advance, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc.
  • the cells of the present invention can be contacted with one or more cell activators of the present invention first, for example, 2-48 hours in advance, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contacted with one or more cell growth factors of the present invention.
  • one or more cell activators of the present invention first, for example, 2-48 hours in advance, for example, 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc.
  • the cells of the present invention can be contacted with the cell activator of the present invention and one or more cell growth factors of the present invention at substantially the same time.
  • the TIL of the present invention can be contacted with the cell activator of the present invention and one or more cell growth factors of the present invention at substantially the same time.
  • the TIL of the present invention can be contacted with the cell activator of the present invention and one or more cell growth factors of the present invention at substantially the same time.
  • the cell growth factor of the present invention can be selected from one or more of the following groups: IL-2, IL-7, IL-12, IL-15, IL-21, interferon- ⁇ , and their functionally active fragments.
  • the cell growth factor of the present invention can contain IL-2 and/or its functionally active fragments.
  • the functionally active fragments of IL-2 can contain fragments of IL-2 that can bind to the IL-2 receptor of the cell known in the art.
  • the cell growth factor of the present invention can contain IL-2 and/or its functionally active fragments, IL-7 and/or its functionally active fragments, and IL-15 and/or its functionally active fragments.
  • contacting the cells of the invention with one or more cell growth factors of the invention may comprise adding the cell growth factors of the invention to the cell culture medium of the cells of the invention.
  • the initial concentration of the cell growth factors of the invention in the cell culture medium of the cells of the invention may be
  • the initial concentration of IL-2 of the present invention in the cell culture medium of the cells of the present invention can be at least about 300-9000 IU/mL, such as at least about 300 IU/mL, at least about 350 IU/mL, at least about 400 IU/mL, at least about 500 IU/mL, at least about 600 IU/mL, at least about 700 IU/mL, at least about 800 IU/mL, at least about 900 IU/mL, at least about 1000 IU/mL, at least about 1100 IU/mL, at least about 1200 IU/mL, at least about 1300 IU/mL, at least about 1400 IU/mL, at least about 1500 IU/mL
  • the cells of the present invention can reduce the amount of cytokines when contacted with IL-2, IL-7 and IL-15, relative to contacting only with IL-2.
  • the amount of IL-2 added can be reduced under the condition of adding IL-7 and IL-15.
  • the concentration of IL-7 can be about 1 to 1000ng/mL, preferably about 1-100ng/mL.
  • the concentration of IL-15 can be about 1 to 1000ng/mL, preferably about 1-100ng/mL.
  • it can be reduced to the commonly used range in the art of various immune cells, for example, reduced to 50-10% of the commonly used range in the art, such as 50%, 20% or 10%.
  • the amount of IL-2 added to TCR-T can be 30-300IU/mL.
  • the amount of IL-2 added to TIL the commonly used range in the art can be 300-9000IU/mL (e.g., 1000-9000IU/mL).
  • the method of the present invention may further comprise: in at least one stage of the in vitro expansion of the present invention, the cells of the present invention may be co-cultured with feeder cells.
  • the cells of the present invention may be contacted with one or more cell activators and/or one or more cell growth factors and co-cultured with the feeder cells of the present invention.
  • a single stage of in vitro expansion of the present invention may refer to the same stage.
  • the in vitro amplification of the present invention can be performed in the first stage of the present invention, the second stage of the present invention, or the third stage of the present invention.
  • the TIL of the present invention in the first stage in vitro expansion of the present invention, can be contacted with one or more cell activators and/or one or more cell growth factors and co-cultured with the feeder cells of the present invention.
  • the TIL of the present invention in the second stage in vitro expansion of the present invention, can be contacted with one or more cell activators and/or one or more cell growth factors of the present invention and co-cultured with the feeder cells of the present invention.
  • the TIL of the present invention in the third stage in vitro expansion of the present invention, can be contacted with one or more cell activators and/or one or more cell growth factors of the present invention and co-cultured with the feeder cells of the present invention.
  • the cells of the present invention can be contacted with one or more cell activators and/or one or more cell growth factors of the present invention for a certain period of time, and then co-cultured with the feeder cells of the present invention.
  • the TIL of the present invention in the first stage of in vitro expansion of the present invention, can be contacted with one or more cell activators and/or one or more cell growth factors of the present invention for a certain period of time, and then co-cultured with the feeder cells of the present invention.
  • the TIL of the present invention in the second stage of in vitro expansion of the present invention, can be contacted with one or more cell activators and/or one or more cell growth factors of the present invention for a certain period of time, and then co-cultured with the feeder cells of the present invention.
  • the TIL of the present invention in the third stage of in vitro expansion of the present invention, can be contacted with one or more cell activators and/or one or more cell growth factors of the present invention for a certain period of time, and then co-cultured with the feeder cells of the present invention.
  • the cells of the present invention can be contacted with one or more cell activators and/or one or more cell growth factors of the present invention for a certain period of time before being co-cultured with the feeder cells of the present invention.
  • the certain period of time of the present invention can be at least about 1 hour.
  • the certain period of time of the present invention can be at least about 1-72 hours, such as at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39
  • a period of time of the present invention can be about 2 hours to about 72 hours.
  • the certain time of the present invention can be about 6 hours to about 7 hours, about 6 hours to about 8 hours, about 6 hours to about 9 hours, about 6 hours to about 10 hours, about 6 hours to about 11 hours, about 6 hours to about 12 hours, about 6 hours to about 13 hours, about 6 hours to about 14 hours, about 6 hours to about 15 hours, about 6 hours to about 16 hours, about 6 hours to about 17 hours, about 6 hours to about 18 hours, about 6 hours to about 19 hours, about 6 hours to about 20 hours, about 6 hours to about 21 hours, about 6 hours to about 22 hours, about 6 hours to about 23 hours, about 6 hours to about 24 hours, about 6 hours to about 36 hours, about 6 hours to about 48 hours, about 6 hours to about 60 hours, or about 6 hours to about 72 hours.
  • the certain time of the present invention can be about 12 hours to about 13 hours, about 12 hours to about 14 hours, about 12 hours to about 15 hours, about 12 hours to about 16 hours, about 12 hours to about 17 hours, about 12 hours to about 18 hours, about 12 hours to about 19 hours, about 12 hours to about 20 hours, about 12 hours to about 21 hours, about 12 hours to about 22 hours, about 12 hours to about 23 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, or about 12 hours to about 72 hours.
  • the certain time of the present invention can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours.
  • the feeder cells of the present invention may include antigen presenting cells.
  • the feeder cells of the present invention may include one or more selected from the following group: peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • the feeder cells of the present invention may be peripheral mononuclear cells.
  • the feeder cells of the present invention may be irradiated feeder cells.
  • the feeder cells of the present invention can be isolated artificial antigen presenting cells (aAPCs), which can include cells expressing HLA-A/B/C, CD64, CD80, ICOS-L and/or CD58, and can be modified to express one or more cell activators of the present invention.
  • the feeder cells of the present invention can be irradiated, for example, can be irradiated with gamma rays, or can be irradiated with X rays.
  • co-culturing the cells of the invention with the feeder cells of the invention may comprise contacting the surface of the feeder cells of the invention with the surface of the cells of the invention.
  • co-culturing the cells of the invention with the feeder cells of the invention comprises adding the feeder cells of the invention to the cell culture medium of the cells of the invention.
  • the feeder cells of the present invention can be added to the cell culture medium of the cells of the present invention at a ratio of about 40:1 to about 400:1.
  • the feeder cells of the present invention can be added to the cell culture medium of the cells of the present invention at a ratio of about 40:1 to about 400:1, about 40:1 to about 300:1, about 40:1 to about 200:1, about 40:1 to about 100:1, about 40:1 to about 90:1, about 40:1 to about 80:1, about 40:1 to about 70:1, about 40:1 to about 60:1, about 40:1 to about 50:1, about 50:1 to about 400:1,
  • the feeder cells of the invention are added to the cell culture medium of the cells of the invention at a ratio of about 60:1 to about 400:1, at about 70:1 to about 400:1, at about 80:1 to about 400:1, at about 90:1 to about 400:1, at about 100:1 to about 400:1, at about 200:1 to about 400:1, or at about 300:1 to about 400:1 of feeder
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and/or weakening the activity of members of at least two families selected from the group consisting of peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family in the second TIL population; wherein, a third TIL population is obtained through step (B).
  • TIL tumor infiltrating lymphocytes
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), comprising: (A) contacting a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with one or more T cell growth factors, wherein a second TIL population is obtained through step (A); (B) reducing the expression and/or activity of members of at least two families selected from the group consisting of peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and CBL family in the second TIL population, and contacting the second TIL population with a T cell activator and/or a T cell growth factor, wherein a third TIL population is obtained through step (B); and (C) co-culturing the third TIL population with feeder cells, wherein a fourth TIL population is obtained
  • TIL
  • the first stage in vitro amplification of the present invention can be arbitrarily replaced with step (A) in the method of the above aspect.
  • the second stage in vitro amplification of the present invention can be arbitrarily replaced with step (B) in the method of the above aspect.
  • the TIL of the present invention that has been amplified in vitro in the first stage can be arbitrarily replaced with the second TIL group obtained by step (A) in the method of the above aspect.
  • the TIL of the present invention that has been amplified in vitro in the second stage can be arbitrarily replaced with the third TIL group obtained by step (B) in the method of the above aspect.
  • the third stage in vitro amplification of the present invention can be arbitrarily replaced with any additional step (C) in the method of the above aspect.
  • the TIL of the present invention that has been amplified in vitro in the third stage can be arbitrarily replaced with the fourth TIL group obtained by any additional step (C) in the method of the above aspect.
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with a plurality of cell growth factors; wherein a second TIL population is obtained through step (A); (B) contacting the second TIL population with a plurality of cell growth factors, with a plurality of cell activators, with a STAT inhibitor (SSI) selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family
  • SSI STAT inhibitor
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro with a cell growth factor; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with a cell growth factor, with a cell activator, reducing the expression and/or weakening the activity of members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family and the CBL family, and co-culturing the TIL with feeder cells, wherein the members of at least two families selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI)
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the method for obtaining TIL cells from a subject's tissue sample can be to obtain an in situ tumor sample or a metastatic tumor sample from a patient during surgery, the weight of which can be at least about 1g, or multiple pieces of tissue can be combined.
  • Tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and/or peritoneal effusion are transported in a sample transport fluid, such as a commercially commonly used tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid at about 2-8°C and processed within 48 hours.
  • Tissue blocks can be mechanically broken into pieces of about 1-27 cubic millimeters in size, transferred into a breathable culture bag or Grex, and cultured for about 3-14 days with cell serum-free culture medium and IL-2 at a concentration of 300-9000IU/mL (e.g., 1000-9000IU/mL, e.g., 6000IU/mL).
  • the cells in the culture medium are collected and transferred into a breathable culture bag, or a Grex, or a Xuri device.
  • the serum-free culture medium of the cells can be supplemented with the CD28 antibody, CD3 antibody and CD28 antibody of the present invention, magnetic beads (e.g., Dynabeads) comprising CD3 antibody and CD28 antibody and/or nanomatrices (e.g., transACT) comprising CD3 antibody and CD28 antibody, IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, e.g., 6000 IU/mL), and a peptidase selected from the C64 family, the ZC3H12 family, the STAT induction
  • the expression and/or activity of members of at least two families of the STAT inhibitor (SSI) family and the CBL family are reduced (members selected from the peptidase C64 family may include TNFAIP3, members of the ZC3H12 family may include ZC3H12A, members of the STAT-induced STAT inhibitor (SSI) family may include SOCS1, and members of the CBL family may include
  • Cells in the culture medium can be collected using a cell processing system, washed, frozen, and tested.
  • the final product CD3 ratio can be greater than 80%, the cell survival rate can be greater than 50%, and cells greater than 80% can be memory effector cells and effector cells.
  • IFN- ⁇ can be secreted, and/or the activated cell ratio can be increased.
  • the present invention provides a cell, and the cell of the present invention can be cultured according to the culture method of the present invention.
  • the cell provided by the present invention can include one or one batch of cells cultured by the culture method of the present invention.
  • the cell provided by the present invention can include multiple or multiple batches of cells cultured by the culture method of the present invention and combined in any proportion.
  • the cells amplified using the method of the present invention can be administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition can be a suspension of cells in a sterile buffer.
  • Cells amplified using the PBMCs of the present invention can be administered by any suitable route known in the art.
  • cells can be administered with a single intra-arterial or intravenous infusion, and the infusion can last for about 30 to 60 minutes.
  • Other suitable routes of administration can include intraperitoneal, intrathecal, and intralymphatic administration.
  • any suitable dose of cells can be administered.
  • about 1 ⁇ 10 9 to about 13.7 ⁇ 10 10 cells may be administered, preferably about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 cells.
  • about 1 ⁇ 10 9 to about 12 ⁇ 10 10 cells may be administered.
  • about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 cells may be administered.
  • about 3 ⁇ 10 10 to about 12 ⁇ 10 10 cells may be administered.
  • about 4 ⁇ 10 10 to about 10 ⁇ 10 10 cells may be administered.
  • about 5 ⁇ 10 10 to about 8 ⁇ 10 10 cells may be administered.
  • the therapeutically effective dose may be about 1 ⁇ 10 9 to about 13.7 ⁇ 10 10 , preferably about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 . In some embodiments, the therapeutically effective dose may be about 1 ⁇ 10 9 to about 12 ⁇ 10 10 cells. In some embodiments, the therapeutically effective dose may be about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 cells. In some embodiments, the therapeutically effective dose may be about 3 ⁇ 10 10 to about 12 ⁇ 10 10 cells. In some embodiments, the therapeutically effective dose may be about 4 ⁇ 10 10 to about 10 ⁇ 10 10 cells.
  • the therapeutically effective dose may be about 5 ⁇ 10 10 to about 8 ⁇ 10 10 cells. In some embodiments, the therapeutically effective dose may be about 6 ⁇ 10 10 to about 8 ⁇ 10 10 cells. In some embodiments, the therapeutically effective dose may be about 7 ⁇ 10 10 to about 8 ⁇ 10 10 cells.
  • the number of cells provided in the composition of the present invention can be about 1 ⁇ 10 6 -9 ⁇ 10 13 , for example, about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , about 6 ⁇ 10 6 , about 7 ⁇ 10 6 , about 8 ⁇ 10 6 , about 9 ⁇ 10 6 , about 1 ⁇ 10 7 , about 2 ⁇ 10 7 , about 3 ⁇ 10 7 , about 4 ⁇ 10 7 , about 5 ⁇ 10 7 , about 6 ⁇ 10 7 , about 7 ⁇ 10 7 , about 8 ⁇ 10 7 , about 9 ⁇ 10 7 , about 1 ⁇ 10 8 , about 2 ⁇ 10 8 , about 3 ⁇ 10 8 , about 4 ⁇ 10 8 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 8 , about 8 ⁇ 10 8 , about 9 ⁇ 10 8 , about 1 ⁇ 10 9 , about 2 ⁇ 10 9 , about 3 ⁇ 10 9 , about 4 ⁇ 10 9 , about 5 ⁇ 10 8 ,
  • the number of cells provided in the compositions of the present invention can range from about 1 ⁇ 10 6 to 5 ⁇ 10 6 , about 5 ⁇ 10 6 to 1 ⁇ 10 7 , about 1 ⁇ 10 7 to 5 ⁇ 10 7 , about 5 ⁇ 10 7 to 1 ⁇ 10 8 , about 1 ⁇ 10 8 to 5 ⁇ 10 8 , about 5 ⁇ 10 8 to 1 ⁇ 10 9 , about 1 ⁇ 10 9 to 5 ⁇ 10 9 , about 5 ⁇ 10 9 to 1 ⁇ 10 10 , about 1 ⁇ 10 10 to 5 ⁇ 10 10 , about 5 ⁇ 10 10 to 1 ⁇ 10 11 , about 5 ⁇ 10 11 to 1 ⁇ 10 12 , about 1 ⁇ 10 12 to 5 ⁇ 10 12 , about 5 ⁇ 10 12 to 1 ⁇ 10 13 , about 1 ⁇ 10 13 to 5 ⁇ 10 13 , or about 5 ⁇ 10 13 to 9 ⁇ 10 13 .
  • the concentration of cells provided in the compositions of the invention can be less than about 100-0.0001% w/w, w/v or v/v of the composition, such as about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2 0.002%, about 0.001%, about 0.0009%, about 0.0008%, about 0.0007%, about 0.0006%, about 0.0005%, about 0.0004%, about 0.0003%, about 0.0002%, or about 0.0001% w/w, w/v, or v/v.
  • the cells provided in the compositions of the invention may be present at a concentration greater than about 90-0.0001% w/w, w/v, or v/v of the composition, such as about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17%, about 16.75%, about 16.50%, about 16.25%, about 17.
  • a concentration greater than about 90-0.0001% w/w, w/v, or v/v of the composition such as about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%,
  • the cells provided in the compositions of the invention may be present in a concentration ranging from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v, or v/v of the composition.
  • the cells provided in the compositions of the invention may be present in a concentration ranging from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w/w, w/v, or v/v of the composition.
  • the amount of cells provided in the composition of the present invention can be equal to or less than about 10-0.0001 g, such as about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5g, about 7.0g, about 6.5g, about 6.0g, about 5.5g, about 5.0g, about 4.5g, about 4.0g, about 3.5g, about 3.0g, about 2.5g, about 2.0g, about 1.5g, about 1.0g, about 0.95g, about 0.9g, about 0.85g, about 0.8g, about 0.75g, about 0.7g, about 0.65g, about 0.6g, about 0.55g, about 0.5g, about 0.45g, about 0.4g, about 0.35g, about 0.3g, about 0.25g, about 0.2g, about 0.15g, about 0.1g, about 0.09g, about 0.08g, about 0.07g, about 0.06g, about 0.05g, about 0.04g, about 0.03g, about
  • the amount of cells provided in the composition of the present invention can be greater than about 0.0001-10 g, such as about 0.0001 g, about 0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006 g, about 0.0007 g, about 0.0008 g, about 0.0009 g, about 0.001 g, about 0.0015 g, about 0.002 g, about 0.0025 g, about 0.003g, about 0.0035g, about 0.004g, about 0.0045g, about 0.005g, about 0.0055g, about 0.006g, about 0.0065g, about 0.007g, about 0.0075g, about 0.008g, about 0.0085g, about 0.009g, about 0.0095g, about 0.01g, about 0.015g, about 0.02g, about 0.025g, about 0.03g, about 0.035g, about 0.04g, about 0.045g, about 0.05g,
  • the cells can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, the cells can be administered in multiple doses.
  • the dose can be once, twice, three times, four times, five times, six times, or more than six times per year.
  • the dose can be once a month, once every two weeks, once a week, or once every 2 days. In some embodiments, the administration of the cells can be continuous administration.
  • the present invention provides a pharmaceutical composition, which in some embodiments may comprise the cell of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a kit, which may include a cell activator, a cell growth factor and/or a feeder cell of the cell culture method of the present invention and an instruction manual recording the steps of the cell culture method of the present invention.
  • the present invention provides a kit, which may include a cell of the present invention and/or a pharmaceutical composition of the present invention.
  • the present invention provides a method of affecting the growth of cells, such as tumor cells, which may include administering cells of the present invention and/or pharmaceutical compositions of the present invention to a subject.
  • affecting tumor growth may include reducing the volume of the tumor to about 99-0.1% of the volume before administration, such as about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2% or about 0.1%.
  • the present invention provides the use of the cell of the present invention and/or the pharmaceutical composition of the present invention in the preparation of a drug, and the drug of the present invention can be used to prevent and/or treat a disease and/or symptom.
  • the disease and/or symptom of the present invention can include a tumor.
  • the tumor of the present invention is selected from a solid tumor.
  • the tumor of the present invention can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.
  • the present invention provides a method for preventing and/or treating a disease and/or symptom, which may include administering to a subject a cell of the present invention and/or a pharmaceutical composition of the present invention.
  • the disease and/or symptom of the present invention may include a tumor.
  • the tumor of the present invention is selected from a solid tumor.
  • the tumor of the present invention may be selected from the following groups: One or more of: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer, and kidney cancer.
  • the present invention provides a TIL of the present invention and/or a pharmaceutical composition of the present invention, which can be used to prevent and/or treat a disease and/or symptom.
  • the disease and/or symptom of the present invention can include a tumor.
  • the tumor of the present invention is selected from a solid tumor.
  • the tumor of the present invention can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.
  • PBMC blood sample
  • a culture bag with a breathable surface, such as a culture bag (Origen)
  • 300mL of thawed complete culture medium can be arbitrarily selected from X-vivo 15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 at a concentration of 300-9000IU/mL (e.g.
  • the non-suspended tumor tissue block Take the non-suspended tumor tissue block, use a 20mL syringe to remove the internal piston, connect it to the culture bag, and use a pipette to transfer about 1g of tissue block into the culture bag through the syringe. Place the culture bag in a carbon dioxide incubator for culture. Clean the scissors and forceps, and perform preliminary disinfection with 75% alcohol, then sterilize after ultrasonic cleaning to obtain the first TIL group.
  • the medium is replenished or half-replaced every 3-7 days to ensure cell nutrition.
  • Complete culture medium is used, and the complete culture medium can arbitrarily select X-vivo 15 culture medium or other commercial T cell culture medium, such as T cell culture medium of brands such as Stem Cell, Lonza, Thermo, and Miltenyi, and essential amino acids and antibiotics can be added, and IL-2 (double heron and/or tetracycline) at a concentration of 300-9000IU/mL (e.g., 1000-9000IU/mL, such as 6000IU/mL) is added.
  • 3-14 days of step (A) for example, sampling and counting can be performed on the 13th or 14th day. If the number of cells is between 5 ⁇ 10 5 and 5 ⁇ 10 8 , the harvesting step of step (A) is entered.
  • step (A) Collect the cells at the end of in vitro expansion in step (A), centrifuge, discard the culture medium, wash the cells once with PBS or saline, obtain TIL (second TIL population) expanded in vitro in step (A), and take samples and count to retain about 5 ⁇ 10 5 to 2 ⁇ 10 8 cells for subsequent in vitro expansion steps; take about 5 ⁇ 10 5 cells for quality control testing; the remaining cells are added to the freezing solution and frozen as frozen preREP TIL in vitro cells.
  • TIL second TIL population expanded in vitro in step (A)
  • step (A) Continue to culture the TILs (second TIL population) expanded in vitro in step (A), or resuscitate the frozen preREP TIL cells in vitro and perform TIL activation in step (B).
  • Complete culture medium is used.
  • the complete culture medium can be arbitrarily selected from X-vivo 15 culture medium or other commercial T cell culture medium, such as T cell culture medium of brands such as Stem Cell, Lonza, Thermo, Miltenyi Biotec, etc., and essential amino acids and antibiotics can be added.
  • the cell density is adjusted to 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL, and suspended in a 24-well culture plate, 1 mL/well, and IL-2 is added at a concentration of 300-9000 IU/mL (e.g. 1000-9000 IU/mL, such as 6000 IU/mL).
  • T cell activators can be added to the culture medium of each TIL cell population at the same time, such as CD3 agonists and/or CD28 agonists, for example, about 30 ng/mL of CD3 antibody (Miltenyi Biotech, OKT3), about 30 ng/mL of CD28 antibody (Merck, 15E8), magnetic beads (diameter about 1 to 10 ⁇ m Dynabeads, Thermo Fisher) added at a ratio of about 1:2-2:1 to TIL, and/or transACT (diameter about 100 to 500 nm, Miltenyi) added at a ratio of about 1:100-1:2000 to TIL. Cultivate for about 0-4 days to obtain a third TIL population.
  • CD3 agonists and/or CD28 agonists for example, about 30 ng/mL of CD3 antibody (Miltenyi Biotech, OKT3), about 30 ng/mL of CD28 antibody (Merck, 15E8), magnetic beads (diameter about 1 to
  • the sgRNA targeting each target selected from the present invention is synthesized, thawed and added to nuclease-free water to a concentration of about 100 ⁇ M.
  • About 2 ⁇ L of gRNA (50 ⁇ M) is added to the P3 buffer after incubation at 95 ° C for 2 minutes for annealing, and 0.3-1 ⁇ L of Cas9 (such as Kai Xia, Ke Rui or Acro, 10 mg / mL) is added, and incubated at 25 ° C for 10 minutes to form a ribonucleoprotein complex (RNP).
  • Cas9 such as Kai Xia, Ke Rui or Acro, 10 mg / mL
  • the above RNP is electroporated with about 1 ⁇ 10 6 cells of the third TIL group by a Lonza electroporator.
  • the electroporation procedure can be human T cell stim (EO115).
  • the fourth TIL group is obtained by culturing for about 0-4 days.
  • Feeder cells are added to the fourth TIL cell group for culture.
  • the contact time between TIL and feeder cells needs to be a certain time Tn after the contact between TIL and IL-2 and T cell activator in step (B) (for example, Tn can be 0 hours to 12 days, such as 24 hours or 48 hours).
  • Tn can be 0 hours to 12 days, such as 24 hours or 48 hours.
  • the culture medium is sampled and counted every 1-3 days, and the medium is replenished or half-replaced according to the cell status until the total number of cells is greater than 1 ⁇ 10 9 or the in vitro expansion culture in step (D) is about 5 to about 14 days, and the in vitro expansion culture in step (D) is terminated.
  • step (D) Take the cells amplified in step (D), discard the culture medium supernatant after centrifugation, and wash three times with PBS or saline or compound electrolyte solution to obtain TIL amplified in step (D) (fifth TIL population), take samples and count them during the third washing, and according to the counting results, discard the supernatant after the last centrifugation, take 3 ⁇ 10 6 cells for quality control detection; add all the remaining cells to the freezing solution, adjust the cell density to 1-3 ⁇ 10 8 cells/mL for freezing.

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Abstract

本发明属于生物医药领域，提供了一种修饰的细胞及其用途，具体涉及一种培养细胞的方法，其包含使所述细胞的肽酶C64家族成员如TNFAIP3表达降低和/或活性减弱。本发明还提供了使用上述培养的细胞预防和/或治疗肿瘤的方法。

Description

一种修饰的细胞及其用途 技术领域
本发明涉及生物医学领域，具体的涉及一种修饰的细胞及其用途。
背景技术
目前，免疫治疗是一种治疗预后不良患者的有效方法。但是免疫治疗中使用的免疫细胞存在体内回输后细胞功能不强或增殖、存续能力弱的问题。因此如何提供一种修饰的免疫细胞，以及一种稳健可靠的免疫细胞的培养方法是亟待解决的问题。
发明内容
本发明提供了一种培养细胞的方法，该方法具有以下一种或多种的优势：增强的靶细胞杀伤能力、增强的细胞增殖能力、增强的细胞因子释放能力、增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
一方面，本发明提供了一种培养细胞的方法，所述方法包含：使所述细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的家族成员和/或其功能活性片段的表达降低和/或活性减弱。
另一方面，本发明提供了一种细胞，所述细胞经过本发明的方法获得。
另一方面，本发明提供了一种药物组合物，其包含本发明的细胞，以及任选的药学上可接受的载体。
另一方面，本发明提供了一种影响细胞生长的方法，其包含施用 本发明的细胞和/或本发明的药物组合物。
另一方面，本发明提供了本发明的细胞和/或本发明的药物组合物在制备药物中的应用，所述药物用于预防和/或治疗疾病和/或症状。
本领域技术人员能够从下文的详细描述中容易地洞察到本发明的其它方面和优势。下文的详细描述中仅显示和描述了本发明的示例性实施方式。如本领域技术人员将认识到的，本发明的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本发明所涉及发明的精神和范围。相应地，本发明的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本发明所涉及发明的特点和优势。对附图简要说明如下：
图1A显示本发明提供的相对于起始密码子的人TNFAIP3基因编辑靶向区段，例如，可以是具有约3个以上的转录因子结合数的一段连续区域；以及可以是该基因的外显子区域或距离外显子约20bp的内含子区域。
图1B显示本发明提供的相对于起始密码子的人ZC3H12A基因编辑靶向区段，例如，可以是具有约3个以上的转录因子结合数的一段连续区域；以及可以是该基因的外显子区域或距离外显子约20bp的内含子区域。
图1C显示本发明提供的相对于起始密码子的人SOCS1基因编辑靶向区段，例如，可以是具有约3个以上的转录因子结合数的一段连续区域；以及可以是该基因的外显子区域或距离外显子约20bp的内含子区域。
图1D显示本发明提供的相对于起始密码子的人CBLB基因编辑靶向区段，例如，可以是具有约3个以上的转录因子结合数的一段连续区域；以及可以是该基因的外显子区域或距离外显子约20bp的内含 子区域。
图2A显示无刺激培养基组中TNFAIP3基因编辑的TCR-T扩增倍数。
图2B-2C显示TransACT刺激组中TNFAIP3基因编辑的TCR-T扩增倍数。
图2D-2G显示不同供者来源的TCR-T细胞在TNFAIP3基因编辑后的靶细胞杀伤能力。图2D、2F展示各时间点杀伤曲线，以及图2E、2G展示试验终点各个试验组的杀伤情况，均高于未编辑的NT组。
图2H-2K显示TNFAIP3基因编辑的TCR-T细胞的各种细胞因子释放能力。
图3A显示无刺激培养基组中ZC3H12A基因编辑的TCR-T扩增倍数。
图3B显示TransACT刺激组中ZC3H12A基因编辑的TCR-T扩增倍数。
图3C-3F显示ZC3H12A基因编辑的TCR-T细胞的靶细胞杀伤能力。图3C、3E展示各时间点杀伤曲线，以及图3D、3F展示试验终点各个试验组的杀伤情况，均高于未编辑的NT组。
图3G-3J显示ZC3H12A基因编辑的TCR-T细胞的各种细胞因子释放能力。
图4A显示无刺激培养基组中SOCS1基因编辑的TCR-T扩增倍数。
图4B显示TransACT刺激组中SOCS1基因编辑的TCR-T扩增倍数。
图4C显示无刺激培养基组中SOCS1基因编辑的来源于不同供者TIL扩增倍数。
图4D显示TransACT刺激组中SOCS1基因编辑的来源于供者306的TIL扩增倍数。
图4E-4H显示SOCS1基因编辑的TCR-T细胞的靶细胞杀伤能力。
图4I显示SOCS1基因编辑的TIL细胞的靶细胞杀伤能力。
图4J显示未刺激组SOCS1基因编辑的TCR-T细胞的细胞因子表达。
图4K显示CD3抗体刺激组SOCS1基因编辑的TCR-T细胞的细胞因子表达。
图4L显示与A375靶细胞共培养的SOCS1基因编辑的TCR-T细胞的细胞因子释放能力。
图4M显示未刺激组SOCS1基因编辑的TIL细胞的细胞因子表达。
图4N显示TransACT刺激组SOCS1基因编辑的TIL细胞的细胞因子表达。
图4O显示SOCS1基因编辑后的TIL细胞具有更高的干性细胞比例。
图4P显示SOCS1基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图5A-5B显示无刺激培养基组中CBLB基因编辑的TCR-T扩增倍数。
图5C-5D显示TransACT刺激组中CBLB基因编辑的TCR-T扩增倍数。
图5E-5H显示CBLB基因编辑的TCR-T细胞的靶细胞杀伤能力。图5E、5G展示各时间点杀伤曲线，以及图5F、5H展示试验终点各个试验组的杀伤情况，均高于未编辑的NT组。
图5I-5L显示CBLB基因编辑的TCR-T细胞的各种细胞因子释放能力。
图6A显示无刺激培养基组中CBLB和ZC3H12A组合基因编辑的TIL扩增倍数。
图6B显示无刺激培养基组中SOCS1和CBLB组合基因编辑的TIL扩增倍数。
图6C显示无刺激培养基组中SOCS1和TNFAIP3组合基因编辑的TIL扩增倍数。
图6D显示CD3抗体刺激组中SOCS1和TNFAIP3组合基因编辑的TIL扩增倍数。
图6E显示无刺激培养基组中SOCS1和ZC3H12A组合基因编辑的TIL扩增倍数。
图6F显示CD3抗体刺激组中SOCS1和ZC3H12A组合基因编辑的TIL扩增倍数。
图6G显示无刺激培养基组中TNFAIP3和CBLB组合基因编辑的TIL扩增倍数。
图6H显示无刺激培养基组中TNFAIP3和ZC3H12A组合基因编辑的TIL扩增倍数。
图6I显示CD3抗体刺激组中TNFAIP3和ZC3H12A组合基因编辑的TIL扩增倍数。
图6J显示无刺激培养基组中TNFAIP3和SOCS1组合基因编辑的TIL扩增倍数。
图6K显示TransACT抗体刺激组中TNFAIP3和SOCS1组合基因编辑的TIL扩增倍数。
图7A显示CBLB和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7B显示CBLB和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7C显示SOCS1和CBLB组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7D显示SOCS1和CBLB组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7E显示SOCS1和CBLB组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7F显示SOCS1和TNFAIP3组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7G显示SOCS1和TNFAIP3组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7H显示SOCS1和TNFAIP3组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7I显示SOCS1和TNFAIP3组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7J显示SOCS1和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7K显示SOCS1和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7L显示SOCS1和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7M显示TNFAIP3和CBLB组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7N显示TNFAIP3和CBLB组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7O显示TNFAIP3和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7P显示TNFAIP3和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7Q显示TNFAIP3和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7R显示TNFAIP3和ZC3H12A组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7S显示TNFAIP3和SOCS1组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7T显示TNFAIP3和SOCS1组合基因编辑的TIL细胞的对自体肿瘤类器官的杀伤能力。
图8A显示CBLB和ZC3H12A组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图8B显示SOCS1和CBLB组合基因编辑后的TIL细胞具有更高的中心记忆T细胞比例。
图8C显示SOCS1和CBLB组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图8D显示SOCS1和TNFAIP3组合基因编辑后的TIL细胞具有更高的中心记忆T细胞比例。
图8E显示SOCS1和TNFAIP3组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图8F显示SOCS1和ZC3H12A组合基因编辑后的TIL细胞具有更高的中心记忆T细胞比例。
图8G-8H显示SOCS1和ZC3H12A组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图8I显示TNFAIP3和CBLB组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图8J显示TNFAIP3和ZC3H12A组合基因编辑后的TIL细胞具有更高的幼稚T细胞比例。
图8K显示TNFAIP3和ZC3H12A组合基因编辑后的TIL细胞具有更高的中心记忆T细胞比例。
图8L-8M显示TNFAIP3和ZC3H12A组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图8N显示TNFAIP3和SOCS1组合基因编辑后的TIL细胞具有更高的干性细胞比例。
图8O显示TNFAIP3和SOCS1组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图9A显示CD3抗体刺激组的CBLB和ZC3H12A组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9B显示无刺激Medium组的SOCS1和CBLB组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9C显示CD3抗体刺激组的SOCS1和CBLB组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9D显示无刺激Medium组的SOCS1和TNFAIP3组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9E显示CD3抗体刺激组的SOCS1和TNFAIP3组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9F显示无刺激Medium组的SOCS1和ZC3H12A组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9G显示CD3抗体刺激组的SOCS1和ZC3H12A组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9H显示无刺激Medium组的TNFAIP3和CBLB组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9I显示CD3抗体刺激组的TNFAIP3和CBLB组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9J-9K显示无刺激Medium组的TNFAIP3和ZC3H12A组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9L显示CD3抗体刺激组的TNFAIP3和ZC3H12A组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9M显示与自体肿瘤类器官共培养的TNFAIP3和SOCS1组合基因编辑后的TIL细胞具有更高的细胞因子释放能力。
图10显示对于来源于供者504的TIL细胞的凋亡检测结果。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本发明的其他优点及效果。
术语定义
在本发明中，术语“CBL家族成员”通常是指具有SH3域的家族成员蛋白或其功能活性片段。例如，包含CBL家族成员可以包含CBLB。例如，CBL家族成员的UniProt编号可以是Q13191。本发明的CBL家族成员还可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的CBL家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“STAT诱导的STAT抑制剂(SSI)家族成员”通常是指具有SH2域的家族成员蛋白或其功能活性片段。例如，包含STAT诱导的STAT抑制剂(SSI)家族成员可以包含SOCS1。例如， STAT诱导的STAT抑制剂(SSI)家族成员的UniProt编号可以是O15524。本发明的STAT诱导的STAT抑制剂(SSI)家族成员还可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的STAT诱导的STAT抑制剂(SSI)家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“肽酶C64家族成员”通常是指具有泛素结合域的家族成员蛋白或其功能活性片段。例如，包含肽酶C64家族成员可以包含TNFAIP3。例如，肽酶C64家族成员的UniProt编号可以是P21580。本发明的肽酶C64家族成员还可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的肽酶C64家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“ZC3H12家族成员”通常是指具有C3H1型锌指结构域的家族成员蛋白或其功能活性片段。例如，包含ZC3H12家族成员可以包含ZC3H12A。例如，ZC3H12家族成员的UniProt编号可以是Q5D1E8。本发明的ZC3H12家族成员还可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的ZC3H12家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“IKAROS锌指蛋白家族成员”通常是指具有锌指结构域的家族成员蛋白或其功能活性片段。例如，包含IKAROS锌指蛋白家族成员可以包含IKZF1。例如，IKAROS锌指蛋白家族成员的UniProt编号可以是Q13422。本发明的IKAROS锌指蛋白家族成员还 可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的IKAROS锌指蛋白家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“肿瘤坏死因子α诱导蛋白3(TNFAIP3)”通常是指一种信号通路的抑制分子。例如，TNFAIP3可以使得NF-κB通路的信号转导物质泛素化。例如，TNFAIP3的UniProt登录号可以是P21580。本发明中，TNFAIP3可以涵盖未加工的TNFAIP3、任何形式加工的TNFAIP3、TNFAIP3的变体或包含TNFAIP3的功能活性片段的物质。
在本发明中，术语“GTP酶激活蛋白1家族成员”通常是指具有GTP酶激活域的家族成员蛋白或其功能活性片段。例如，包含GTP酶激活蛋白1家族成员可以包含RASA2。例如，GTP酶激活蛋白1家族成员的UniProt编号可以是Q15283。本发明的GTP酶激活蛋白1家族成员还可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的GTP酶激活蛋白1家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“FGF结合蛋白家族成员”通常是指具有FGF结合域的家族成员蛋白或其功能活性片段。例如，包含FGF结合蛋白家族成员可以包含FIBP。例如，FGF结合蛋白家族成员的UniProt编号可以是O43427。本发明的FGF结合蛋白家族成员还可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的FGF结合蛋白家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“Mediator(MED)家族成员”通常是指具有CDK8结合域的家族成员蛋白或其功能活性片段。例如，包含Mediator(MED)家族成员可以包含MED12。例如，Mediator(MED)家族成员的UniProt编号可以是Q93074。本发明的Mediator(MED)家族成员还可以涵盖其功能活性片段，不限于人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体、其活性片段、或其在细胞中发生的加工和/或修饰后产生的包含该功能活性片段的物质。例如，本发明的Mediator(MED)家族成员可以包含其功能活性片段以及其它任意的结构域。
在本发明中，术语“免疫细胞”通常是指参与进行先天性和适应性免疫应答的细胞。例如，可以包括但不限于淋巴细胞(诸如T细胞(包括胸腺细胞)和B细胞)、自然杀伤(NK)细胞、NKT细胞、巨噬细胞、单核细胞、嗜酸性粒细胞、嗜碱性粒细胞、嗜中性粒细胞、树突状细胞和肥大细胞。在一些实施方案中，修饰的免疫效应细胞是T细胞，诸如CD4+T细胞、CD8+T细胞(也称为细胞毒性T细胞或CTL)、调控性T细胞(Treg)、Th1细胞、Th2细胞、Th17细胞αβT细胞和/或γδT细胞。例如，本发明的免疫细胞还包含来源于干细胞分化的免疫细胞。例如，本发明的免疫细胞还包含来源于多能干细胞分化的免疫细胞。例如，获取本发明的干细胞可以是通过诱导产生。例如，本发明的上述干细胞可以包含诱导的多能干细胞(iPSC)。
在本发明中，术语“嵌合抗原受体”通常是指一种工程化抗原受体。例如，CAR可以包含经由铰链和跨膜结构域与包含信号传导结构域的细胞质结构域融合的细胞外抗原结合结构域。在一些实施方案中，CAR细胞外结构域可以以MHC非依赖性方式与由靶细胞表达的抗原结合，从而导致细胞的活化和增殖。在一些实施方案中，CAR的细胞外结构域可以识别与抗体或其抗原结合片段融合的标签。例如，可以使得单个CAR构建体可以通过用一种抗体取代另一种抗体来靶向多种不同的抗原。在一些实施方案中，CAR的细胞外结构域可以包含来源于抗体 的抗原结合片段。可用于本公开的抗原结合结构域可以包括例如scFv、抗体、抗体的抗原结合区、重链/轻链的可变区和/或单链抗体。
在本发明中，术语“T细胞受体”通常是指一种工程化抗原受体。例如，TCR可以包含已从识别特定靶抗原的T细胞群体中分离并克隆的TCRα和/或TCRβ链。例如，TCRα和/或TCRβ基因(即TRAC和TRBC)可以从分离自患有特定恶性肿瘤的个体的T细胞群体或已分离自用特异性肿瘤抗原或肿瘤细胞免疫的人源化小鼠的T细胞群体中克隆而来。工程化TCR可以通过与其内源对应物相同的机制识别抗原(例如，通过识别在靶细胞表面上表达的主要组织相容性复合物(MHC)蛋白的背景下呈递的其同源抗原)，从而可以导致TCR工程化细胞的活化和增殖。
在本发明中，术语“基因调控系统”通常是指调节目标基因表达或活性的系统。例如，基因调控系统可以包含基因调控分子。例如，基因调控系统可以调节基因的表达或活性，如使该基因处于失活或激活的状态、使该基因的数量增加或降低、使该基因处于转录量提高或降低的状态和/或使该基因的转录产物处于失活或激活的状态；例如，基因调控系统可以调节基因的表达或活性，如使单个细胞中该基因的表达产物的量提高或降低和/或使表达该基因的表达产物的细胞的数量提高或降低。
在本发明中，术语“指导核酸分子”通常是指一种可以用于基因编辑的核酸分子。例如，指导核酸分子可以提供核苷酸插入或删除的信息，指导编辑过程。例如，指导核酸分子可以是指导RNA或向导RNA(guide RNA，gRNA)。例如，“gRNA”可以是指结合到Cas蛋白并使Cas蛋白靶向靶DNA内特定位置的RNA分子。例如，其中gRNA与DNA靶向序列之间的杂交促进CRISPR复合物的形成，可以不必要求完全互补性，例如只要存在引起杂交并促进CRISPR复合物形成的充分互补性即可。
在本发明中，术语“酶蛋白”通常是指一种具有酶活性的蛋白。例如，酶蛋白可以是指Cas蛋白。例如，Cas蛋白可以包含至少一个RNA识别或结合结构域，该结构域可以与gRNA相互作用。Cas蛋白还可以包含核酸酶结构域(例如，DNA酶或RNA酶结构域)、DNA结合结构域、解旋酶结构域、蛋白-蛋白相互作用结构域、二聚化结构域和/或其他结构域。核酸酶结构域可以具有用于核酸切割的催化活性。切割可以包括核酸分子共价键的断裂。Cas蛋白可以为野生型蛋白(即，自然界存在的蛋白)、经修饰的Cas蛋白(即，Cas蛋白变体)或者野生型或经修饰的Cas蛋白的片段。Cas蛋白还可以是野生型或经修饰的Cas蛋白的活性变体或片段。本发明中，Cas蛋白可以涵盖未加工的Cas蛋白、任何形式加工的Cas蛋白、Cas蛋白的变体或包含Cas蛋白的功能活性片段的物质。
在本发明中，术语“核糖核蛋白复合物”通常是指一种蛋白与核酸形成的复合体。例如，核糖核蛋白复合物中的蛋白可以具有核酸酶活性。例如，核糖核蛋白复合物可以在其中核酸的指导下，对目标序列进行切割。例如，核糖核蛋白复合物可以是Cas蛋白与gRNA形成的复合物。
在本发明中，术语“脂质纳米颗粒(LNP)”通常是指一种脂质-核酸颗粒或核酸-脂质颗粒。例如，LNP表示由脂质(例如，阳离子脂质、非阳离子脂质和防止颗粒聚集的缀合脂质)和核酸制成的颗粒，其中核酸(例如，mRNA、gRNA、siRNA、aiRNA、miRNA、ssDNA、dsDNA、ssRNA、短发夹RNA(shRNA)、dsRNA、自我扩增RNA或质粒，包括由其转录干扰RNA或mRNA的质粒)包封在脂质中。例如，蛋白可以包封在LNP中，例如可以将本领域已知的Cas蛋白包封在LNP中。例如，LNP中的脂质包含(1)“简单脂质”，其包括脂肪和油以及蜡；(2)“复合脂质”，其包括磷脂和糖脂；以及(3)“衍生的脂质”如类固醇。例如，LNP中的脂质也可以包含脂质衍生物，例如与蛋白或多肽共价或非共价结合的脂质。例如，LNP中的组分还可以包含多肽组分，其 中多肽组分可以替代传统LNP中的一种或多种脂质组分而保持或者提高LNP的递送能力。
在本发明中，术语“外显子”通常是指基因上可以被表达为蛋白质的部分。例如，外显子可以是指在蛋白质生物合成过程中具有被表达为蛋白质的能力。例如，剪切目标基因的外显子序列可以降低目标基因的活性或功能。
在本发明中，术语“内含子”通常是指DNA中不编码所表达蛋白质的部分或全部的区段。通常在内源条件下，内含子被转录成RNA分子，但在被翻译成蛋白质之前其被从内源RNA中剪切掉。例如，靶向内含子的位置进行编辑，可以降低目标基因的活性或功能。例如，靶向内含子与外显子交界处，如距离外显子上游或下游约0bp至约100bp，优选约0bp至约20bp的内含子区域进行编辑，可以降低目标基因的活性或功能。
在本发明中，术语“起始密码子”通常是指在基因上可以界定蛋白合成(mRNA翻译)的起始相邻核苷酸的单元('密码子')。例如，靶向起始密码子上游0bp至1500bp，优选起始密码子上游0bp至100bp的区域进行编辑，可以降低目标基因的活性或功能。
在本发明中，术语“原型间隔序列毗邻基序(PAM)”通常是指靶序列后的短序列。例如，Cas9对靶DNA的位点特异性切割时，PAM序列可以用于决定切割的位置。例如，确定了PAM的区域，本领域技术人员可以容易确定合适的靶序列位置，以及可以容易设计出用于切割目标序列的gRNA序列。
在本发明中，术语“表达降低”通常是指产物或其基因的表达量的降低和/或能够表达所述产物的细胞比例下降(例如至少约5-100％)。例如，可以是细胞中该基因表达的产物的量降低或包含该基因表达的 产物的细胞比例降低，或者分泌该基因表达的产物的细胞比例降低。例如，可以通过检测细胞的基因组中该基因的敲除量，间接表示该基因的表达降低。例如，可以通过检测一个细胞群中该基因被敲除的细胞所占的比例，间接表示该基因的表达降低。
在本发明中，术语“活性”通常是指物质的生物学功能。例如，基因的活性可以是指该基因的转录和/或翻译状态。例如，基因的活性减弱(例如至少约5-100％)可以是指该基因的转录功能减弱、该基因无法被正常转录或该基因转录产物的功能被抑制。
在本发明中，术语“CD80”通常是指一种细胞刺激分子。例如，CD80可以是CD28的配体。例如，CD80可以见于GenBank登记号P33681。本发明的CD80蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含CD80的功能活性片段的物质。例如，本发明的CD80可以包含CD80的功能活性片段以及其它任意的结构域。
在本发明中，术语“CD86”通常是指一种细胞刺激分子。例如，CD86可以是CD28的配体。例如，CD86可以见于GenBank登记号P42081。本发明的CD86蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含CD86的功能活性片段的物质。例如，本发明的CD86可以包含CD86的功能活性片段以及其它任意的结构域。
在本发明中，术语“分泌”通常是指一种物质可以定位于细胞的胞外。例如，分泌的物质可以在细胞内合成之后，被运送到细胞的胞外空间。例如，可以通过酶联免疫吸附剂测定或其它检测方法检测一种物质是否是分泌的物质。
在本发明中，术语“T细胞受体”或“TCR”通常是指响应于抗原的呈递参与T细胞的活化的膜蛋白的复合体。TCR可以负责识别结合至主要组织相容性复合体分子的抗原。TCR可以由alpha(α)和beta(β)链的 异二聚体组成，或由gamma和delta(γ/δ)链构成。TCR可以以α/β和γ/δ形式存在，其是结构上相似的，但是具有独特的解剖学位置和功能。例如，TCR可以是在表达TCR的任何细胞上被修饰的TCR。例如，TCR的种类可以通过TCR亚型分析试剂进行分析。
在本发明中，术语“克隆多样性”通常是指某种物质具有多种克隆型。例如，TCR的克隆多样性可以是指TCR可以具有不同序列结构和/或抗原识别能力。例如，TCR具有的多样性常用β链亚型来区分，可以包括Vβ23、Vβ7.2、Vβ5.2、Vβ11、Vβ16、Vβ3等，当一个T细胞群具有更多的β链亚型时，可以认为该T细胞群具有更高的克隆多样性。
在本发明中，“CD4+细胞”通常是指CD4阳性的细胞，例如可以是T细胞。术语“CD4+细胞”，“CD4阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD4的抗体对细胞染色和使用荧光激活细胞分选。例如，已有的数据可以证明，CD4+细胞比例的提高可以使得细胞群分泌IFN和/或TNF的能力提高，并可以提高T细胞群的促进肿瘤抑制的效果。例如，请见Tay,R.E.,Richardson,E.K.等人(2020).Cancer Gene Therapy,1-13.但是，本领域缺少一种提高CD4+细胞比例的方法，本发明可以提供一种影响CD4+细胞比例的方法。
在本发明中，“CD8+细胞”通常是指CD8阳性的细胞，例如可以是T细胞。术语“CD8+细胞”，“CD8阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD8的抗体对细胞染色和使用荧光激活细胞分选。
在本发明中，术语“IC50值”或“IC50值”通常是指目标物获得生物学过程50％抑制需要的浓度。可以使用Cheng-Prusoff方程(Biochem.Pharmacol.(1973)22:3099)将IC50值换算成绝对抑制常数(Ki)。
在本发明中，术语“KD值”或“KD值”通常是指解离常数，其可通过表面等离子体共振进行测定。通常，表面等离子体共振分析使用BIAcore系统(Pharmacia Biosensor，Piscataway，NJ)，通过表面等离子体共振(SPR)，测量配体(固定化于生物传感器基质上的物质)和分析物(溶液中的物质)之间的实时结合相互作用。也可以通过固定化分析物(生物传感器基质上的物质)和呈递配体，进行表面等离子体分析。
在本发明中，术语“编码”通常是指能够根据基本上确定的规则，由一种分子的结构或组成信息，直接或间接推断出与其相关的另一类分子的结构或组成信息。例如，可以根据氨基酸的序列推断出其核苷酸序列，例如根据脱氧核糖核酸转录互补核酸的特性，包括能翻译成多肽的核酸。例如，脱氧核糖核酸可编码从脱氧核糖核酸转录的RNA。脱氧核糖核酸可类似地编码从脱氧核糖核酸所转录的RNA翻译的多肽。
在本发明中，术语“小分子化合物”通常是指肽、肽模拟物、氨基酸、氨基酸类似物、多核苷酸、多核苷酸类似物、核苷酸、核苷酸类似物、分子量小于约10,000克/摩尔的有机或无机物(即包括异源有机物和有机金属化合物)、分子量小于约5,000克/摩尔的有机或无机物、分子量小于约1,000克/摩尔的有机或无机物、分子量小于约500克/摩尔的有机或无机物，以及这类药物的盐、酯和其它药学上可接受的形式。
在本发明中，术语“NK细胞”也称为“自然杀伤细胞”，通常是指一种细胞质中具有大颗粒的细胞。NK细胞由骨髓淋巴样干细胞发育而成，可以依赖于骨髓或胸腺微环境分化、发育。在本发明中，TIL细胞中的NK细胞的比例可以通过本发明的方法加以改变。
在本发明中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、 非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab')2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合CD3)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。
在本发明中，术语“抗原结合片段”通常指具有特异结合抗原能力的一个或多个多肽片段。在本发明中，所述抗原结合片段可以包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv和/或dAb。
在本发明中，术语“表达”通常是指编码目标多肽的基因在细胞内发生的转录和/或翻译过程。可以通过测量存在于细胞中的相应mRNA的量来确定宿主细胞中编码目标多肽的基因的转录水平。例如，可通 过PCR或通过RNA杂交对编码目标多肽的基因转录的mRNA进行定量测量。可以通过多种方法测量编码目标多肽的基因的翻译水平，例如通过ELISA，通过多肽生物活性测试，或通过蛋白质印迹或放射免疫测试法。在本发明中，术语“表达”通常也可以是指产物发生的转录和/或翻译过程。例如，细胞因子的表达可以是细胞转录和/或翻译该细胞因子的过程。例如，细胞因子的表达可以通过检测存在于细胞中的相应mRNA的量或检测通过细胞生产的该细胞因子的量，或两者进行确定。
在本发明中，术语“一个阶段的体外扩增”、“单个阶段的体外扩增”、或“第一阶段体外扩增”等中的“阶段”通常是指TIL在体外经过的一段扩增过程。在一种实施方式中，每一个阶段之间可以是通过TIL细胞数量的变化来划分的，在一种实施方式中，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1-50倍时，例如至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的条件来划分的。在一种实施方式中，当细胞培养基中添加了或补充添加了T细胞激活剂和/或T细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，当TIL细胞进行了离心和/或细胞洗涤后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。在一种实施方式中，当TIL细胞体外培养约1-100天后，例如约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天、约30天、约40天、约50天或约100 天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
在本发明中，术语“第一阶段体外扩增”通常是指从组织中获得初级TIL后，使用T细胞生长因子进行扩增的阶段。在一种实施方式中，本发明的组织可以选自以下组：肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液，本发明的胸腔积液可以是有转移癌的患者的胸腔积液。在一种实施方式中，本发明的扩增可以是自体或者异体进行的体内扩增，或者可以是体外扩增。本发明的第一阶段体外扩增也可以称为preREP(快速扩增前)阶段。例如，源自肿瘤组织且未经体外扩增的TIL可以称为第一TIL群。例如，在两步骤法划分的本发明的培养方式中经过第一阶段体外扩增获得的TIL可以称为第二TIL群。
在本发明中，术语“第二阶段体外扩增”通常是指从受试者体内取出的组织并进行扩增后，再次进行扩增的阶段。在一种实施方式中，与经第一阶段体外扩增的TIL相比，本发明的经第二阶段体外扩增的TIL细胞数量增加，例如，可以增加至少约10倍(或至少约20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在一种实施方式中，第二阶段体外扩增可以与第一阶段体外扩增的培养条件不同，例如加入的培养物质可以不同。例如，在两步骤法划分的本发明的培养方式中第二阶段体外扩增也可以称为REP(快速扩增)阶段。例如，在两步骤法划分的本发明的培养方式中经过第二阶段体外扩增获得的TIL可以称为第三TIL群。
在本发明中，术语“体内”通常是指发生在受试者体内的事件。
在本发明中，术语“体外”通常是指在受试者体外发生的事件。
在本发明中，术语“离体”通常是指涉及对已从受试者体内移除的细胞、组织和/或器官进行治疗或进行手术的事件。在一种实施方式中， 该细胞、组织和/或器官可以通过手术或治疗方法返回到受试者的身体。
在本发明中，术语“分泌能力”通常是指细胞表达多肽或蛋白并将本发明的多肽或蛋白转移到细胞外环境的能力。
在本发明中，术语“辐照”通常是指通过射线对物质进行的处理。例如，在一种实施方式中，辐照可以是指通过X射线、α射线、β射线或γ射线对物质进行辐照。
在本发明中，术语“工程化细胞”通常是指将DNA或RNA形式的额外遗传物质加入细胞的总遗传物质而被基因修饰的细胞。在一种实施方式中，工程化细胞可以经过基因修饰以表达本发明的T细胞激活剂和/或T细胞生长因子的TIL。
在本发明中，术语“共培养”通常是指将两个或更多个不同群体的细胞在它们之间有一定程度的接触的情况下培养。本发明的两个或更多个不同群体的细胞的“接触”，在一种实施方式中可以通过直接接触，即其中一个群体的细胞与另一个群体的细胞直接物理接触。或者在一种实施方式中可以通过共用培养基所介导的间接接触。本发明的共用的培养基可以含有由共培养细胞的至少一个群体所产生和释放的代谢产物，并用于培养另一个群体的细胞。
在本发明中，术语“接触”通常是指两个或更多个不同类型的物质以任何顺序、任何方式以及任何时长接触在一起。在一种实施方式中可以通过直接接触，例如可以将一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子加入TIL细胞的培养基，例如可以将包含一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子的培养基加入和/或替换TIL细胞的培养基，例如，可以将包含一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子的培养基用于TIL细胞的培养；在一种实施方式中可以通过间接接触，例如可以将饲养细胞产生和释放的代谢 产物，用于培养TIL细胞。
在本发明中，术语“同时接触”、“共同接触”、“与...接触同时”、“同时”和“共同”通常是指向受试者和/或细胞施用两种以上物质，使得物质同时存在于受试者和/或细胞培养的环境中。同时接触可以包括以不同的组合物同时施用、以不同的组合物在不同时间施用，或以其中存在两种以上活性药物成分的组合物施用。例如，本发明中“同时接触”通常可以是指基本上同时接触。
在本发明中，术语“扩增”通常是指在一段时间内细胞的数量增加若干倍。在一种实施方式中细胞的数量可以增加至少约3倍(或4、5、6、7、8或9倍)，在一种实施方式中细胞的数量可以增加至少约10倍(或20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在本发明中，术语“经扩增”通常是指本发明的细胞经过上述一种或多种扩增。
在本发明中，术语“聚合物”通常是指由连接在一起的单独化学部分组成的分子，本发明的聚合物部分可相同或不同。在一种实施方式中，术语“聚合物”可以指尾尾相连而形成线性分子的单独化学部分，以及以分支(如“多臂”或“星型”)结构形式连接在一起的单独化学部分。在一种实施方式中聚合物可以包括例如多糖、葡聚糖、水凝胶、聚乙二醇、或泊洛沙姆。泊洛沙姆是非离子三嵌段共聚物，其具有聚氧丙烯(聚(环氧丙烷))中央疏水链，侧连两条聚氧乙烯(聚(环氧乙烷))亲水链。本发明包含的物质可以与本文所描述的或本领域已知的任何聚合物一起配制，或与它们一起给予。
在本发明中，术语“嵌合抗体(chimeric antibody)”通常是指鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，可以建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，可以根据需 要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，可以在真核系统或原核系统中表达嵌合抗体分子。
在本发明中，术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，通常是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库。
在本发明中，术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区可以都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本发明所述抗体或配体可以为全人源单克隆抗体。全人抗体制备的相关技术可以为：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
在本发明中，术语“CDR”通常是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一可以由Kabat E.A.等人，Chothia等人和MacCallum等人提供。如本发明中使用的，CDR的Kabat定义可以应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR1、CDR2和CDR3(CDR H1、CDR H2、CDR H3或H1、H2、H3)。
在本发明中，术语“IL-2”或“IL2”通常是指称为白细胞介素2的T 细胞生长因子，并包括所有形式的IL-2，可以包括在一种实施方式中人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体，或其活性片段。编码IL-2基因的GeneID可以为3558。
在本发明中，术语“抗原呈递细胞”、“抗原递呈细胞”、或“APC”通常是指，在其表面上展示与主要组织相容性复合物(MHC)复合的外源抗原的免疫系统细胞，如辅助细胞(例如，B细胞、树突细胞等)。T细胞可以使用其T细胞受体(TCR)识别这些复合物。APC可以加工抗原并将其递呈至T细胞。在一种实施方式中，抗原呈递细胞可以包括选自以下组：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。
在本发明中，术语“TIL特性”通常是指TIL细胞经过本发明培养方法获得的特性。TIL特性的变化可以包含：增加的TIL细胞数量，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的体外肿瘤细胞杀伤能力，提高的体内肿瘤杀伤能力，提高的T细胞受体(TCR)克隆多样性和提高的组织中TIL细胞数量，或它们的任何组合。本发明的变化可以是提高或者降低。
在本发明中，术语“存续”通常是指细胞在体外和/或受试者体内的存在。例如，TIL细胞存续能力的增加，可以是指TIL细胞在体内存在的时间增加。例如，存续能力增加可以是指细胞在受试者组织内，例如肿瘤、脾脏、骨髓、肺组织及血液中存在的时间的增加。例如，存续能力增加可以是培养基中撤去IL-2之后，TIL细胞存续能力的增加。
在本发明中，术语“人工抗原呈递细胞”通常是指人工构建的用于呈递外源抗原的免疫细胞，例如，呈递外源抗原的方式可以是人工抗原呈递细胞的表面包含外源抗原与主要组织相容性复合物(MHC)的复合物。在一个实施方案中，可以包括分离的人工抗原呈递细胞(aAPC)，其可以包含表达HLA-A/B/C(编码其的基因GeneID可以为3105、3106或3107)、CD64(编码其的基因GeneID可以为2209)、CD80(编码其的 基因GeneID可以为941)、ICOS-L(编码其的基因GeneID可以为23308)和CD58(编码其的基因GeneID可以为965)的细胞，并可以被修饰以表达一种以上T细胞激活剂。
在本发明中，术语“融合蛋白”通常是指含有第一多肽或蛋白质或其片段、类似物或衍生物的氨基酸序列和异源多肽或蛋白质(即，不同于第一多肽或蛋白质或其片段、类似物或衍生物的第二多肽或蛋白质或其片段、类似物或衍生物，或者通常不是第一多肽或蛋白质或其片段、类似物或衍生物的一部分)的氨基酸序列的多肽或蛋白质。在某些情形中，融合蛋白可包含与异源蛋白、多肽或肽融合的预防性或治疗性药物。其中，本发明的异源蛋白、多肽或肽可以是或不是不同类型的预防性或治疗性药物。例如，可将具有免疫调节活性的两种不同蛋白质、多肽或肽融合到一起形成融合蛋白。在某些情形中，与异源蛋白、多肽或蛋白质融合前的初始多肽或蛋白质的活性相比，融合蛋白可以保留或提高了活性。
在本发明中，术语“杀伤能力”通常是指通过使本发明的细胞接触有效量的物质从而杀伤靶细胞来实现。在一个实施方案中，本发明的物质可以是TIL细胞。本发明的杀伤可以包括通过自身或者促进其它细胞或物质的CDC、凋亡、ADCC和/或吞噬作用，或通过两种或更多种这些机制的组合以杀伤细胞。
在本发明中，术语“施用”或“给药”通常是指通过本领域已知的任意途径，将物质递送给有此需要的受试者。药用载体和制剂或组合物也是本领域众所周知的。给药途径可以包括：静脉内的、肌肉内的、真皮内的、皮下的、透皮的、粘膜的、瘤内的和/或粘膜的。
在本发明中，术语“试剂盒”通常是指一起被包装在容器、接受器或其它容器中的两种或更多种组分，其中一种对应于本发明的物质。例如，包含本发明的TIL细胞。
在本发明中，术语“受试者”通常是指细胞或动物，可以是哺乳动物，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型，例如肿瘤动物模型，和本领域技术人员已知的其它动物模型。
在本发明中，术语“饲养细胞(feeder)”通常是指可以用于支持培养另一种目的细胞生长的培养细胞。例如，可以通过体外生长和分泌至少一种因子至培养基。在一种实施方式中，饲养细胞可以包括抗原呈递细胞。
在本发明中，术语“特异性结合”通常是指识别特异性靶点物质，但是基本不识别或结合样品中其它分子的结合物质。例如，如果一种结合物质可以特异性结合来自一个物种的本发明的特异性靶点物质，则本发明的结合物质还可以特异性结合来自其它的一个或多个物种的本发明的靶点物质或同源靶点物质。这种种间反应性本身可以不会改变结合物质作为特异性的分类。在某些情形中，特异性结合至靶点物质的结合物质还可以结合至靶点物质的不同等位形式。
在本发明中，术语“完整的培养过程”通常是指将细胞从患者体内分离的肿瘤组织中分离开始，经过一次或一次以上的扩增，最终获得可以施用于受试者的细胞的完整过程。
在本发明中，术语“细胞培养基”通常是指细胞例如哺乳动物细胞在其中生长的营养液。细胞培养基的配制在本领域中是熟知的。典型地，细胞培养基包括缓冲液、盐、碳水化合物、氨基酸、维生素以及必要的微量元素。细胞培养基可以含有或不含有血清、蛋白胨和/或蛋白质。细胞培养基可以补充有另外的组分或浓度增加的组分，如氨基 酸、盐、糖、维生素、激素、生长因子、缓冲液、抗生素、脂质、微量元素等，这取决于有待培养的细胞的要求和/或所希望的细胞培养参数。
在本发明中，术语“药物组合物”或“药物制剂”通常是指一种制备物，本发明的制备物可以允许有效成分的生物活性有效，并且可以不含有对于将会施用该制剂的受试者不可接受地有毒的额外组分。这类制剂是无菌的。“可药用的”赋形剂(载体、添加物)是可以合理地施用至受试哺乳动物以提供有效剂量的所用有效成分的那些赋形剂。
在本发明中，术语“肿瘤浸润淋巴细胞”或“TIL”通常是指最初作为白细胞获得的细胞群，本发明的细胞已经离开受试者的血流并迁移到肿瘤中。TIL可以包括但不限于CD8+细胞毒性T细胞(淋巴细胞)、Th1和Th17CD4+T细胞、天然杀伤细胞、树突细胞和M1巨噬细胞。TIL可以包括初级TIL和次级TIL。“初级TIL”可以是从受试者组织样品获得的那些TIL细胞，“次级TIL”可以是本发明中已扩增或经扩增的任何TIL群。在一些实施方式中，本发明的肿瘤浸润淋巴细胞可以是未经分离纯化的，或者可以是与肿瘤细胞相互浸润的。在一种实施方式中，本发明的TIL可以是指TIL群。
在本发明中，术语“中心记忆T细胞”通常是指具有长期记忆性的，并能够接受抗原再刺激的T细胞。中心记忆T细胞可以具有CD45RO+CD62L+的表型，例如可以是通过CD45RO+和CD62L+来鉴定中心记忆T细胞。中心记忆T细胞可以相比普通T细胞具有更强的抗肿瘤生长的能力。
在本发明中，术语“调节性T细胞”通常是指一类控制体内自身免疫反应性的T细胞亚群。调节性T细胞可以具有CD4+CD25+Foxp3+的表型，例如可以是通过CD4+、CD25+和Foxp3+来鉴定调节性T细胞。调节性T细胞可以具有抑制T细胞的抗肿瘤生长的能力。
在本发明中，术语“活化T细胞”通常是指经过活化而可以具有抗肿瘤生长的能力的T细胞。活化T细胞可以具有PD-1+(PD1+)、LAG-3+(LAG3+)或CD28+的表型，例如可以是通过PD-1+、LAG-3+或CD28+来鉴定活化T细胞。活化T细胞可以具有抗肿瘤生长的能力。
在本发明中，术语“肿瘤特异性T细胞”通常是指可以特异性抗肿瘤生长的T细胞。肿瘤特异性T细胞可以具有CD103+CD39+的表型，例如，可以是通过CD103+和CD39+来鉴定肿瘤特异性T细胞。肿瘤特异性T细胞可以相比普通T细胞具有更特异性的抗肿瘤生长的能力。
在本发明中，术语“干细胞样T细胞”通常是指可以具有自我增殖和/或分化的潜能的一类T细胞。例如，在本发明中具有分化潜力和/或持续增殖能力的细胞可以认为是干细胞样细胞。例如，幼稚(naive)T细胞(CD45RO-CD62L+)可以认为是干细胞样细胞。例如，幼稚(naive)T细胞可以具有CD45RO-CD62L+的表型。例如可以是通过CD45RO-CD62L+来鉴定干细胞样T细胞。例如可以是通过CD39-CD69-来鉴定干细胞样T细胞。例如，干细胞样T细胞可以具有TCF1+的表型，例如可以是通过TCF1+来鉴定干细胞样T细胞。干细胞样T细胞可以相比普通T细胞具有更强和/或更长期的抗肿瘤生长的能力。
在本发明中，术语肿瘤“碎片”通常是指从受试者体内取出肿瘤组织后，可以通过机械破碎、酶解和/或其它破碎方法，形成的肿瘤碎片。
在本发明中，术语“组合物”或“药物组合物”通常是指至少一种细胞以及至少一种和任选多于一种的其他药学上可接受的化学组分如运载体、稳定剂、稀释剂、分散剂、助悬剂、增稠剂和/或赋形剂的混合物。
在本发明中，术语“药学上可接受的载体”通常是指不干扰活性成 分的一种或多种非毒性材料。例如，药学上可接受的载体可以不干扰扰活性成分的生物活性；例如，药学上可接受的载体可以不干扰扰活性成分所具有的生物活性的有效性。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选的其他治疗剂。这类药学上可接受的制剂还可以含有适合于给予人的相容的固体或液体填料、稀释剂或包封物质。可以用于在此所描述的配制品中的其他设想的载体、赋形剂和/或添加剂可以包括：例如，调味剂、抗微生物剂、增甜剂、抗氧化剂、抗静电剂、脂质、蛋白质赋形剂(如血清白蛋白、明胶、酪蛋白)、成盐平衡离子(如钠)等等。适合用于在此所描述的配制品中的这些和另外已知的药物载体、赋形剂和/或添加剂是本领域中已知的。本发明中，“药学上可接受的载体(carrier)”可以理解为不包含基因工程用到的核酸形式的载体(vector)。
在本发明中，术语“功能活性片段”通常是指具有全长蛋白质或核酸的部分区域，但保留或部分保留全长蛋白质或核酸的生物活性或功能的片段。例如，功能活性片段可以保留或部分保留全长蛋白质结合另一种分子的能力。
在本发明中，术语“T细胞激活剂”通常是指与T细胞上的相应结合受体结合，并介导T细胞共刺激反应的物质。T细胞激活剂可以是T细胞产生有效免疫应答所需的除抗原受体之外的物质。T细胞激活剂可以是指T细胞共刺激分子。例如，本发明的T细胞激活剂可以包含其变体、同源物或包含其功能活性片段的任何物质。T细胞激活剂可以包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号淋巴细胞活化分子(SLAM蛋白)、NK细胞活化受体、BTLA(编码其的基因GeneID可以为151888)、Toll配体受体、OX40(编码其的基因GeneID可以为7293)、CD2(编码其的基因GeneID可以为914)、CD7(编码其的基因GeneID可以为924)、CD27(编码其的基因GeneID可以为939)、CD28(编码其的基因GeneID可以为940)、CD30(编码其的基因GeneID可以为943)、CD40(编码其的基因 GeneID可以为958)、CDS、ICAM-1(编码其的基因GeneID可以为3383)、LFA-1(CD11a/CD18)(编码其的基因GeneID可以为3689)、4-1BB(CD137)(编码其的基因GeneID可以为3604)、B7-H3(编码其的基因GeneID可以为80381)、ICOS(CD278)(编码其的基因GeneID可以为29851)、GITR(编码其的基因GeneID可以为8784)、BAFFR(编码其的基因GeneID可以为115650)、LIGHT(编码其的基因GeneID可以为8740)、HVEM(LIGHTR)(编码其的基因GeneID可以为8764)、KIRDS2(编码其的基因GeneID可以为100132285)、SLAMF7(编码其的基因GeneID可以为57823)、NKp80(KLRF1)(编码其的基因GeneID可以为51348)、NKp44(编码其的基因GeneID可以为9436)、NKp30(编码其的基因GeneID可以为259197)、NKp46(编码其的基因GeneID可以为9437)、CD19(编码其的基因GeneID可以为930)、CD4(编码其的基因GeneID可以为920)、CD8α(编码其的基因GeneID可以为925)、CD8β(编码其的基因GeneID可以为926)、IL-2Rβ、IL-2Rγ、IL7Rα(编码其的基因GeneID可以为3575)、ITGA4(编码其的基因GeneID可以为3676)、VLA1(编码其的基因GeneID可以为3672)、CD49a(编码其的基因GeneID可以为3672)、IA4(编码其的基因GeneID可以为3732)、CD49D(编码其的基因GeneID可以为3676)、ITGA6(编码其的基因GeneID可以为3655)、VLA-6(编码其的基因GeneID可以为3655)、CD49f(编码其的基因GeneID可以为3655)、ITGAD(编码其的基因GeneID可以为3681)、CD11d(编码其的基因GeneID可以为3681)、ITGAE(编码其的基因GeneID可以为3682)、CD103(编码其的基因GeneID可以为3682)、ITGAL(编码其的基因GeneID可以为3683)、CD11a(编码其的基因GeneID可以为3683)、LFA-1(编码其的基因GeneID可以为3683)、ITGAM(编码其的基因GeneID可以为3684)、CD11b(编码其的基因GeneID可以为3684)、ITGAX(编码其的基因GeneID可以为3687)、CD11c(编码其的基因GeneID可以为3687)、ITGB1(编码其的基因GeneID可以为3688)、CD29(编码其的基因GeneID可以为3688)、ITGB2(编码其的基因GeneID可以为3689)、CD18(编码其的基因GeneID可以为3689)、LFA-1(编码其的基因GeneID 可以为3689)、ITGB7(编码其的基因GeneID可以为3695)、NKG2D(编码其的基因GeneID可以为22914)、NKG2C(编码其的基因GeneID可以为3822)、TNFR2(编码其的基因GeneID可以为7133)、TRANCE/RANKL(编码其的基因GeneID可以为8600)、DNAM1(CD226)(编码其的基因GeneID可以为10666)、SLAMF4(CD244、2B4)(编码其的基因GeneID可以为51744)、CD84(编码其的基因GeneID可以为8832)、CD96(Tactile)(编码其的基因GeneID可以为10225)、CEACAM1(编码其的基因GeneID可以为634)、CRTAM(编码其的基因GeneID可以为56253)、Ly9(CD229)(编码其的基因GeneID可以为4063)、CD160(BY55)(编码其的基因GeneID可以为11126)、PSGL1(编码其的基因GeneID可以为6404)、CD100(SEMA4D)(编码其的基因GeneID可以为10507)、CD69(编码其的基因GeneID可以为969)、SLAMF6(NTB-A、Ly108)(编码其的基因GeneID可以为114836)、SLAM(SLAMF1、CD150、IPO-3)(编码其的基因GeneID可以为6504)、BLAME(SLAMF8)(编码其的基因GeneID可以为56833)、SELPLG(CD162)(编码其的基因GeneID可以为6404)、LTBR(编码其的基因GeneID可以为4055)、LAT(编码其的基因GeneID可以为27040)、GADS(编码其的基因GeneID可以为9402)、SLP-76(编码其的基因GeneID可以为3937)、PAG/Cbp(编码其的基因GeneID可以为55824)、CD19a、特异性结合CD3的配体、特异性结合CD28的配体、特异性结合HVEM的配体、特异性结合CD40L的配体、特异性结合OX40的配体和特异性结合4-1BB的配体。共刺激胞内信号传导结构域可以是指T细胞激活剂的胞内部分。胞内信号传导结构域可以包含从中衍生的分子的完整胞内部分或完整天然胞内信号传导结构域或其功能性片段。
在本发明中，术语“T细胞生长因子”通常是指引起细胞增殖的生物活性多肽或小分子化合物。例如，本发明的T细胞生长因子可以包含其变体、同源物或包含其功能活性片段的任何物质。在一种实施方式中，T细胞生长因子可以选自以下组的一种或多种：IL-2(编码其的 基因GeneID可以为3558)、IL-4(编码其的基因GeneID可以为3565)、IL-6(编码其的基因GeneID可以为3569)、IL-7(编码其的基因GeneID可以为3574)、IL-10(编码其的基因GeneID可以为3586)、IL-12(编码其的基因GeneID可以为3592或3593)、IL-15(编码其的基因GeneID可以为3600)、IL-21(编码其的基因GeneID可以为59067)、TNF-α(编码其的基因GeneID可以为100137091)、γ干扰素(编码其的基因GeneID可以为3458)、GZMB(编码其的基因GeneID可以为3002)、CD107a(编码其的基因GeneID可以为6499)等等。
在本发明中，术语“基本上同时”通常是指接触过程的一段时间内TIL可以与两种以上的物质同时接触，但是可以不限于在整个接触过程中TIL总是与两种以上的物质同时接触。在一种实施方式中，基本上同时可以是指一段时间内TIL可以与至少10-95％，例如至少10％、20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％的两种以上的物质的每种物质同时接触。
在本发明中，术语“树突状细胞”通常是指存在于体内、体外、离体或宿主或受试者内的或可衍生自造血干细胞或单核细胞的抗原递呈细胞。树突状细胞及其前体可以从各种淋巴器官例如脾脏、淋巴结以及骨髓和外周血分离。本发明的树突状细胞可以具有特征形态，例如在树突细胞体的多个方向上延伸的薄层(板状伪足)。通常，树突细胞可以表达高水平的MHC和共刺激(例如B7-1和B7-2)分子。树突状细胞可以在体外诱导T细胞的抗原特异性分化，并且能够在体外和体内引发原代T细胞应答。
在本发明中，术语“体外扩增”通常是指经过培养以产生细胞的数量的变化，经扩增的细胞也可以产生细胞的数量和/或比例变化，分泌能力变化，杀伤能力变化或表达能力的变化，或它们的任何组合。本发明的变化可以是提高或者降低。在本发明中，体外扩增可以是为了扩增目的；为了检测TIL细胞的功能，例如检测TIL细胞释放细胞因 子能力，而对TIL细胞进行的操作步骤(例如向TIL细胞的培养基中加入一种以上物质以检测TIL细胞释放细胞因子能力)，可以不属于本发明的体外扩增。
在本发明中，术语“外周单个核细胞”或“外周血单个核细胞”通常是指外周血中具有单个核的细胞。例如，在本发明中，本发明的外周血单个核细胞可以包括淋巴细胞、单核细胞和/或树突状细胞。
在本发明中，术语“细胞因子”通常是指由一个细胞群释放的对另一个细胞起细胞间调节剂作用的蛋白。本发明的细胞因子可以是淋巴细胞因子(lymphokines)、单核细胞因子(monokines)和多肽激素。本发明的细胞因子可以包括白细胞介素(ILs)如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-15、IL-21和/或IL-12。在本发明中，术语细胞因子可以包括来自天然来源或来自重组细胞培养物的蛋白，天然序列细胞因子的生物活性等价物，以及其功能活性片段。
在本发明中，术语“直径”通常是指本发明物质的截面的直径。例如，当本发明的物质不是球形时，则术语“直径”通常是指本发明物质的最大截面的最大直径和/或平均直径。确定物质的直径的方法可以是本领域通用的方法，例如透射电子显微镜。
在本发明中，术语“肿瘤”通常是指任何新的病理性的组织增生。本发明的肿瘤可能是良性的，也可能是恶性的。本发明的肿瘤可能是实体的，也可能是血液的。术语“肿瘤”可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
在本发明中，术语“肿瘤组织”通常是指来自对象中的肿瘤，包括对象中的任何实体肿瘤和/或非实体肿瘤的任何组织的样品。
在本发明中，术语“T细胞亚群比例”通常是指根据不同T细胞亚群占TIL细胞或TIL群中的比例。例如，本发明不同的T细胞亚群具有不同的免疫活性和/或分化能力。例如，本发明的T细胞亚群可以根据T细胞表面标志物进行区分。例如，中心记忆T细胞可以具有CD45RO+CD62L+的表型。例如，幼稚T细胞可以具有CD45RO-CD62L+的表型。例如，调节性T细胞可以具有CD4+CD25+Foxp3+的表型。例如，活化T细胞可以具有CD25+、CD28+、PD-1+或41BB+的表型。例如，肿瘤特异性T细胞可以具有CD103+CD39+的表型。例如，干细胞样T细胞可以具有TCF1+的表型。
在本发明中，术语“TIL细胞数量”通常是指本发明的TIL细胞中细胞数量。在本发明中，TIL细胞数量可以是指本发明任一阶段获得的TIL群中的细胞数量。例如，TIL细胞数量可以是指源自肿瘤组织且未经体外扩增的第一TIL群的细胞数量。例如，TIL细胞数量可以是指经第一阶段体外扩增的第二TIL群的细胞数量。例如，TIL细胞数量可以是指经第二阶段体外扩增的第三TIL群的细胞数量。例如，TIL细胞数量可以是指本发明任意一种培养方法最终获得的TIL的细胞。在本发明中，TIL细胞数量可以通过本领域常用的方法测量，例如可以包括但不限于细胞计数板手动细胞计数和/或自动细胞计数器计数。
在本发明中，术语“约”和“大约”通常是指在统计上有意义的数值范围内。这样的范围可以在给定值或范围的一个数量级内，可以包括在50％内，优选包括在20％内，更优选包括在10％内，最优选包括在5％内。术语“约”或“大约”所包含的可允许变化可以取决于所研究的特定系统，并且本领域普通技术人员可以容易地理解。
在本发明中，术语“以上”、“以下”、“至多”和“至少”包括本数。
发明详述
TNFAIP3敲除
本发明提供了使所述细胞的肽酶C64家族成员和/或其功能活性片段的表达降低和/或活性减弱的方法。
一方面，本发明提供一种培养细胞的方法，使所述细胞的肽酶C64家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，所述肽酶C64家族成员可以包含泛素结合域。例如，所述肽酶C64家族成员可以包含TNFAIP3。
例如，本发明的目标基因可以是编码肽酶C64家族成员和/或其功能活性片段的基因。例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞可以显示出改善的细胞特性。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的至少一种目标基因的表达降低和/或活性减弱的细胞。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的目标基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的细胞。
例如，所述细胞包含免疫细胞。例如，所述细胞包含免疫效应细胞。例如，所述细胞包含免疫效应T细胞、免疫效应NK细胞、免疫效应NKT细胞。例如，所述细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
例如，所述细胞包含单核细胞、巨噬细胞和/或树突状细胞。
例如，本发明的细胞还包含来源于干细胞分化的细胞。例如，本发明的细胞还包含来源于多能干细胞分化的细胞。例如，获取本发明的干细胞可以是通过诱导产生。例如，本发明的上述干细胞可以包含诱导的多能干细胞(iPSC)、胚胎干细胞、骨髓干细胞、脐带血干细 胞和/或外周血干细胞。
例如，本发明的“干细胞”还包括多能细胞，多潜能细胞，前体细胞和祖细胞。例如，干细胞可以从获自骨髓组织的造血或间充质干细胞、获自胎盘组织的胎盘干细胞、获自胚胎组织的胚胎干细胞或获自胎儿的生殖组织的胚胎生殖细胞获得。示例性多能干细胞还可以通过与多能性相关的某些转录因子的表达将其重编程为多能状态而由体细胞产生；这些细胞被称为“诱导性多能干细胞”或“iPSC”。
例如，所述细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。例如，“未修饰的细胞”或“未改造的细胞”可以是指其中基因组未被修饰并且不包含基因调控系统或包含对照基因调控系统(例如，空载体对照、非靶向gRNA、干扰siRNA等)的细胞或细胞群体。例如，所述细胞包含αβT细胞和/或γδT细胞。例如，所述细胞包含肿瘤浸润淋巴细胞(TIL)。例如，所述TIL为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
例如，本发明的TIL可以为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。例如，可以通过将肿瘤组织处理成肿瘤碎片获得本发明的TIL。例如，本发明的肿瘤碎片的体积约为1-27立方毫米。例如，本发明的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约14立方毫米、约15立方毫米、约16立方毫米、约17立方毫米、约18立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或 约27立方毫米。
例如，所述细胞包含展示在细胞表面上的工程化免疫受体。例如，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。例如，所述细胞包含嵌合抗原受体和/或T细胞受体。
一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，可以包含：使所述TIL中包含肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以使所述TIL中包含肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片 段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第三阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。
例如，与肽酶C64家族成员的表达和/或活性未改变的细胞相比，使所述肽酶C64家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
例如，本发明的改善的细胞数量是指与肽酶C64家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述肽酶C64家族成员的表达降低和/或活性减弱的本发明细胞的细胞数量可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，增加的活细胞比例可以表现为细胞活率的增加。例如，本发明的增加的活细胞比例可以是指与肽酶C64家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述肽酶C64家族成员的表达降低和/或活性减弱的本发明细胞的活细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的细胞因子分泌能力可以是指细胞的选自以下组的细胞因子分泌能力提高：IL-2、IL-6、CD107a、GZMB、TNF-α和IFN-γ。例如，本发明的提高的细胞因子分泌能力可以是指与肽酶C64家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述肽酶C64家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的细胞因子分泌能力可以是指与肽酶C64家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述肽酶C64家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约 4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的肿瘤细胞杀伤能力可以是指与肽酶C64家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述肽酶C64家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的肿瘤细胞杀伤能力可以是指与肽酶C64家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述肽酶C64家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本发明的细胞的肿瘤细胞杀伤率可以通过IncuCyte系统或者CFSE和DAPI染色法测量。例如，本发明的细胞的肿瘤细胞杀伤可以是指细胞杀伤实体瘤细胞的能力。
例如，本发明的改善的细胞亚群比例可以包含选自以下组的一种或多种：增加的CD8+细胞比例，增加的中心记忆细胞和/或幼稚细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和增加的干细胞样细胞比例。
例如，在细胞中CD8+细胞、中心记忆细胞和/或幼稚细胞、活化 细胞、肿瘤特异性细胞和/或干细胞样细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的降低的耗竭细胞比例可以是细胞中PD-1+、LAG-3+、TIM-3+、和/或CD39+细胞的比例的增加。例如，本发明的减少的调节性细胞的比例可以是细胞中CD4+CD25+Foxp3+细胞的比例的减少。例如，本发明的减少的凋亡细胞的比例可以是细胞中CD95+caspass3+细胞和/或CD95+DR5+细胞的比例的减少。
例如，在细胞中耗竭细胞、调节性细胞和/或凋亡细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本发明的培养方法可以包含对于细胞的基因编辑步骤。例如，其包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至 少一个阶段的所述体外扩增中，可以将基因调控系统引入所述细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目标基因。例如，所述基因调控系统可以破坏所述细胞的基因组中的所述目标基因的区域或其片段。例如，使用所述基因调控系统后，细胞中的所述目标基因所在的DNA区域或其片段被剪切而该目标基因的表达能力降低或该目标基因的活性被抑制。例如，所述基因调控系统对目标基因的编辑效果可以是长期的、持续的。其中本发明所述基因组区域根据人类参考基因组hg38版本确定的。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所述酶蛋白特异性剪切目的基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。例如，可以将编码gRNA和Cas蛋白的多核苷酸引入、或各自独立地单独引入靶细胞。
例如，本发明使细胞的至少一种目标基因的表达降低和/或活性减弱可以包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述细胞中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。例如，所述指导核酸分子可以包含指导RNA(gRNA)。例如，可以将包含编码gRNA和Cas蛋白的多核苷酸的复合物引入所述细胞中。例如，可以将包含gRNA和Cas蛋白的复合物引入所述细胞中。
例如，所述gRNA可以用于与所述目标基因的序列结合。例如，所述gRNA与所述目标基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目标基因的序列。例如，所述gRNA与所述目标基因的序列的结合可以使得gRNA的 CRISPR系统特异性剪切所述目标基因。
例如，本发明的编辑靶标区域可以是启动子前的一段区域。例如，本发明的编辑靶标区域可以是转录因子结合力高的区域。例如，本发明的编辑靶标区域可以是具有特定数目的转录因子结合数的区域。例如，本发明的编辑靶标区域可以是具有约3个或以上的转录因子结合数的一段连续区域。
例如，当基因编辑系统包含CRISPR/Cas9时，本发明的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是AGG、TGG、GGG或CGG。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的5’端上游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5’端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，当基因编辑系统包含CRISPR/Cas12时，本发明的指导核酸分子靶向的区域上游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是NTTN、TTYN、VTTV、TRTV、TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)3’端下游约15至约25个核苷酸组成的序列结合：NTTN、TTYN、VTTV、TRTV、TTTV、TATV、 TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。
例如，当本发明的基因编辑系统包含野生型Cas12a(也可以称为Cpf1，如AsCas12a，FnCas12a，LbCas12a，BbCas12a，CMaCas12a和OsCas12a)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：NTTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如enAsCas12a(突变位点E174R、S542R和K548R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTYN(TTTN/TTCN)，VTTV(ATTV/CTTV/GTTV)，或TRTV(TATV/TGTV)，其中N可以为A、T、C或G，Y可以为T或C，V可以为A、C或G，R可以为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如opAsCas12a(突变位点：E174R和S542R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV(TTTA，TTTC，或TTTG)，其中V可以为A、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如AsCas12aUltra(突变位点：M537R和F870L)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV，TATV，或TYCV，其中V可以为A、C或G，Y可以为T或C。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如hfCas12Max(突变位点：N243R/E336R/D892R)和Cas12Max(突变位点：N243R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TNN、或NTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，所述指导核酸分子可以包含能够结合编码肽酶C64家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码肽酶C64家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。
例如，靶序列可以是选自表1A所示的基因组坐标定义的区域或其片段。
例如，本发明的靶序列可以是TNFAIP3的OUT结构功能域。例如，本发明的靶序列可以是TNFAIP3的锌指结构功能域。例如，本发明的靶序列可以是chr6:137871529-137871637、chr6:137874734-137874807、chr6:137874895-137874943、chr6:137875008-137875040、chr6:137875046-137875149、chr6:137875614-137875650、chr6:137875666-137875724、chr6:137875789-137875816、chr6:137875844-137876075、chr6:137879031-137879264、chr6:137879337-137879460、chr6:137879958-137880090、chr6:137880155-137880268、chr6:137880275-137880377、chr6:137880940-137881384。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域与选自以下组的靶序列互补：SEQ ID NO：107-212、1562-2532。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：1-106、591-1561、7267-7324、7419、7420中所示的序列。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：7267-7324、7419、7420中所示的序列。
例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例可以降低和/或单个细胞中所述目的基因的表达量可以下降。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低至少约5％。例如，表达所述编码肽酶C64家族成员和/或其功能活性片段的基因的产物的细胞比例降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，表达所述编码肽酶C64家族成员和/或其功能活性片段的基因的产物的细胞比例可以从可以观测的细胞比例到0％。例如，表达所述编码肽酶C64家族成员和/或其功能活性片段的基因的产物的细胞比例可以降低到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述编码肽酶C64家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述编码肽酶C64家族成员和/或其功能活性片段的基因的产物的细胞比例可以为至多约95％。例如，表达所述编码肽酶C64家族成员和/或其功能活性片段的基因的产物的细胞比例的细胞比例可以为至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述编码肽酶C64家族成员和/或其功能活性 片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以下降至少约5％。例如，单个细胞中所述目的基因的表达量可以下降至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目的基因的表达量可以从可以观测的量到0％。例如，，单个细胞中所述目的基因的表达量可以下降到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以为所述目标基因的表达和/或活性未改变的细胞的至多约95％。例如，细胞中单个细胞中所述编码肽酶C64家族成员和/或其功能活性片段的基因(例如编码TNFAIP3的基因)表达量可以为所述编码肽酶C64家族成员和/或其功能活性片段的表达和/或活性未改变的细胞的至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
例如，本发明的方法包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述细胞的肽酶C64家族成员的表达降低和/或活性减弱。
例如，使所述源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL的肽酶C64家族成员的表达降低和/或活性减弱。
例如，所述第一阶段体外扩增进行至少约7天。例如，所述第二阶段体外扩增进行至少约7天。
例如，可以在单个阶段的本发明的体外扩增中，使所述细胞与所述一种或多种细胞激活剂接触以及使所述细胞中包含肽酶C64家族成员和/或其功能活性片段的表达和/或活性降低。例如，细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，在单个阶段的所述体外扩增中，使本发明的细胞的肽酶C64家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第一阶段体外扩增中，可以使本发明的TIL与本发明的肽酶C64家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL的肽酶C64家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL的肽酶C64家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。
例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞 基本上同时使肽酶C64家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先使肽酶C64家族成员的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先与本发明的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使肽酶C64家族成员的表达降低和/或活性减弱。
例如，在本发明第一阶段体外扩增中，可以使本发明的TIL基本上同时使肽酶C64家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL基本上同时使肽酶C64家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL基本上同时使肽酶C64家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群肽酶C64家族成员的表达降低和/或活性减弱；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本发明的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本发明的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本发明的经第一阶段体 外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本发明的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本发明的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本发明的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种细胞生长因子接触、与多种细胞激活剂接触、使肽酶C64家族成员的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与细胞生长因子接触、与细胞激活剂接触、使肽酶C64家族成员的表达降低和/或活性减弱且使所述TIL与饲养细胞共培养，肽酶C64家族成员可以包含TNFAIP3；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织、胸腔积液和/或腹腔积液在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约 2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，细胞无血清培养基可以添加本发明的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2以及使肽酶C64家族成员的表达降低和/或活性减弱(肽酶C64家族成员可以包含TNFAIP3，例如可以通过用携带包含本发明的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)进行转导使所述TIL中编码肽酶C64家族成员的基因的细胞比例为约95％或以下)，活化本发明的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的细胞可以为记忆效应细胞和效应细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化细胞比例上调的特征。
ZC3H12A敲除
1.一种培养细胞的方法，所述方法包含：使所述细胞的ZC3H12家族成员和/或其功能活性片段的表达降低和/或活性减弱。
2.根据实施方案1所述的方法，其中所述细胞包含免疫细胞。
3.根据实施方案2所述的方法，其中所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
4.根据实施方案2-3中任一项所述的方法，其中所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
5.根据实施方案2-4中任一项所述的方法，其中所述免疫细胞来源于干细胞分化的免疫细胞。
6.根据实施方案5所述的方法，其中所述干细胞包含诱导的多能干细胞(iPSC)。
7.根据实施方案2-6中任一项所述的方法，其中所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。
8.根据实施方案2-7中任一项所述的方法，其中所述免疫细胞包含αβT细胞和/或γδT细胞。
9.根据实施方案2-8中任一项所述的方法，其中所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
10.根据实施方案9所述的方法，其中所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
11.根据实施方案10所述的方法，其中所述碎片的体积为约1立方毫米至约27立方毫米。
12.根据实施方案2-11中任一项所述的方法，其中所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
13.根据实施方案12所述的方法，其中所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
14.根据实施方案2-13中任一项所述的方法，其中所述免疫细胞 包含嵌合抗原受体和/或T细胞受体。
15.根据实施方案1-14中任一项所述的方法，其中使所述细胞的ZC3H12家族成员的表达降低和/或活性减弱包含抑制核酸酶的功能。
16.根据实施方案1-15中任一项所述的方法，其中与ZC3H12家族成员的表达和/或活性未改变的细胞相比，使所述ZC3H12家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
17.根据实施方案16所述的方法，其中所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
18.根据实施方案17所述的方法，其中所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
19.根据实施方案1-18中任一项所述的方法，其中所述ZC3H12家族成员包含C3H1型锌指结构域。
20.根据实施方案1-19中任一项所述的方法，其中所述ZC3H12家族成员包含ZC3H12A。
21.根据实施方案1-20中任一项所述的方法，其中使所述细胞的ZC3H12家族成员的表达降低和/或活性减弱包含将基因调控系统引入所述细胞中。
22.根据实施方案21所述的方法，其中所述基因调控系统能够在 DNA水平破坏所述ZC3H12家族成员。
23.根据实施方案21-22中任一项所述的方法，其中所述基因调控系统包含指导核酸分子和酶蛋白。
24.根据实施方案23所述的方法，其中使所述ZC3H12家族成员的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)、包含gRNA与Cas蛋白的LNP，或者包含编码gRNA与编码Cas蛋白的核酸的LNP引入所述细胞中。
25.根据实施方案23-24中任一项所述的方法，其中所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
26.根据实施方案23-25中任一项所述的方法，其中所述指导核酸分子包含指导RNA(gRNA)。
27.根据实施方案23-26中任一项所述的方法，其中所述指导核酸分子能够与所述ZC3H12家族成员的序列结合。
28.根据实施方案23-27中任一项所述的方法，其中所述指导核酸分子能够与选自表1B所示的基因组坐标定义的区域或其片段结合。
29.根据实施方案23-28中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：281-348、3116-3698。
30.根据实施方案23-29中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、CGG和GGG。
31.根据实施方案23-30中任一项所述的方法，其中所述指导核酸分子包括靶向结构域，所述靶向结构域包含如SEQ ID NO：213-280、2533-3115、7325-7345、7416、7417中任一项所示的序列。
32.根据实施方案1-31中任一项所述的方法，其中与ZC3H12家族成员的表达和/或活性未改变的细胞相比，使所述ZC3H12家族成员的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低和/或单个细胞中所述目的基因的表达量下降。
33.根据实施方案1-32中任一项所述的方法，其中使所述ZC3H12家族成员的表达降低和/或活性减弱获得的细胞中，表达所述目的基因的细胞比例为约95％或以下。
34.一种细胞，所述细胞经过实施方案1-33中任一项所述的方法获得。
35.一种组合物，其包含实施方案34所述的细胞。
36.一种药物组合物，其包含实施方案34所述的细胞和/或实施方案35所述的组合物，以及任选的药学上可接受的载体。
37.一种影响细胞生长的方法，其包含施用实施方案34所述的细胞、实施方案35所述的组合物和/或实施方案36所述的药物组合物。
38.实施方案34所述的细胞、实施方案35所述的组合物和/或实施方案36所述的药物组合物在制备药物中的应用，其中所述药物用于预防和/或治疗疾病和/或症状。
39.根据实施方案38所述的应用，其中所述疾病和/或症状包含肿瘤。
40.根据实施方案38-39中任一项所述的应用，其中所述疾病和/或症状包含实体瘤。
41.根据实施方案38-40中任一项所述的应用，其中所述疾病和/或症状包含选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
本发明提供了使所述细胞的ZC3H12家族成员和/或其功能活性片段的表达降低和/或活性减弱的方法。
一方面，本发明提供一种培养细胞的方法，使所述细胞的ZC3H12家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，所述ZC3H12家族成员可以包含C3H1型锌指结构域。例如，所述ZC3H12家族成员可以包含ZC3H12A。
例如，本发明的目标基因可以是编码ZC3H12家族成员和/或其功能活性片段的基因。例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞可以显示出改善的细胞特性。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的至少一种目标基因的表达降低和/或活性减弱的细胞。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的目标基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的细胞。
例如，所述细胞包含免疫细胞。例如，所述细胞包含免疫效应细胞。例如，所述细胞包含免疫效应T细胞、免疫效应NK细胞、免疫效应NKT细胞。例如，所述细胞包含吞噬细胞、淋巴细胞、中性粒细 胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
例如，所述细胞包含单核细胞、巨噬细胞和/或树突状细胞。
例如，本发明的细胞还包含来源于干细胞分化的细胞。例如，本发明的细胞还包含来源于多能干细胞分化的细胞。例如，获取本发明的干细胞可以是通过诱导产生。例如，本发明的上述干细胞可以包含诱导的多能干细胞(iPSC)、胚胎干细胞、骨髓干细胞、脐带血干细胞和/或外周血干细胞。
例如，本发明的“干细胞”还包括多能细胞，多潜能细胞，前体细胞和祖细胞。例如，干细胞可以从获自骨髓组织的造血或间充质干细胞、获自胎盘组织的胎盘干细胞、获自胚胎组织的胚胎干细胞或获自胎儿的生殖组织的胚胎生殖细胞获得。示例性多能干细胞还可以通过与多能性相关的某些转录因子的表达将其重编程为多能状态而由体细胞产生；这些细胞被称为“诱导性多能干细胞”或“iPSC”。
例如，所述细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。例如，“未修饰的细胞”或“未改造的细胞”可以是指其中基因组未被修饰并且不包含基因调控系统或包含对照基因调控系统(例如，空载体对照、非靶向gRNA、干扰siRNA等)的细胞或细胞群体。例如，所述细胞包含αβT细胞和/或γδT细胞。例如，所述细胞包含肿瘤浸润淋巴细胞(TIL)。例如，所述TIL为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
例如，本发明的TIL可以为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。例如，可以通过将 肿瘤组织处理成肿瘤碎片获得本发明的TIL。例如，本发明的肿瘤碎片的体积约为1-27立方毫米。例如，本发明的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约14立方毫米、约15立方毫米、约16立方毫米、约17立方毫米、约18立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。
例如，所述细胞包含展示在细胞表面上的工程化免疫受体。例如，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。例如，所述细胞包含嵌合抗原受体和/或T细胞受体。
一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，可以包含：使所述TIL中包含ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以使所述TIL中包含ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且 在本发明的第一阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第三阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中ZC3H12家族成员 和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。
例如，与ZC3H12家族成员的表达和/或活性未改变的细胞相比，使所述ZC3H12家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
例如，本发明的改善的细胞数量是指与ZC3H12家族成员的表达 和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述ZC3H12家族成员的表达降低和/或活性减弱的本发明细胞的细胞数量可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，增加的活细胞比例可以表现为细胞活率的增加。例如，本发明的增加的活细胞比例可以是指与ZC3H12家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述ZC3H12家族成员的表达降低和/或活性减弱的本发明细胞的活细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的细胞因子分泌能力可以是指细胞的选自以下组的细胞因子分泌能力提高：IL-2、IL-6、CD107a、GZMB、TNF-α和IFN-γ。例如，本发明的提高的细胞因子分泌能力可以是指与ZC3H12家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述ZC3H12家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的细胞因子分 泌能力可以是指与ZC3H12家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述ZC3H12家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的肿瘤细胞杀伤能力可以是指与ZC3H12家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述ZC3H12家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的肿瘤细胞杀伤能力可以是指与ZC3H12家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述ZC3H12家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本发明的细胞的肿瘤细胞杀伤率可以通过IncuCyte系统或者CFSE和DAPI染色法测量。例如，本发明的细胞 的肿瘤细胞杀伤可以是指细胞杀伤实体瘤细胞的能力。
例如，本发明的改善的细胞亚群比例可以包含选自以下组的一种或多种：增加的CD8+细胞比例，增加的中心记忆细胞和/或幼稚细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和增加的干细胞样细胞比例。
例如，在细胞中CD8+细胞、中心记忆细胞和/或幼稚细胞、活化细胞、肿瘤特异性细胞和/或干细胞样细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的降低的耗竭细胞比例可以是细胞中PD-1+、LAG-3+、TIM-3+、和/或CD39+细胞的比例的增加。例如，本发明的减少的调节性细胞的比例可以是细胞中CD4+CD25+Foxp3+细胞的比例的减少。例如，本发明的减少的凋亡细胞的比例可以是细胞中CD95+caspass3+细胞和/或CD95+DR5+细胞的比例的减少。
例如，在细胞中耗竭细胞、调节性细胞和/或凋亡细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4 ％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本发明的培养方法可以包含对于细胞的基因编辑步骤。例如，其包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以将基因调控系统引入所述细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目标基因。例如，所述基因调控系统可以破坏所述细胞的基因组中的所述目标基因的区域或其片段。例如，使用所述基因调控系统后，细胞中的所述目标基因所在的DNA区域或其片段被剪切而该目标基因的表达能力降低或该目标基因的活性被抑制。例如，所述基因调控系统对目标基因的编辑效果可以是长期的、持续的。其中本发明所述基因组区域根据人类参考基因组hg38版本确定的。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所述酶蛋白特异性剪切目的基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。例如，可以将编码gRNA和Cas蛋白的多核苷酸引入、或各自独立地单独引入靶细胞。
例如，本发明使细胞的至少一种目标基因的表达降低和/或活性减弱可以包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述细胞中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。例如，所述指导核酸分子可以包含指导RNA (gRNA)。例如，可以将包含编码gRNA和Cas蛋白的多核苷酸的复合物引入所述细胞中。例如，可以将包含gRNA和Cas蛋白的复合物引入所述细胞中。
例如，所述gRNA可以用于与所述目标基因的序列结合。例如，所述gRNA与所述目标基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目标基因的序列。例如，所述gRNA与所述目标基因的序列的结合可以使得gRNA的CRISPR系统特异性剪切所述目标基因。
例如，本发明的编辑靶标区域可以是启动子前的一段区域。例如，本发明的编辑靶标区域可以是转录因子结合力高的区域。例如，本发明的编辑靶标区域可以是具有特定数目的转录因子结合数的区域。例如，本发明的编辑靶标区域可以是具有约3个或以上的转录因子结合数的一段连续区域。
例如，当基因编辑系统包含CRISPR/Cas9时，本发明的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是AGG、TGG、GGG或CGG。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的5’端上游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5’端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，当基因编辑系统包含CRISPR/Cas12时，本发明的指导核酸分子靶向的区域上游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是NTTN、TTYN、VTTV、TRTV、 TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)3’端下游约15至约25个核苷酸组成的序列结合：NTTN、TTYN、VTTV、TRTV、TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。
例如，当本发明的基因编辑系统包含野生型Cas12a(也可以称为Cpf1，如AsCas12a，FnCas12a，LbCas12a，BbCas12a，CMaCas12a和OsCas12a)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：NTTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如enAsCas12a(突变位点E174R、S542R和K548R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTYN(TTTN/TTCN)，VTTV(ATTV/CTTV/GTTV)，或TRTV(TATV/TGTV)，其中N可以为A、T、C或G，Y可以为T或C，V可以为A、C或G，R可以为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如opAsCas12a(突变位点：E174R和S542R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV(TTTA，TTTC，或TTTG)，其中V可以为A、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如AsCas12aUltra(突变位点：M537R和F870L)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV，TATV，或TYCV，其中V可以为A、C或G，Y可以为T或C。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如hfCas12Max(突变位点：N243R/E336R/D892R)和Cas12Max(突变位点：N243R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TNN、或NTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，所述指导核酸分子可以包含能够结合编码ZC3H12家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所 述指导核酸分子可以包含能够结合编码ZC3H12家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。
例如，靶序列可以是选自表1B所示的基因组坐标定义的区域或其片段。
例如，本发明的靶序列可以是ZC3H12A的C3H1型锌指结构功能域。例如，本发明的靶序列可以是R_22_chr1:37482635-37482714。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域与选自以下组的靶序列互补：SEQ ID NO：281-348、3116-3698。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：213-280、2533-3115、7325-7345、7416、7417中所示的序列。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：7325-7345、7416、7417中所示的序列。
例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例可以降低和/或单个细胞中所述目的基因的表达量可以下降。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低至少约5％。例如，表达所述编码ZC3H12家族成员和/或其功能活性片段的基因的产物的细胞比例降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，表达所述编码ZC3H12家族成员和/或其功能活性片段的基因的产物的细胞比例可以从可以观测的细胞比例到0％。例如，表达所述编码ZC3H12家族成员和/或其功能活性片段的基因的产物的细胞比例可以降低到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述编码ZC3H12家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述编码ZC3H12家族成员和/或其功能活性片段的基因的产物的细胞比例可以为至多约95％。例如，表达所述编码ZC3H12家族成员和/或其功能活性片段的基因的产物的细胞比例的细胞比例可以为至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述编码ZC3H12家族成员和/或其功能活性 片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以下降至少约5％。例如，单个细胞中所述目的基因的表达量可以下降至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目的基因的表达量可以从可以观测的量到0％。例如，，单个细胞中所述目的基因的表达量可以下降到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以为所述目标基因的表达和/或活性未改变的细胞的至多约95％。例如，细胞中单个细胞中所述编码ZC3H12家族成员和/或其功能活性片段的基因(例如编码ZC3H12A的基因)表达量可以为所述编码ZC3H12家族成员和/或其功能活性片段的表达和/或活性未改变的细胞的至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
例如，本发明的方法包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述细胞的ZC3H12家族成员的表达降低和/或活性减弱。
例如，使所述源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL的ZC3H12家族成员的表达降低和/或活性减弱。
例如，所述第一阶段体外扩增进行至少约7天。例如，所述第二阶段体外扩增进行至少约7天。
例如，可以在单个阶段的本发明的体外扩增中，使所述细胞与所述一种或多种细胞激活剂接触以及使所述细胞中包含ZC3H12家族成员和/或其功能活性片段的表达和/或活性降低。例如，细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，在单个阶段的所述体外扩增中，使本发明的细胞的ZC3H12家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第一阶段体外扩增中，可以使本发明的TIL与本发明的ZC3H12家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL的ZC3H12家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL的ZC3H12家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。
例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞 基本上同时使ZC3H12家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先使ZC3H12家族成员的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先与本发明的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使ZC3H12家族成员的表达降低和/或活性减弱。
例如，在本发明第一阶段体外扩增中，可以使本发明的TIL基本上同时使ZC3H12家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL基本上同时使ZC3H12家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL基本上同时使ZC3H12家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群ZC3H12家族成员的表达降低和/或活性减弱；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本发明的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本发明的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本发明的经第一阶段体 外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本发明的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本发明的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本发明的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种细胞生长因子接触、与多种细胞激活剂接触、使ZC3H12家族成员的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与细胞生长因子接触、与细胞激活剂接触、使ZC3H12家族成员的表达降低和/或活性减弱且使所述TIL与饲养细胞共培养，ZC3H12家族成员可以包含ZC3H12A；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织、胸腔积液和/或腹腔积液在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约 2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，细胞无血清培养基可以添加本发明的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2以及使ZC3H12家族成员的表达降低和/或活性减弱(ZC3H12家族成员可以包含ZC3H12A，例如可以通过用携带包含本发明的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)进行转导使所述TIL中编码ZC3H12家族成员的基因的细胞比例为约95％或以下)，活化本发明的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的细胞可以为记忆效应细胞和效应细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化细胞比例上调的特征。
SOCS1敲除
1.一种培养细胞的方法，所述方法包含：使所述细胞的STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达降低和/或活性减弱。
2.根据实施方案1所述的方法，其中所述细胞包含免疫细胞。
3.根据实施方案2所述的方法，其中所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
4.根据实施方案2-3中任一项所述的方法，其中所述免疫细胞包 含单核细胞、巨噬细胞和/或树突状细胞。
5.根据实施方案2-4中任一项所述的方法，其中所述免疫细胞来源于干细胞分化的免疫细胞。
6.根据实施方案5所述的方法，其中所述干细胞包含诱导的多能干细胞(iPSC)。
7.根据实施方案2-6中任一项所述的方法，其中所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。
8.根据实施方案2-7中任一项所述的方法，其中所述免疫细胞包含αβT细胞和/或γδT细胞。
9.根据实施方案2-8中任一项所述的方法，其中所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
10.根据实施方案9所述的方法，其中所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
11.根据实施方案10所述的方法，其中所述碎片的体积为约1立方毫米至约27立方毫米。
12.根据实施方案2-11中任一项所述的方法，其中所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
13.根据实施方案12所述的方法，其中所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
14.根据实施方案2-13中任一项所述的方法，其中所述免疫细胞包含嵌合抗原受体和/或T细胞受体。
15.根据实施方案1-14中任一项所述的方法，其中使所述细胞的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱包含抑制细胞因子信号负调节的功能。
16.根据实施方案1-15中任一项所述的方法，其中与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
17.根据实施方案16所述的方法，其中所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
18.根据实施方案17所述的方法，其中所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
19.根据实施方案1-18中任一项所述的方法，其中所述STAT诱导的STAT抑制剂(SSI)家族成员包含SH2域。
20.根据实施方案1-19中任一项所述的方法，其中所述STAT诱导的STAT抑制剂(SSI)家族成员包含SOCS1。
21.根据实施方案1-20中任一项所述的方法，其中使所述细胞的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱 包含将基因调控系统引入所述细胞中。
22.根据实施方案21所述的方法，其中所述基因调控系统能够在DNA水平破坏所述STAT诱导的STAT抑制剂(SSI)家族成员。
23.根据实施方案21-22中任一项所述的方法，其中所述基因调控系统包含指导核酸分子和酶蛋白。
24.根据实施方案23所述的方法，其中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)、包含gRNA与Cas蛋白的LNP，或者包含编码gRNA与编码Cas蛋白的核酸的LNP引入所述细胞中。
25.根据实施方案23-24中任一项所述的方法，其中所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
26.根据实施方案23-25中任一项所述的方法，其中所述指导核酸分子包含指导RNA(gRNA)。
27.根据实施方案23-26中任一项所述的方法，其中所述指导核酸分子能够与所述STAT诱导的STAT抑制剂(SSI)家族成员的序列结合。
28.根据实施方案23-27中任一项所述的方法，其中所述指导核酸分子能够与选自表1C所示的基因组坐标定义的区域或其片段结合。
29.根据实施方案23-28中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：399-448、4393-5086。
30.根据实施方案23-29中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、CGG和GGG。
31.根据实施方案23-30中任一项所述的方法，其中所述指导核酸分子包括靶向结构域，所述靶向结构域包含如SEQ ID NO：349-398、3699-4392、7346-7375、7418中任一项所示的序列。
32.根据实施方案1-31中任一项所述的方法，其中与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低和/或单个细胞中所述目的基因的表达量下降。
33.根据实施方案1-32中任一项所述的方法，其中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱获得的细胞中，表达所述目的基因的细胞比例为约95％或以下。
34.一种细胞，所述细胞经过实施方案1-33中任一项所述的方法获得。
35.一种组合物，其包含实施方案34所述的细胞。
36.一种药物组合物，其包含实施方案34所述的细胞和/或实施方案35所述的组合物，以及任选的药学上可接受的载体。
37.一种影响细胞生长的方法，其包含施用实施方案34所述的细胞、实施方案35所述的组合物和/或实施方案36所述的药物组合物。
38.实施方案34所述的细胞、实施方案35所述的组合物和/或实施方案36所述的药物组合物在制备药物中的应用，其中所述药物用于预防和/或治疗疾病和/或症状。
39.根据实施方案38所述的应用，其中所述疾病和/或症状包含肿瘤。
40.根据实施方案38-39中任一项所述的应用，其中所述疾病和/或症状包含实体瘤。
41.根据实施方案38-40中任一项所述的应用，其中所述疾病和/或症状包含选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
本发明提供了使所述细胞的STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达降低和/或活性减弱的方法。
一方面，本发明提供一种培养细胞的方法，使所述细胞的STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，所述STAT诱导的STAT抑制剂(SSI)家族成员可以包含SH2域。例如，所述STAT诱导的STAT抑制剂(SSI)家族成员可以包含SOCS1。
例如，本发明的目标基因可以是编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因。例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞可以显示出改善的细胞特性。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的至少一种目标基因的表达降低和/或活性 减弱的细胞。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的目标基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的细胞。
例如，所述细胞包含免疫细胞。例如，所述细胞包含免疫效应细胞。例如，所述细胞包含免疫效应T细胞、免疫效应NK细胞、免疫效应NKT细胞。例如，所述细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
例如，所述细胞包含单核细胞、巨噬细胞和/或树突状细胞。
例如，本发明的细胞还包含来源于干细胞分化的细胞。例如，本发明的细胞还包含来源于多能干细胞分化的细胞。例如，获取本发明的干细胞可以是通过诱导产生。例如，本发明的上述干细胞可以包含诱导的多能干细胞(iPSC)、胚胎干细胞、骨髓干细胞、脐带血干细胞和/或外周血干细胞。
例如，本发明的“干细胞”还包括多能细胞，多潜能细胞，前体细胞和祖细胞。例如，干细胞可以从获自骨髓组织的造血或间充质干细胞、获自胎盘组织的胎盘干细胞、获自胚胎组织的胚胎干细胞或获自胎儿的生殖组织的胚胎生殖细胞获得。示例性多能干细胞还可以通过与多能性相关的某些转录因子的表达将其重编程为多能状态而由体细胞产生；这些细胞被称为“诱导性多能干细胞”或“iPSC”。
例如，所述细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。例如，“未修饰的细胞”或“未改造的细胞”可以是指其中基因组未被修饰并且不包含基因调控系统或包含对照基因调控系统(例如，空载体对照、非靶向gRNA、干扰siRNA等)的细胞或细胞群体。例如，所述细胞包含αβT细胞和/或γδT细胞。例如， 所述细胞包含肿瘤浸润淋巴细胞(TIL)。例如，所述TIL为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
例如，本发明的TIL可以为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。例如，可以通过将肿瘤组织处理成肿瘤碎片获得本发明的TIL。例如，本发明的肿瘤碎片的体积约为1-27立方毫米。例如，本发明的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约14立方毫米、约15立方毫米、约16立方毫米、约17立方毫米、约18立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。
例如，所述细胞包含展示在细胞表面上的工程化免疫受体。例如，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。例如，所述细胞包含嵌合抗原受体和/或T细胞受体。
一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，可以包含：使所述TIL中包含STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以使所述TIL中包含STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL 中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第三阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述 TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。
例如，与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
例如，本发明的改善的细胞数量是指与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱的本发明细胞的细胞数量可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，增加的活细胞比例可以表现为细胞活率的增加。例如，本发明的增加的活细胞比例可以是指与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/ 或活性减弱的本发明细胞的活细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的细胞因子分泌能力可以是指细胞的选自以下组的细胞因子分泌能力提高：IL-2、IL-6、CD107a、GZMB、TNF-α和IFN-γ。例如，本发明的提高的细胞因子分泌能力可以是指与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的细胞因子分泌能力可以是指与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的肿瘤细胞杀伤能力可以是指与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的肿瘤细胞杀伤能力可以是指与STAT诱导的STAT抑制剂(SSI)家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本发明的细胞的肿瘤细胞杀伤率可以通过IncuCyte系统或者CFSE和DAPI染色法测量。例如，本发明的细胞的肿瘤细胞杀伤可以是指细胞杀伤实体瘤细胞的能力。
例如，本发明的改善的细胞亚群比例可以包含选自以下组的一种或多种：增加的CD8+细胞比例，增加的中心记忆细胞和/或幼稚细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和增加的干细胞样细胞比例。
例如，在细胞中CD8+细胞、中心记忆细胞和/或幼稚细胞、活化 细胞、肿瘤特异性细胞和/或干细胞样细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的降低的耗竭细胞比例可以是细胞中PD-1+、LAG-3+、TIM-3+、和/或CD39+细胞的比例的增加。例如，本发明的减少的调节性细胞的比例可以是细胞中CD4+CD25+Foxp3+细胞的比例的减少。例如，本发明的减少的凋亡细胞的比例可以是细胞中CD95+caspass3+细胞和/或CD95+DR5+细胞的比例的减少。
例如，在细胞中耗竭细胞、调节性细胞和/或凋亡细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本发明的培养方法可以包含对于细胞的基因编辑步骤。例如，其包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至 少一个阶段的所述体外扩增中，可以将基因调控系统引入所述细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目标基因。例如，所述基因调控系统可以破坏所述细胞的基因组中的所述目标基因的区域或其片段。例如，使用所述基因调控系统后，细胞中的所述目标基因所在的DNA区域或其片段被剪切而该目标基因的表达能力降低或该目标基因的活性被抑制。例如，所述基因调控系统对目标基因的编辑效果可以是长期的、持续的。其中本发明所述基因组区域根据人类参考基因组hg38版本确定的。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所述酶蛋白特异性剪切目的基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。例如，可以将编码gRNA和Cas蛋白的多核苷酸引入、或各自独立地单独引入靶细胞。
例如，本发明使细胞的至少一种目标基因的表达降低和/或活性减弱可以包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述细胞中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。例如，所述指导核酸分子可以包含指导RNA(gRNA)。例如，可以将包含编码gRNA和Cas蛋白的多核苷酸的复合物引入所述细胞中。例如，可以将包含gRNA和Cas蛋白的复合物引入所述细胞中。
例如，所述gRNA可以用于与所述目标基因的序列结合。例如，所述gRNA与所述目标基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目标基因的序列。例如，所述gRNA与所述目标基因的序列的结合可以使得gRNA的 CRISPR系统特异性剪切所述目标基因。
例如，本发明的编辑靶标区域可以是启动子前的一段区域。例如，本发明的编辑靶标区域可以是转录因子结合力高的区域。例如，本发明的编辑靶标区域可以是具有特定数目的转录因子结合数的区域。例如，本发明的编辑靶标区域可以是具有约3个或以上的转录因子结合数的一段连续区域。
例如，当基因编辑系统包含CRISPR/Cas9时，本发明的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是AGG、TGG、GGG或CGG。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的5’端上游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5’端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，当基因编辑系统包含CRISPR/Cas12时，本发明的指导核酸分子靶向的区域上游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是NTTN、TTYN、VTTV、TRTV、TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)3’端下游约15至约25个核苷酸组成的序列结合：NTTN、TTYN、VTTV、TRTV、TTTV、TATV、 TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。
例如，当本发明的基因编辑系统包含野生型Cas12a(也可以称为Cpf1，如AsCas12a，FnCas12a，LbCas12a，BbCas12a，CMaCas12a和OsCas12a)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：NTTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如enAsCas12a(突变位点E174R、S542R和K548R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTYN(TTTN/TTCN)，VTTV(ATTV/CTTV/GTTV)，或TRTV(TATV/TGTV)，其中N可以为A、T、C或G，Y可以为T或C，V可以为A、C或G，R可以为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如opAsCas12a(突变位点：E174R和S542R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV(TTTA，TTTC，或TTTG)，其中V可以为A、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如AsCas12aUltra(突变位点：M537R和F870L)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV，TATV，或TYCV，其中V可以为A、C或G，Y可以为T或C。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如hfCas12Max(突变位点：N243R/E336R/D892R)和Cas12Max(突变位点：N243R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TNN、或NTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，所述指导核酸分子可以包含能够结合编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约 19个、或约15至约17个核苷酸组成的靶序列。
例如，靶序列可以是选自表1C所示的基因组坐标定义的区域或其片段。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域与选自以下组的靶序列互补：SEQ ID NO：399-448、4393-5086。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：349-398、3699-4392、7346-7375、7418中所示的序列。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：7346-7375、7418中所示的序列。
例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例可以降低和/或单个细胞中所述目的基因的表达量可以下降。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低至少约5％。例如，表达所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因的产物的细胞比例降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，表达所述编码STAT诱导的STAT抑 制剂(SSI)家族成员和/或其功能活性片段的基因的产物的细胞比例可以从可以观测的细胞比例到0％。例如，表达所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因的产物的细胞比例可以降低到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因的产物的细胞比例可以为至多约95％。例如，表达所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因的产物的细胞比例的细胞比例可以为至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以下降至少约5％。例如，单个细胞中所述目的基因的表达量可以下降至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约 40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目的基因的表达量可以从可以观测的量到0％。例如，，单个细胞中所述目的基因的表达量可以下降到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以为所述目标基因的表达和/或活性未改变的细胞的至多约95％。例如，细胞中单个细胞中所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的基因(例如编码SOCS1的基因)表达量可以为所述编码STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性未改变的细胞的至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
例如，本发明的方法包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述细胞的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱。
例如，使所述源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤 转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱。
例如，所述第一阶段体外扩增进行至少约7天。例如，所述第二阶段体外扩增进行至少约7天。
例如，可以在单个阶段的本发明的体外扩增中，使所述细胞与所述一种或多种细胞激活剂接触以及使所述细胞中包含STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达和/或活性降低。例如，细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，在单个阶段的所述体外扩增中，使本发明的细胞的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第一阶段体外扩增中，可以使本发明的TIL与本发明的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。
例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞基本上同时使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小 时、或提前48小时等，再与本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先与本发明的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱。
例如，在本发明第一阶段体外扩增中，可以使本发明的TIL基本上同时使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL基本上同时使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL基本上同时使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本发明的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本发明的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本发明的经第一阶段体外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本发明的经第二阶段体 外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本发明的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本发明的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种细胞生长因子接触、与多种细胞激活剂接触、使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与细胞生长因子接触、与细胞激活剂接触、使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱且使所述TIL与饲养细胞共培养，STAT诱导的STAT抑制剂(SSI)家族成员可以包含SOCS1；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织、胸腔积液和/或腹腔积液在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方 毫米大小，转移入透气培养袋或Grex中，加入细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，细胞无血清培养基可以添加本发明的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2以及使STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱(STAT诱导的STAT抑制剂(SSI)家族成员可以包含SOCS1，例如可以通过用携带包含本发明的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)进行转导使所述TIL中编码STAT诱导的STAT抑制剂(SSI)家族成员的基因的细胞比例为约95％或以下)，活化本发明的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的细胞可以为记忆效应细胞和效应细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化细胞比例上调的特征。
CBLB敲除
1.一种培养细胞的方法，所述方法包含：使所述细胞的CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。
2.根据实施方案1所述的方法，其中所述细胞包含免疫细胞。
3.根据实施方案2所述的方法，其中所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
4.根据实施方案2-3中任一项所述的方法，其中所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
5.根据实施方案2-4中任一项所述的方法，其中所述免疫细胞来源于干细胞分化的免疫细胞。
6.根据实施方案5所述的方法，其中所述干细胞包含诱导的多能干细胞(iPSC)。
7.根据实施方案2-6中任一项所述的方法，其中所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。
8.根据实施方案2-7中任一项所述的方法，其中所述免疫细胞包含αβT细胞和/或γδT细胞。
9.根据实施方案2-8中任一项所述的方法，其中所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
10.根据实施方案9所述的方法，其中所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
11.根据实施方案10所述的方法，其中所述碎片的体积为约1立方毫米至约27立方毫米。
12.根据实施方案2-11中任一项所述的方法，其中所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
13.根据实施方案12所述的方法，其中所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
14.根据实施方案2-13中任一项所述的方法，其中所述免疫细胞 包含嵌合抗原受体和/或T细胞受体。
15.根据实施方案1-14中任一项所述的方法，其中使所述细胞的CBL家族成员的表达降低和/或活性减弱包含抑制E3泛素蛋白连接酶的功能。
16.根据实施方案1-15中任一项所述的方法，其中与CBL家族成员的表达和/或活性未改变的细胞相比，使所述CBL家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
17.根据实施方案16所述的方法，其中所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
18.根据实施方案17所述的方法，其中所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
19.根据实施方案1-18中任一项所述的方法，其中所述CBL家族成员包含SH3域。
20.根据实施方案1-19中任一项所述的方法，其中所述CBL家族成员包含CBLB。
21.根据实施方案1-20中任一项所述的方法，其中使所述细胞的CBL家族成员的表达降低和/或活性减弱包含将基因调控系统引入所述细胞中。
22.根据实施方案21所述的方法，其中所述基因调控系统能够在DNA水平破坏所述CBL家族成员。
23.根据实施方案21-22中任一项所述的方法，其中所述基因调控系统包含指导核酸分子和酶蛋白。
24.根据实施方案23所述的方法，其中使所述CBL家族成员的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)、包含gRNA与Cas蛋白的LNP，或者包含编码gRNA与编码Cas蛋白的核酸的LNP引入所述细胞中。
25.根据实施方案23-24中任一项所述的方法，其中所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
26.根据实施方案23-25中任一项所述的方法，其中所述指导核酸分子包含指导RNA(gRNA)。
27.根据实施方案23-26中任一项所述的方法，其中所述指导核酸分子能够与所述CBL家族成员的序列结合。
28.根据实施方案23-27中任一项所述的方法，其中所述指导核酸分子能够与选自表1D所示的基因组坐标定义的区域或其片段结合。
29.根据实施方案23-28中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：520-590、6177-7266。
30.根据实施方案23-29中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、CGG和GGG。
31.根据实施方案23-30中任一项所述的方法，其中所述指导核酸分子包括靶向结构域，所述靶向结构域包含如SEQ ID NO：449-519、5087-6176、7376-7413、7414、7415中任一项所示的序列。
32.根据实施方案1-31中任一项所述的方法，其中与CBL家族成员的表达和/或活性未改变的细胞相比，使所述CBL家族成员的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低和/或单个细胞中所述目的基因的表达量下降。
33.根据实施方案1-32中任一项所述的方法，其中使所述CBL家族成员的表达降低和/或活性减弱获得的细胞中，表达所述目的基因的细胞比例为约95％或以下。
34.一种细胞，所述细胞经过实施方案1-33中任一项所述的方法获得。
35.一种组合物，其包含实施方案34所述的细胞。
36.一种药物组合物，其包含实施方案34所述的细胞和/或实施方案35所述的组合物，以及任选的药学上可接受的载体。
37.一种影响细胞生长的方法，其包含施用实施方案34所述的细胞、实施方案35所述的组合物和/或实施方案36所述的药物组合物。
38.实施方案34所述的细胞、实施方案35所述的组合物和/或实施方案36所述的药物组合物在制备药物中的应用，其中所述药物用于预防和/或治疗疾病和/或症状。
39.根据实施方案38所述的应用，其中所述疾病和/或症状包含肿 瘤。
40.根据实施方案38-39中任一项所述的应用，其中所述疾病和/或症状包含实体瘤。
41.根据实施方案38-40中任一项所述的应用，其中所述疾病和/或症状包含选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
本发明提供了使所述细胞的CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱的方法。
一方面，本发明提供一种培养细胞的方法，使所述细胞的CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，所述CBL家族成员可以包含SH3域。例如，所述CBL家族成员可以包含CBLB。
例如，本发明的目标基因可以是编码CBL家族成员和/或其功能活性片段的基因。例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞可以显示出改善的细胞特性。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的至少一种目标基因的表达降低和/或活性减弱的细胞。在一种实施方式中，目标基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的目标基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的细胞。
例如，所述细胞包含免疫细胞。例如，所述细胞包含免疫效应细胞。例如，所述细胞包含免疫效应T细胞、免疫效应NK细胞、免疫 效应NKT细胞。例如，所述细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
例如，所述细胞包含单核细胞、巨噬细胞和/或树突状细胞。
例如，本发明的细胞还包含来源于干细胞分化的细胞。例如，本发明的细胞还包含来源于多能干细胞分化的细胞。例如，获取本发明的干细胞可以是通过诱导产生。例如，本发明的上述干细胞可以包含诱导的多能干细胞(iPSC)、胚胎干细胞、骨髓干细胞、脐带血干细胞和/或外周血干细胞。
例如，本发明的“干细胞”还包括多能细胞，多潜能细胞，前体细胞和祖细胞。例如，干细胞可以从获自骨髓组织的造血或间充质干细胞、获自胎盘组织的胎盘干细胞、获自胚胎组织的胚胎干细胞或获自胎儿的生殖组织的胚胎生殖细胞获得。示例性多能干细胞还可以通过与多能性相关的某些转录因子的表达将其重编程为多能状态而由体细胞产生；这些细胞被称为“诱导性多能干细胞”或“iPSC”。
例如，所述细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。例如，“未修饰的细胞”或“未改造的细胞”可以是指其中基因组未被修饰并且不包含基因调控系统或包含对照基因调控系统(例如，空载体对照、非靶向gRNA、干扰siRNA等)的细胞或细胞群体。例如，所述细胞包含αβT细胞和/或γδT细胞。例如，所述细胞包含肿瘤浸润淋巴细胞(TIL)。例如，所述TIL为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
例如，本发明的TIL可以为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或 腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。例如，可以通过将肿瘤组织处理成肿瘤碎片获得本发明的TIL。例如，本发明的肿瘤碎片的体积约为1-27立方毫米。例如，本发明的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约14立方毫米、约15立方毫米、约16立方毫米、约17立方毫米、约18立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。
例如，所述细胞包含展示在细胞表面上的工程化免疫受体。例如，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。例如，所述细胞包含嵌合抗原受体和/或T细胞受体。
一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，可以包含：使所述TIL中包含CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以使所述TIL中包含CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且 未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第三阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低，且在本 发明的第二阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第二阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低，且在本发明的第三阶段体外扩增中，可以使所述TIL中CBL家族成员和/或其功能活性片段的表达和/或活性降低。
例如，与CBL家族成员的表达和/或活性未改变的细胞相比，使所述CBL家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
例如，本发明的改善的细胞数量是指与CBL家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述CBL家族成员的表达降低和/或活性减弱的本发明细胞的细胞数量可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，增加的活细胞比例可以表现为细胞活率的增加。例如，本发明的增加的活细胞比例可以是指与CBL家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述CBL家族成员的表达降低和/或活性减弱的本发明细胞的活细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的细胞因子分泌能力可以是指细胞的选自以下组的细胞因子分泌能力提高：IL-2、IL-6、CD107a、GZMB、TNF-α和IFN-γ。例如，本发明的提高的细胞因子分泌能力可以是指与CBL家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述CBL家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的细胞因子分泌 能力可以是指与CBL家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述CBL家族成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的提高的肿瘤细胞杀伤能力可以是指与CBL家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述CBL家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的肿瘤细胞杀伤能力可以是指与CBL家族成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述CBL家族成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本发明的细胞的肿瘤细胞杀伤率可以通过IncuCyte系统或者CFSE和DAPI染色法测量。例如，本发明的细胞的肿瘤细胞 杀伤可以是指细胞杀伤实体瘤细胞的能力。
例如，本发明的改善的细胞亚群比例可以包含选自以下组的一种或多种：增加的CD8+细胞比例，增加的中心记忆细胞和/或幼稚细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和增加的干细胞样细胞比例。
例如，在细胞中CD8+细胞、中心记忆细胞和/或幼稚细胞、活化细胞、肿瘤特异性细胞和/或干细胞样细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的降低的耗竭细胞比例可以是细胞中PD-1+、LAG-3+、TIM-3+、和/或CD39+细胞的比例的增加。例如，本发明的减少的调节性细胞的比例可以是细胞中CD4+CD25+Foxp3+细胞的比例的减少。例如，本发明的减少的凋亡细胞的比例可以是细胞中CD95+caspass3+细胞和/或CD95+DR5+细胞的比例的减少。
例如，在细胞中耗竭细胞、调节性细胞和/或凋亡细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4 ％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本发明的培养方法可以包含对于细胞的基因编辑步骤。例如，其包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以将基因调控系统引入所述细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目标基因。例如，所述基因调控系统可以破坏所述细胞的基因组中的所述目标基因的区域或其片段。例如，使用所述基因调控系统后，细胞中的所述目标基因所在的DNA区域或其片段被剪切而该目标基因的表达能力降低或该目标基因的活性被抑制。例如，所述基因调控系统对目标基因的编辑效果可以是长期的、持续的。其中本发明所述基因组区域根据人类参考基因组hg38版本确定的。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所述酶蛋白特异性剪切目的基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。例如，可以将编码gRNA和Cas蛋白的多核苷酸引入、或各自独立地单独引入靶细胞。
例如，本发明使细胞的至少一种目标基因的表达降低和/或活性减弱可以包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述细胞中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。例如，所述指导核酸分子可以包含指导RNA (gRNA)。例如，可以将包含编码gRNA和Cas蛋白的多核苷酸的复合物引入所述细胞中。例如，可以将包含gRNA和Cas蛋白的复合物引入所述细胞中。
例如，所述gRNA可以用于与所述目标基因的序列结合。例如，所述gRNA与所述目标基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目标基因的序列。例如，所述gRNA与所述目标基因的序列的结合可以使得gRNA的CRISPR系统特异性剪切所述目标基因。
例如，本发明的编辑靶标区域可以是启动子前的一段区域。例如，本发明的编辑靶标区域可以是转录因子结合力高的区域。例如，本发明的编辑靶标区域可以是具有特定数目的转录因子结合数的区域。例如，本发明的编辑靶标区域可以是具有约3个或以上的转录因子结合数的一段连续区域。
例如，当基因编辑系统包含CRISPR/Cas9时，本发明的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是AGG、TGG、GGG或CGG。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的5’端上游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5’端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，当基因编辑系统包含CRISPR/Cas12时，本发明的指导核酸分子靶向的区域上游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是NTTN、TTYN、VTTV、TRTV、 TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)3’端下游约15至约25个核苷酸组成的序列结合：NTTN、TTYN、VTTV、TRTV、TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。
例如，当本发明的基因编辑系统包含野生型Cas12a(也可以称为Cpf1，如AsCas12a，FnCas12a，LbCas12a，BbCas12a，CMaCas12a和OsCas12a)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：NTTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如enAsCas12a(突变位点E174R、S542R和K548R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTYN(TTTN/TTCN)，VTTV(ATTV/CTTV/GTTV)，或TRTV(TATV/TGTV)，其中N可以为A、T、C或G，Y可以为T或C，V可以为A、C或G，R可以为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如opAsCas12a(突变位点：E174R和S542R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV(TTTA，TTTC，或TTTG)，其中V可以为A、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如AsCas12aUltra(突变位点：M537R和F870L)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV，TATV，或TYCV，其中V可以为A、C或G，Y可以为T或C。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如hfCas12Max(突变位点：N243R/E336R/D892R)和Cas12Max(突变位点：N243R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TNN、或NTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，所述指导核酸分子可以包含能够结合编码CBL家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导 核酸分子可以包含能够结合编码CBL家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。
例如，靶序列可以是选自表1D所示的基因组坐标定义的区域或其片段。例如，本发明的靶序列可以是CBLB的PTB结构功能域。例如，本发明的靶序列可以是CBLB的锌指结构功能域。例如，本发明的靶序列可以是CBLB的UBA结构功能域。例如，本发明的靶序列可以是chr3:105658893-105659215、chr3:105720184-105720231、chr3:105720234-105720278、chr3:105720300-105720327、chr3:105720333-105720375、chr3:105723918-105723956、chr3:105724025-105724052、chr3:105724100-105724131、chr3:105724134-105724219、chr3:105724232-105724310、chr3:105733918-105733970、chr3:105733988-105734073、chr3:105734082-105734109、chr3:105737162-105737268、chr3:105740404-105740449、chr3:105740470-105740551、chr3:105740563-105740646、chr3:105740707-105740734、chr3:105745796-105745885、chr3:105745896-105745947、chr3:105745963-105746060、chr3:105749514-105749557、chr3:105749570-105749653、chr3:105749657-105749701、chr3:105749751-105749827、chr3:105749860-105749909、chr3:105751528-105751641、chr3:105751674-105751705、chr3:105754228-105754312、chr3:105754319-105754376、chr3:105754386-105754419、chr3:105754544-105754599、chr3:105776307-105776394、chr3:105776402-105776557、 chr3:105776632-105776662、chr3:105824008-105824362、chr3:105839348-105839393、chr3:105839418-105839485、chr3:105839533-105839704、chr3:105853309-105853347、chr3:105853445-105853472、chr3:105853571-105853598、chr3:105867293-105867352、chr3:105867358-105867389。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域与选自以下组的靶序列互补：SEQ ID NO：520-590、6177-7266。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：449-519、5087-6176、7376-7413、7414、7415中所示的序列。
例如，所述指导核酸分子可以包括靶向结构域，所述靶向结构域可以包含如SEQ ID NO：7376-7413、7414、7415中所示的序列。
例如，与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例可以降低和/或单个细胞中所述目的基因的表达量可以下降。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低至少约5％。例如，表达所述编码CBL家族成员和/或其功能活性片段的基因的产物的细胞比例降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例 如，表达所述编码CBL家族成员和/或其功能活性片段的基因的产物的细胞比例可以从可以观测的细胞比例到0％。例如，表达所述编码CBL家族成员和/或其功能活性片段的基因的产物的细胞比例可以降低到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述编码CBL家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中表达所述编码CBL家族成员和/或其功能活性片段的基因的产物的细胞比例可以为至多约95％。例如，表达所述编码CBL家族成员和/或其功能活性片段的基因的产物的细胞比例的细胞比例可以为至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述编码CBL家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中与目标基因的表达和/或活性未改变的细胞相比，使所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以下降至少约5％。例如，单个细胞中所述目的基因的表达量可以下降至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至 少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目的基因的表达量可以从可以观测的量到0％。例如，，单个细胞中所述目的基因的表达量可以下降到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。
例如，本发明的方法中所述细胞的至少一种目标基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以为所述目标基因的表达和/或活性未改变的细胞的至多约95％。例如，细胞中单个细胞中所述编码CBL家族成员和/或其功能活性片段的基因(例如编码CBLB的基因)表达量可以为所述编码CBL家族成员和/或其功能活性片段的表达和/或活性未改变的细胞的至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
例如，本发明的方法包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述细胞的CBL家族成员的表达降低和/或活性减弱。
例如，使所述源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL的CBL 家族成员的表达降低和/或活性减弱。
例如，所述第一阶段体外扩增进行至少约7天。例如，所述第二阶段体外扩增进行至少约7天。
例如，可以在单个阶段的本发明的体外扩增中，使所述细胞与所述一种或多种细胞激活剂接触以及使所述细胞中包含CBL家族成员和/或其功能活性片段的表达和/或活性降低。例如，细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，在单个阶段的所述体外扩增中，使本发明的细胞的CBL家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第一阶段体外扩增中，可以使本发明的TIL与本发明的CBL家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL的CBL家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL的CBL家族成员的表达降低和/或活性减弱且与本发明的一种或多种细胞激活剂接触。
例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞基本上同时使CBL家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先使CBL家族成员的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先与本发明的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使CBL家族成员的表达降低和/或活性减弱。
例如，在本发明第一阶段体外扩增中，可以使本发明的TIL基本上同时使CBL家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL基本上同时使CBL家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL基本上同时使CBL家族成员的表达降低和/或活性减弱以及本发明的一种或多种细胞激活剂接触。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群CBL家族成员的表达降低和/或活性减弱；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本发明的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本发明的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本发明的经第一阶段体外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本发明的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本发明的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本发明的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积 液且未经体外扩增的第一TIL群与多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种细胞生长因子接触、与多种细胞激活剂接触、使CBL家族成员的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与细胞生长因子接触、与细胞激活剂接触、使CBL家族成员的表达降低和/或活性减弱且使所述TIL与饲养细胞共培养，CBL家族成员可以包含CBLB；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织、胸腔积液和/或腹腔积液在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，细胞无血清培养基可以添加本发明的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2以及使CBL家族成员的表达降低和/或活性减弱(CBL家族成员可以包含CBLB，例如可以通过用携带包含本发明的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)进行 转导使所述TIL中编码CBL家族成员的基因的细胞比例为约95％或以下)，活化本发明的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的细胞可以为记忆效应细胞和效应细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化细胞比例上调的特征。
靶点组合敲除
1.一种培养细胞的方法，所述方法包含：使所述细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族成员和/或其功能活性片段的表达降低和/或活性减弱。
2.根据实施方案1所述的方法，其中所述细胞包含免疫细胞。
3.根据实施方案2所述的方法，其中所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
4.根据实施方案2-3中任一项所述的方法，其中所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
5.根据实施方案2-4中任一项所述的方法，其中所述免疫细胞来源于干细胞分化的免疫细胞。
6.根据实施方案5所述的方法，其中所述干细胞包含诱导的多能干细胞(iPSC)、胚胎干细胞、骨髓干细胞、脐带血干细胞和/或外周血干细胞。
7.根据实施方案2-6中任一项所述的方法，其中所述免疫细胞包 含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。
8.根据实施方案2-7中任一项所述的方法，其中所述免疫细胞包含αβT细胞和/或γδT细胞。
9.根据实施方案2-8中任一项所述的方法，其中所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
10.根据实施方案9所述的方法，其中所述TIL为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
11.根据实施方案10所述的方法，其中所述碎片的体积为约1立方毫米至约27立方毫米。
12.根据实施方案2-11中任一项所述的方法，其中所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
13.根据实施方案12所述的方法，其中所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
14.根据实施方案2-13中任一项所述的方法，其中所述免疫细胞包含嵌合抗原受体和/或T细胞受体。
15.根据实施方案1-14中任一项所述的方法，其中所述使细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族成员和/或其功能活性片段的表达降低和/或活性减弱包含至少2个选自以下组的效果：抑制去泛素化酶和/或锌指核酸酶的功能、抑制核酸酶的功能、抑制细胞因子信号负调节的功能和抑制E3泛素蛋白连接酶的功能。
16.根据实施方案1-15中任一项所述的方法，其中与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族成员和/或其功能活性片段的表达降低和/或活性未改变的细胞相比，使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族成员和/或其功能活性片段的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
17.根据实施方案16所述的方法，其中所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力、提高的体外肿瘤细胞杀伤能力和提高的体内肿瘤杀伤能力。
18.根据实施方案17所述的方法，其中所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
19.根据实施方案1-18中任一项所述的方法，其中所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族的家族成员分别包含泛素结合域、C3H1型锌指结构域、SH2域和SH3域。
20.根据实施方案1-19中任一项所述的方法，其中所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族的家族成员分别包含TNFAIP3、ZC3H12A、SOCS1和CBLB。
21.根据实施方案1-20中任一项所述的方法，其中使所述细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI) 家族和CBL家族的家族成员的表达降低和/或活性减弱包含将基因调控系统引入所述细胞中。
22.根据实施方案21所述的方法，其中所述基因调控系统在DNA水平破坏所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族的家族成员；以及任选地，所述细胞选自BRD4、FAS、FIBP、IKZF1、LAG3、MED12、PD1、RASA2、TIGIT、TIM3、ADNP、NFKBIA、PTPN6和TNIP1的表达降低和/或活性减弱。
23.根据实施方案21-22中任一项所述的方法，其中所述基因调控系统包含指导核酸分子和酶蛋白。
24.根据实施方案23所述的方法，其中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族的家族成员的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的复合物或者包含所述指导核酸分子和编码所述酶蛋白的核酸的复合物、包含gRNA与Cas蛋白的LNP，或者包含编码gRNA与编码Cas蛋白的核酸的LNP引入所述细胞中。
25.根据实施方案23-24中任一项所述的方法，其中所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段，优选地选自Cas 9和Cas 12。
26.根据实施方案23-25中任一项所述的方法，其中所述指导核酸分子包含指导RNA(gRNA)。
27.根据实施方案23-26中任一项所述的方法，其中所述指导核酸分子与所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个的家族成员的序列结合。
28.根据实施方案23-27中任一项所述的方法，其中所述指导核酸分子与选自以下组所示的原型间隔序列毗邻基序(PAM)5’端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、CGG和GGG，或者与选自以下组所示的原型间隔序列毗邻基序(PAM)3’端下游约15至约25个核苷酸组成的序列结合：NTTN、TTYN、VTTV、TRTV、TTTV、TATV、TYCV、TNN、和NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。
29.根据实施方案23-28中任一项所述的方法，其中所述指导核酸分子与选自表2A-2D或表3A-3D所示的基因组坐标定义的区域或其片段结合。
30.根据实施方案23-29中任一项所述的方法，其中所述指导核酸分子与选自以下组所示的至少2个区域或其片段结合：SEQ ID NO：107-212、1562-2532，SEQ ID NO：281-348、3116-3698，SEQ ID NO：399-448、4393-5086，和SEQ ID NO：520-590、6177-7266。
31.根据实施方案23-30中任一项所述的方法，其中所述指导核酸分子包括靶向结构域，所述靶向结构域包含如SEQ ID NO：1-106、591-1561、7267-7324、7419、7420，SEQ ID NO：213-280、2533-3115、7325-7345、7416、7417，SEQ ID NO：349-398、3699-4392、7346-7375、7418，和SEQ ID NO：449-519、5087-6176、7376-7413、7414、7415中至少2个所示的序列。
32.根据实施方案1-31中任一项所述的方法，其中与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族的家族成员的表达和/或活性未改变的细胞相比，使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族的家族成员的表达降低和/或活性减弱获得的细胞中表达目的基因的细胞比例降低和/或单个细胞中目的基因的表达量下降。
33.根据实施方案1-32中任一项所述的方法，其中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族的家族成员的表达降低和/或活性减弱获得的细胞中，表达目的基因的细胞比例为约95％以下。
34.一种细胞，所述细胞经过实施方案1-33中任一项所述的方法获得。
35.一种药物组合物，其包含实施方案34所述的细胞，以及任选的药学上可接受的载体。
36.一种影响细胞生长的方法，其包含施用实施方案34所述的细胞和/或实施方案35所述的药物组合物。
37.实施方案34所述的细胞和/或实施方案35所述的药物组合物在制备药物中的应用，所述药物用于预防和/或治疗疾病和/或症状。
38.一种药物，其用于预防和/或治疗疾病和/或症状，包含实施方案34所述的细胞和/或实施方案35所述的药物组合物作为活性成分。
39.一种预防和/或治疗疾病和/或症状的方法，其包含给需要的受试者施用实施方案34所述的细胞和/或实施方案35所述的药物组合物。
40.实施方案34所述的细胞和/或实施方案35所述的药物组合物，其用途为预防和/或治疗疾病和/或症状。
41.根据实施方案37所述的应用，根据实施方案38所述的药物，根据实施方案39所述的方法，和/或根据实施方案40所述用途的细胞和/或药物组合物，其中所述疾病和/或症状包含肿瘤。
42.根据实施方案37所述的应用，根据实施方案38所述的药物，根据实施方案39所述的方法，和/或根据实施方案40所述用途的细胞和/或药物组合物，其中所述疾病和/或症状包含实体瘤。
43.根据实施方案37所述的应用，根据实施方案38所述的药物，根据实施方案39所述的方法，和/或根据实施方案40所述用途的细胞和/或药物组合物，其中所述疾病和/或症状包含选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
本发明提供了使所述细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达降低和/或活性减弱的方法。
一方面，本发明提供一种培养细胞的方法，使所述细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达降低和/或活性减弱。
例如，所述细胞进一步可以包含任选地选自BRD4、FAS、FIBP、IKZF1、LAG3、MED12、PD1、RASA2、TIGIT、TIM3、ADNP、NFKBIA、PTPN6和TNIP1的表达降低和/或活性减弱。
例如，所述肽酶C64家族成员可以包含泛素结合域。例如，所述肽酶C64家族成员可以包含TNFAIP3。
例如，所述ZC3H12家族成员可以包含C3H1型锌指结构域。例如，所述ZC3H12家族成员可以包含ZC3H12A。
例如，所述STAT诱导的STAT抑制剂(SSI)家族成员可以包含SH2域。例如，所述STAT诱导的STAT抑制剂(SSI)家族成员可以包含SOCS1。
例如，所述CBL家族成员可以包含SH3域。例如，所述CBL家族成员可以包含CBLB。
例如，在本发明的细胞中，使肽酶C64家族和ZC3H12家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使TNFAIP3和ZC3H12A的表达降低和/或活性减弱。
例如，在本发明的细胞中，使肽酶C64家族和STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使TNFAIP3和SOCS1的表达降低和/或活性减弱。
例如，在本发明的细胞中，使肽酶C64家族和CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使TNFAIP3和CBLB的表达降低和/或活性减弱。
例如，在本发明的细胞中，使ZC3H12家族和STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使ZC3H12A和SOCS1的表达降低和/或活性减弱。
例如，在本发明的细胞中，使ZC3H12家族和CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使ZC3H12A和CBLB的表达降低和/或活性减弱。
例如，在本发明的细胞中，使STAT诱导的STAT抑制剂(SSI)家族和CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使SOCS1和CBLB的表达降低和/或活性减弱。
例如，在本发明的细胞中，使肽酶C64家族、ZC3H12家族和STAT诱导的STAT抑制剂(SSI)家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使TNFAIP3、ZC3H12A和SOCS1的表达降低和/或活性减弱。
例如，在本发明的细胞中，使肽酶C64家族、ZC3H12家族和CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使TNFAIP3、ZC3H12A和CBLB的表达降低和/或活性减弱。
例如，在本发明的细胞中，使肽酶C64家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使TNFAIP3、SOCS1和CBLB的表达降低和/或活性减弱。
例如，在本发明的细胞中，使ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使ZC3H12A、SOCS1和CBLB的表达降低和/或活性减弱。
例如，在本发明的细胞中，使肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，使TNFAIP3、ZC3H12A、SOCS1和CBLB的表达降低和/或活性减弱。
例如，本发明的目的基因可以是编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因。例如，与目的基因的表达和/或活性未改变的细胞相比，使所述细胞的至少2个目的基因的表达降低和/或活性减弱获得的细胞可以显示出改善的细胞特性。在一种实 施方式中，目的基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的细胞。在一种实施方式中，目的基因的表达和/或活性未改变的细胞可以是指源自同一供体的且未曾使所述细胞的目的基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的细胞。
在一种实施方式中，未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的相应细胞可以是指源自同一供体的经过同样方式分离的且未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的细胞。在一种实施方式中，未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的相应细胞可以是指源自同一供体的同一肿瘤来源的且未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的细胞。在一种实施方式中，未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的相应细胞可以是指将源自同一供体的同一肿瘤来源的细胞分为两组，其中一组未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的细胞可以为未曾使所述细胞的至少2个目的基因的表达降低和/或活性减弱的相应细胞。例如，至少2个目的基因的表达降低和/或活性减弱可以是指天然的细胞的该目的基因的处于一定程度的表达状态，经过本发明的处理，可以使得该细胞的该目的基因的表达量降低，即该目的基因的表达量降低可以是使天然细胞从表达该目的基因转变为基本不表达该目的基因或表达该目的基因的量降低。
例如，所述细胞包含免疫细胞。例如，所述细胞包含免疫效应细胞。例如，所述细胞包含免疫效应T细胞、免疫效应NK细胞、免疫效应NKT细胞。例如，所述细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
例如，所述细胞包含单核细胞、巨噬细胞和/或树突状细胞。
例如，本发明的细胞还包含来源于干细胞分化的细胞。例如，本发明的细胞还包含来源于多能干细胞分化的细胞。例如，获取本发明的干细胞可以是通过诱导产生。例如，本发明的上述干细胞可以包含诱导的多能干细胞(iPSC)、胚胎干细胞、骨髓干细胞、脐带血干细胞和/或外周血干细胞。
例如，本发明的“干细胞”还包括多能细胞，多潜能细胞，前体细胞和祖细胞。例如，干细胞可以从获自骨髓组织的造血或间充质干细胞、获自胎盘组织的胎盘干细胞、获自胚胎组织的胚胎干细胞或获自胎儿的生殖组织的胚胎生殖细胞获得。示例性多能干细胞还可以通过与多能性相关的某些转录因子的表达将其重编程为多能状态而由体细胞产生；这些细胞被称为“诱导性多能干细胞”或“iPSC”。
例如，所述细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。例如，“未修饰的细胞”或“未改造的细胞”可以是指其中基因组未被修饰并且不包含基因调控系统或包含对照基因调控系统(例如，空载体对照、非靶向gRNA、干扰siRNA等)的细胞或细胞群体。例如，所述细胞包含αβT细胞和/或γδT细胞。例如，所述细胞包含肿瘤浸润淋巴细胞(TIL)。例如，所述TIL为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
例如，本发明的TIL可以为源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。例如，可以通过将肿瘤组织处理成肿瘤碎片获得本发明的TIL。例如，本发明的肿瘤碎片的体积约为1-27立方毫米。例如，本发明的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方 毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约14立方毫米、约15立方毫米、约16立方毫米、约17立方毫米、约18立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。
例如，所述细胞包含展示在细胞表面上的工程化免疫受体。例如，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。例如，所述细胞包含嵌合抗原受体和/或T细胞受体。
一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，可以包含：使所述TIL中包含选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以使所述TIL中包含选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有 或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低，以及在本发明的第二阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使 所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第三阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低，以及在本发明的第二阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低，以及在本发明的第三阶段体外扩增中， 可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第二阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低，以及在本发明的第三阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本发明的源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本发明的第一阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低，及在本发明的第二阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低，以及在本发明的第三阶段体外扩增中，可以使所述TIL中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。
例如，每一个阶段体外扩增之间可以是通过TIL细胞数量的变化 来划分的，例如，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1-1000倍时，例如至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、至少约50倍、至少约100倍、至少约200倍、至少约500倍、或者至少约1000倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段的体外扩增之间也可以是通过TIL细胞培养的条件的变化来划分的。例如，当细胞培养基中添加了或补充添加了细胞激活剂和/或细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了IL-2后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了一种或多种基因调控系统后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了饲养细胞后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当TIL细胞进行了离心和/或细胞洗涤的操作后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。例如，当TIL细胞体外培养约1-100天后，例如约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
例如，其中所述使所述细胞的肽酶C64家族成员的表达降低和/或活性减弱包含抑制去泛素化酶和/或锌指核酸酶的功能。
例如，其中所述使所述细胞的ZC3H12家族成员的表达降低和/或活性减弱包含抑制核酸酶的功能。
例如，其中所述使所述细胞的STAT诱导的STAT抑制剂(SSI)家族成员的表达降低和/或活性减弱包含抑制细胞因子信号负调节的功能。
例如，其中所述使所述细胞的CBL家族成员的表达降低和/或活性减弱包含抑制E3泛素蛋白连接酶的功能。
例如，与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达和/或活性未改变的细胞相比，使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
例如，所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力(即细胞数量)、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力、提高的体外肿瘤细胞杀伤能力和提高的体内肿瘤杀伤能力。
例如，所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
例如，本发明改善的细胞数量是指与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱的本发明细胞的细胞数量可以增加至少约1-50倍，例如至 少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，增加的活细胞比例可以表现为细胞存活率的增加。例如，本发明增加的活细胞比例可以是指与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱的本发明细胞的活细胞比例可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明提高的细胞因子分泌能力可以是指细胞的选自以下组的细胞因子分泌能力提高：IL-2、IL-6、CD107a、GZMB、TNF-α和IFN-γ。例如，本发明提高的细胞因子分泌能力可以是指与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约1-50倍，例如至少约1倍、至少约2倍、至少约 3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明的提高的细胞因子分泌能力可以是指与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱的本发明细胞中分泌细胞因子的细胞比例可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本发明的细胞的细胞因子分泌能力是通过流式细胞术或者CBA法(Cytometric Bead Array)测定。
例如，本发明提高的体外肿瘤细胞杀伤能力和/或提高的体内肿瘤杀伤能力可以是指与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约1-50倍，例如至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20 倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本发明提高的体外肿瘤细胞杀伤能力和/或提高的体内肿瘤杀伤能力可以是指与选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达和/或活性未改变的细胞相比，在至少一个体外扩增阶段中使所述选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱的本发明细胞的肿瘤细胞杀伤率可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本发明细胞的肿瘤细胞杀伤率可以通过IncuCyte系统或者CFSE和DAPI染色法测量。例如，本发明细胞的肿瘤细胞杀伤可以是指细胞杀伤实体瘤细胞的能力。
例如，本发明改善的细胞亚群比例可以包含选自以下组的一种或多种：增加的CD8+细胞比例，增加的中心记忆细胞和/或幼稚细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例(具有CD103+CD39+的表型)，增加的干细胞样细胞比例，降低的耗竭细胞的比例，降低的凋亡细胞的比例。
例如，本发明增加的CD8+细胞比例可以是细胞中CD8阳性细胞的比例的增加。例如，在细胞中CD8+细胞比例可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至 少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明增加的活化细胞比例可以是细胞中CD28+、CD25+和/或41BB+细胞的比例的增加。例如，在细胞中活化细胞比例可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1-50倍，例如至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本发明降低的耗竭细胞比例可以是细胞中PD-1+、LAG-3+、TIM-3+、CD39+、CD38+和/或CD101+细胞的比例的增加。例如，在细胞中耗竭细胞比例可以降低至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少 约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1-50倍，例如至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本发明降低的调节性细胞的比例可以是细胞中CD4+CD25+Foxp3+细胞的比例的减少。例如，在细胞中调节性细胞比例可以减少至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明降低的凋亡细胞的比例可以是细胞中Annexin V+7-AAD+细胞和/或Annexin V+7-AAD-细胞的比例的减少。例如，在细胞中凋亡细胞比例可以减少至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明增加的具有干性的细胞的比例可以是细胞中CD69-CD39-细胞和/或TCF1+细胞的比例的增加。例如，在细胞中具有干性的细胞比例可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明增加的中心记忆细胞比例可以是细胞中CD45RA-CCR7+或者CD45RO+CD62L+细胞的比例的增加。例如，在细胞中中心记忆细胞比例可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明增加的幼稚T细胞比例可以是细胞中CD45RO-CD62L+细胞的比例的增加。例如，在细胞中幼稚细胞比例可以增加至少约100-0.1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少 约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.9％、至少约0.8％、至少约0.7％、至少约0.6％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本发明的培养方法可以包含对于细胞的基因编辑步骤。例如，其包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以将基因调控系统引入所述细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目的基因。例如，所述基因调控系统可以破坏所述细胞的基因组中的所述目的基因的区域或其片段。例如，使用所述基因调控系统后，细胞中的所述目的基因所在的DNA区域或其片段被剪切而该目的基因的表达能力降低或该目的基因的活性被抑制。例如，所述基因调控系统对目的基因的编辑效果可以是长期的、持续的。例如，本发明的细胞中选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的活性被抑制。
其中本发明所述基因组区域根据人类参考基因组hg38版本确定的。
例如，本发明的细胞中TNFAIP3与ZC3H12A的活性被抑制。例如，本发明的表1A和1B分别所示的细胞中TNFAIP3与ZC3H12A的优选亚区域被敲除和/或抑制。例如，本发明的表1A中TNFAIP3 R_1至30中与表1B中ZC3H12A R_1至9中所示的优选靶向亚区域两组合，组成的组合序号1至270的组合被敲除和/或抑制。
例如，本发明的细胞中TNFAIP3与SOCS1的活性被抑制。例如，本发明的表1A和1C分别所示的细胞中TNFAIP3与SOCS1的优选亚 区域被敲除和/或抑制。例如，本发明的表1A中TNFAIP3 R_1至30中与表1C中SOCS1 R_1至10中所示的优选靶向亚区域两组合，组成的组合序号271至570的组合被敲除和/或抑制。
例如，本发明的细胞中TNFAIP3与CBLB的活性被抑制。例如，本发明的表1A和1D分别所示的细胞中TNFAIP3与CBLB的优选亚区域被敲除和/或抑制。例如，本发明的表1A中TNFAIP3 R_1至30中与表1D中CBLB R_1至44中所示的优选靶向亚区域两组合，组成的组合序号571至1890的组合被敲除和/或抑制。
例如，本发明的细胞中ZC3H12A与SOCS1的活性被抑制。例如，本发明的表1B和1C分别所示的细胞中ZC3H12A与SOCS1的优选亚区域被敲除和/或抑制。例如，本发明的表1B中ZC3H12A R_1至9中与表1C中SOCS1 R_1至10中所示的优选靶向亚区域两组合，组成的组合序号1891至1980的组合被敲除和/或抑制。
例如，本发明的细胞中ZC3H12A与CBLB的活性被抑制。例如，本发明的表1B和1D分别所示的细胞中ZC3H12A与CBLB的优选亚区域被敲除和/或抑制。例如，本发明的表1B中ZC3H12A R_1至9中与表1D中CBLB R_1至44中所示的优选靶向亚区域两组合，组成的组合序号1981至2376的组合被敲除和/或抑制。
例如，本发明的细胞中SOCS1与CBLB的活性被抑制。例如，本发明的表1C和1D分别所示的细胞中SOCS1与CBLB的优选亚区域被敲除和/或抑制。例如，本发明的表1C中SOCS1 R_1至10中与表1D中CBLB R_1至44中所示的优选靶向亚区域两组合，组成的组合序号2377至2816的组合被敲除和/或抑制。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所 述酶蛋白特异性剪切目的基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。例如，可以将编码gRNA和Cas蛋白的多核苷酸引入、或各自独立地单独引入靶细胞。
例如，本发明使细胞的至少2种目的基因的表达降低和/或活性减弱可以包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述细胞中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。例如，可以将包含编码gRNA和Cas蛋白的多核苷酸的复合物引入所述细胞中。例如，可以将包含gRNA和Cas蛋白的复合物引入所述细胞中。
例如，所述gRNA可以用于与所述目的基因的序列结合。例如，所述gRNA与所述目的基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目的基因的序列。例如，所述gRNA与所述目的基因的序列的结合可以使得gRNA的CRISPR系统特异性剪切所述目的基因。
例如，本发明的编辑靶标区域可以是起始密码子前的一段区域。例如，本发明的编辑靶标区域可以是转录因子结合力高的区域。例如，本发明的编辑靶标区域可以是具有特定数目的转录因子结合数的区域。例如，本发明的编辑靶标区域可以是具有约3个以上的转录因子结合数的一段连续区域。例如，本发明的编辑靶标区域的基因组坐标可以选自表1A至1D所示的优选靶向亚区域。
例如，本发明所述靶向TNFAIP3的指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：107-212、1562-2532。例如，本发明所述靶向ZC3H12A的指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：281-348、3116-3698。例如，本发明 所述靶向SOCS1的指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：399-448、4393-5086。例如，本发明所述靶向CBLB的指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：520-590、6177-7266。
例如，当基因编辑系统包含CRISPR/Cas9时，本发明的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是AGG、TGG、GGG或CGG。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的5’端上游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5’端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，当基因编辑系统包含CRISPR/Cas12时，本发明的指导核酸分子靶向的区域上游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是NTTN、TTYN、VTTV、TRTV、TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约15至约25个(例如约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)3’端下游约15至约25个核苷酸组成的序列结合：NTTN、TTYN、VTTV、TRTV、TTTV、TATV、TYCV、TNN、或NTN，其中N为A、T、C或G，Y为T或C，V为A、C或G，R为A或G。
例如，当本发明的基因编辑系统包含野生型Cas12a(也可以称为Cpf1，如AsCas12a，FnCas12a，LbCas12a，BbCas12a，CMaCas12a和OsCas12a)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：NTTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如enAsCas12a(突变位点E174R、S542R和K548R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTYN(TTTN/TTCN)，VTTV(ATTV/CTTV/GTTV)，或TRTV(TATV/TGTV)，其中N可以为A、T、C或G，Y可以为T或C，V可以为A、C或G，R可以为A或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如opAsCas12a(突变位点：E174R和S542R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TTTV(TTTA，TTTC，或TTTG)，其中V可以为A、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如AsCas12aUltra(突变位点：M537R和F870L)，本发明的指导核酸分子靶向的 区域上游可以有选自以下的PAM序列：TTTV，TATV，或TYCV，其中V可以为A、C或G，Y可以为T或C。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，当本发明的基因编辑系统包含突变型Cas12a，如hfCas12Max(突变位点：N243R/E336R/D892R)和Cas12Max(突变位点：N243R)，本发明的指导核酸分子靶向的区域上游可以有选自以下的PAM序列：TNN、或NTN，其中N可以为A、T、C或G。例如，当确定了目的基因的PAM区域，本领域人员可以容易确定目的基因PAM的3’端下游约17至约25个(例如约17、约18、约19、约20、约21、约22、约23、约24、或约25个)核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，所述指导核酸分子可以包含能够结合编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约15至约25个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约22至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约20个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶 序列可以是选自表2A-2D所示的基因组坐标定义的区域或其片段。本发明中，仅出于展示方便以及受限于展示空间有限，将部分靶序列以2A-2D展示作为用于双敲的靶序列；但是，本发明中的表3A-3D中选择两种或更多种的靶点的靶序列相互组合，同样可以用于对靶点的双敲或多敲。
例如，所述指导核酸分子可以包含如SEQ ID NO：1-106、591-1561、7267-7324、7419、7420中任一项所示靶向TNFAIP3的sgRNA的靶向结构域、如SEQ ID NO：213-280、2533-3115、7325-7345、7416、7417中任一项所示靶向ZC3H12A的sgRNA的靶向结构域、如SEQ ID NO：349-398、3699-4392、7346-7375、7418中任一项所示靶向SOCS1的sgRNA的靶向结构域、或者如SEQ ID NO：449-519、5087-6176、7376-7413、7414、7415中任一项所示靶向CBLB的sgRNA的靶向结构域。
例如，本发明的细胞中TNFAIP3与ZC3H12A的活性被抑制。例如，本发明的SEQ ID NO：1-106与SEQ ID NO：213-280中所示的guide序列两两组合形成编辑组合序号1至7208的组合被敲除和/或抑制。
例如，本发明的细胞中TNFAIP3与SOCS1的活性被抑制。例如，本发明的SEQ ID NO：1-106与SEQ ID NO：349-398中所示的guide序列两两组合形成编辑组合序号7209至12508的组合被敲除和/或抑制。
例如，本发明的细胞中TNFAIP3与CBLB的活性被抑制。例如，本发明的SEQ ID NO：1-106与SEQ ID NO：449-519中所示的guide序列两两组合形成编辑组合序号12509至20034的组合被敲除和/或抑制。
例如，本发明的细胞中ZC3H12A与SOCS1的活性被抑制。例如，本发明的SEQ ID NO：213-280与SEQ ID NO：349-398中所示的guide 序列两两组合形成编辑组合序号20035至23434的组合被敲除和/或抑制。
例如，本发明的细胞中ZC3H12A与CBLB的活性被抑制。例如，本发明的SEQ ID NO：213-280与SEQ ID NO：449-519中所示的guide序列两两组合形成编辑组合序号23435至28262的组合被敲除和/或抑制。
例如，本发明的细胞中SOCS1与CBLB的活性被抑制。例如，本发明的SEQ ID NO：349-398与SEQ ID NO：449-519中所示的guide序列两两组合形成编辑组合序号28263至31812的组合被敲除和/或抑制。
例如，与目的基因的表达和/或活性未改变的细胞相比，使所述细胞的至少2种目的基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例可以降低和/或单个细胞中所述目的基因的表达量可以下降。
例如，本发明的方法中与目的基因的表达和/或活性未改变的细胞相比，使所述细胞的至少2种目的基因的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低至少约5％。例如，表达所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因的产物的细胞比例降低至少约100-5％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，表达所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI) 家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因的产物的细胞比例可以从可观测到的细胞比例到1％。例如，表达所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因的产物的细胞比例可以降低到至少约100-1％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中所述细胞的至少2种目的基因的表达降低和/或活性减弱获得的细胞中表达所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因的产物的细胞比例可以为至多约95％。例如，表达所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因的产物的细胞比例的细胞比例可以为至多约95-5％，例如至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本发明的方法中与目的基因的表达和/或活性未改变的细胞相比，使所述细胞的至少2种目的基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以下降至少约5％。例如，单个细胞中所述目的基因的表达量可以下降至少约100-5％，例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目的基因的表达量可以从可观测到的表达量到1％。例如，单个细胞中所述目的基因的表达量可以下降到至少约100-1％例如至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、或至少约1％。
例如，本发明的方法中所述细胞的至少2种目的基因的表达降低和/或活性减弱获得的细胞中单个细胞中所述目的基因的表达量可以为所述目的基因的表达和/或活性未改变的细胞的至多约95％。例如，单个细胞中所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的基因(例如编码TNFAIP3、ZC3H12A、SOCS1、CBLB的基因)表达量可以为所述编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性未改变的细胞的至多约95-5％，例如至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、 至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
例如，本发明方法包含：使所述细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱。
例如，使所述源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱。
例如，所述第一阶段体外扩增进行至少约7天。例如，所述第二阶段体外扩增进行至少约7天。
例如，可以在单个阶段的本发明的体外扩增中，使所述细胞与所述一种或多种细胞激活剂接触以及使所述细胞中包含选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员和/或其功能活性片段的表达和/或活性降低。例如，细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，在单个阶段的所述体外扩增中，使本发明细胞的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱且使该细胞与本发明的一种或多种细胞激活剂接触。例如，在本发明第一阶段体外扩增中，可以使本发明TIL的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂 (SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱且使该TIL与本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，可以使本发明TIL的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱且使该TIL与本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，可以使本发明TIL的选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱且使该TIL与本发明的一种或多种细胞激活剂接触。
例如，在单个阶段的体外扩增中，本发明的细胞基本上同时使选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱以及与本发明的一种或多种细胞激活剂接触。例如，在单个阶段的体外扩增中，本发明的细胞先使选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱，例如，可以提前2-48小时，例如提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本发明的一种或多种细胞激活剂接触。例如，在单个阶段的体外扩增中，本发明的细胞先与本发明的一种或多种细胞激活剂接触，例如，可以提前2-48小时，例如提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱。
例如，在本发明第一阶段体外扩增中，本发明的TIL基本上同时使选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱以及与本发明的一种或多种细胞激活剂接触。例如，在本发明第二阶段体外扩增中，本发明的TIL基本上同时使选自肽酶C64家族、ZC3H12 家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱以及与本发明的一种或多种细胞激活剂接触。例如，在本发明第三阶段体外扩增中，本发明的TIL基本上同时使选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱以及与本发明的一种或多种细胞激活剂接触。
TIL细胞培养
例如，本发明的第二阶段体外扩增进行至少约7天。例如，本发明的第二阶段体外扩增可以进行至少约7天、至少约8天、至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本发明的第二阶段体外扩增可以进行约9天至约14天，例如，本发明的第二阶段体外扩增可以进行约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本发明的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。例如，本发明的第一阶段体外扩增可以认为是preREP阶段。
例如，本发明的第一阶段体外扩增进行至少约7天。例如，本发明的第二阶段体外扩增可以进行至少约7天、至少约8天、至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本发明的第二阶段体外扩增可以进行约9天至约14天，例如，本发明的第二阶段体外扩增可以进行约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。
例如，本发明第二阶段体外扩增进行的天数可以是从第二阶段体外扩增的开始时刻进行计算。例如，第二阶段体外扩增开始的当时，可以认为是第二阶段体外扩增进行了约0时。例如，第二阶段体外扩增开始后进行了约24小时，可以认为是第二阶段体外扩增进行了约1天。例如，第二阶段体外扩增开始的当天，可以认为是第二阶段体外扩增进行了约0天。例如，本发明第二阶段体外扩增进行的天数可以是通过第二阶段体外扩增进行的天数进行计算。例如，第二阶段体外扩增开始后的第二天，可以认为是第二阶段体外扩增进行了约1天。
例如，本发明的细胞激活剂可以包含选自以下组的一种或多种：CD80、CD86、B7-H3、4-1BBL、CD27、CD30、CD134、B7h、CD40、LIGHT、以及它们的功能活性片段。例如，本发明的细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，本发明的细胞激活剂可以包含选自以下组：CD3、CD28、HVEM、CD40L、OX40和4-1BB的抗体以及它们的抗原结合片段。例如，本发明的细胞激活剂可以包含CD3激动剂。例如，本发明的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以是Miltenyi Biotech的OKT3，可以是BD的SP34。例如，本发明的细胞激活剂可以包含CD28激动剂。例如，本发明的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以是Merck的15E8。
例如，本发明的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链VL和重链VH，可以包含BD的SP34的轻链VL和重链VH。例如，本发明的细胞激活剂可以包含CD28激动剂。例如，本发明的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链VL和重链VH。例如，本发明的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链LCDR1-3和重链HCDR1-3，可以包含BD的SP34的轻链 LCDR1-3和重链HCDR1-3，本发明的抗CD3的抗体和/或其抗原结合片段可以具有CD3结合能力。例如，本发明的细胞激活剂可以包含CD28激动剂。例如，本发明的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链LCDR1-3和重链HCDR1-3，本发明的抗CD28的抗体和/或其抗原结合片段可以具有CD28结合能力。在本发明中，本发明抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR和/或抗体轻链可变区VL中的至少一个CDR。本发明CDR可以是根据IMGT命名法定义的，本发明CDR可以是根据Chothia定义的，或本发明CDR可以是根据Kabat定义的。
例如，使本发明的细胞与本发明的一种或多种细胞激活剂接触可以包含选自以下组的一种或多种方式：(1)将本发明的细胞激活剂添加至本发明细胞的细胞培养基中；(2)将表达本发明的细胞激活剂的工程化细胞添加至本发明细胞的细胞培养基中；(3)将包含本发明的细胞激活剂的固相介质添加至本发明细胞的细胞培养基中。例如，使本发明的细胞与本发明的一种或多种细胞激活剂接触可以包含将包含本发明的细胞激活剂的固相介质添加至本发明细胞的细胞培养基中。例如，使本发明的细胞与本发明的一种或多种细胞激活剂接触可以包含将包含本发明的CD28抗体与CD3抗体的固相介质添加至本发明细胞的细胞培养基中。
例如，所述细胞激活剂在本发明细胞的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本发明的CD28抗体在本发明细胞的细胞培养基中的初始浓度可以为至少约30ng/mL；例如，本发明的CD3抗体在本发明细胞的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本发明的CD28抗体初始浓度的选择可以与本发明的CD3抗体初始浓度的选择相互独立；例如，本发明的CD28抗体与本发明的CD3抗体在本发明细胞的细胞培养基中的初始浓度可以任意组合。例如，本发明的CD28抗体在本发明细胞的细胞培养基中的初始浓度可以任 意选自约30ng/mL-约300ng/mL。例如，本发明的CD3抗体在本发明细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本发明的CD28抗体在本发明细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，且本发明的CD3抗体在本发明细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，本发明的CD28抗体初始浓度的选择可以与本发明的CD3抗体初始浓度的选择相互独立。例如，本发明的固相介质的直径可以为约500纳米至约10微米。例如，本发明的固相介质的直径可以通过透射电子显微镜测量。例如，本发明的固相介质的直径可以为约1纳米至约500纳米。例如，本发明的固相介质的直径可以为约100纳米至约500纳米。例如，本发明的固相介质的直径可以为约200纳米至约500纳米。例如，本发明的固相介质的直径可以通过透射电子显微镜测量。
例如，本发明的固相介质可以包含聚合物。例如，本发明的固相介质可以包含葡聚糖。
例如，每mg本发明的固相介质包含至少约25μg的本发明的细胞激活剂。
例如，以约100:1-约1:2000，优选约1:100-约1:2000的本发明固相介质与本发明细胞的比例，将包含本发明细胞激活剂的固相介质添加至本发明细胞的细胞培养基中。例如，以约2:1-约1:2的本发明固相介质与本发明细胞的比例，将包含本发明细胞激活剂的固相介质添加至本发明细胞的细胞培养基中。
例如，当本发明的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2的本发明固相介质与本发明细胞的比例，将包含本发明细胞激活剂的固相介质添加至本发明细胞的细胞培养基中。例如，当本发明的固相介质的直径为约100纳米至约500纳米时，可以以约 2:1-约1:2、以约2:1-约1:1、或以约1:1-约1:2的本发明固相介质与本发明细胞的比例，将包含本发明细胞激活剂，例如CD3激动剂和/或CD28激动剂的固相介质添加至本发明细胞的细胞培养基中。
例如，当本发明的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000的本发明固相介质与本发明细胞的比例，将包含本发明细胞激活剂的固相介质添加至本发明细胞的细胞培养基中。例如，当本发明的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000、以约1:200-约1:2000、以约1:300-约1:2000、以约1:400-约1:2000、以约1:500-约1:2000、以约1:600-约1:2000、以约1:700-约1:2000、以约1:800-约1:2000、以约1:900-约1:2000、以约1:1000-约1:2000、以约1:1200-约1:2000以约1:1400-约1:2000、以约1:1600-约1:2000、或以约1:1800-约1:2000的本发明固相介质与本发明细胞的比例，例如可以将包含本发明CD28激动剂和CD3激动剂的固相介质添加至本发明细胞的细胞培养基中。
例如，本发明的方法还可以包含：在至少一个阶段的本发明体外扩增中，使本发明细胞与一种或多种细胞生长因子接触。
例如，在单个阶段的本发明体外扩增中，可以使本发明的细胞与本发明的细胞激活剂接触且与本发明的一种或多种细胞生长因子接触。例如，在本发明第一阶段体外扩增中，可以使本发明的TIL与本发明的细胞激活剂接触且与本发明的一种或多种细胞生长因子接触。例如，在本发明第二阶段体外扩增中，可以使本发明的TIL与本发明的细胞激活剂接触且与本发明的一种或多种细胞生长因子接触。例如，在本发明第三阶段体外扩增中，可以使本发明的TIL与本发明的细胞激活剂接触且与本发明的一种或多种细胞生长因子接触。
例如，在单个阶段的本发明体外扩增中，使本发明细胞基本上同时与本发明细胞激活剂以及本发明一种或多种细胞生长因子接触。例 如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞基本上同时与本发明的一种或多种细胞生长因子以及本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先与本发明的一种或多种细胞生长因子接触，例如，可以提前2-48小时，例如提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本发明的一种或多种细胞激活剂接触。例如，在单个阶段的本发明的体外扩增中，可以使本发明的细胞先与本发明的一种或多种细胞激活剂接触，例如，可以提前2-48小时，例如提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本发明的一种或多种细胞生长因子接触。
例如，在本发明第一阶段体外扩增中，可以使本发明细胞基本上同时与本发明细胞激活剂以及本发明一种或多种细胞生长因子接触。例如，在本发明第二阶段体外扩增中，可以使本发明TIL基本上同时与本发明细胞激活剂以及本发明一种或多种细胞生长因子接触。例如，在本发明第三阶段体外扩增中，可以使本发明TIL基本上同时与本发明细胞激活剂以及本发明一种或多种细胞生长因子接触。
例如，本发明的细胞生长因子可以选自以下组的一种或多种：IL-2、IL-7、IL-12、IL-15、IL-21、γ干扰素、以及它们的功能活性片段。例如，本发明的细胞生长因子可以包含IL-2和/或其功能活性片段。例如，IL-2的功能活性片段可以包含本领域已知的可以与细胞的IL-2受体结合的IL-2的片段。例如，本发明的细胞生长因子可以包含IL-2和/或其功能活性片段、IL-7和/或其功能活性片段和IL-15和/或其功能活性片段。
例如，本发明的细胞与本发明一种或多种细胞生长因子接触可以包含将本发明细胞生长因子添加至本发明细胞的细胞培养基中。例如，本发明的细胞生长因子在本发明细胞的细胞培养基中的初始浓度可以 为至少约300IU/mL。例如，本发明IL-2在本发明细胞的细胞培养基中的初始浓度可以为至少约300-9000IU/mL，例如至少约300IU/mL、至少约350IU/mL、至少约400IU/mL、至少约500IU/mL、至少约600IU/mL、至少约700IU/mL、至少约800IU/mL、至少约900IU/mL、至少约1000IU/mL、至少约1100IU/mL、至少约1200IU/mL、至少约1300IU/mL、至少约1400IU/mL、至少约1500IU/mL、至少约2000IU/mL、至少约2500IU/mL、至少约2600IU/mL、至少约2700IU/mL、至少约2800IU/mL、至少约2900IU/mL、至少约3000IU/mL、至少约3100IU/mL、至少约3200IU/mL、至少约3300IU/mL、至少约3400IU/mL、至少约3500IU/mL、至少约4000IU/mL、至少约4500IU/mL、至少约5000IU/mL、至少约5500IU/mL、至少约6000IU/mL、至少约6500IU/mL、至少约7000IU/mL、至少约7500IU/mL、至少约8000IU/mL、至少约8500IU/mL、或至少约9000IU/mL。
例如，本发明的细胞在与IL-2、IL-7以及IL-15接触，相对于仅与IL-2接触，可以降低细胞因子的用量。例如，加入IL-7和IL-15的条件下可以降低IL-2的加入量。例如，IL-7的浓度可以为约1至1000ng/mL，优选地为约1-100ng/mL。例如，IL-15的浓度可以为约1至1000ng/mL，优选地为约1-100ng/mL。例如，对于IL-2的加入量，可以降低至各种免疫细胞的本领域常用范围，例如降低至本领域常用范围的50-10％，例如50％、20％或10％。例如，TCR-T的IL-2加入量，本领域常用范围可以是30-300IU/mL。例如，TIL的IL-2加入量，本领域常用范围可以是300-9000IU/mL(例如1000-9000IU/mL)。
例如，本发明的方法还可以包含：在至少一个阶段的本发明体外扩增中，可以使本发明细胞与饲养细胞共培养。
例如，在单个阶段的本发明体外扩增中，可以使本发明细胞与一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本发明饲养细胞共培养，例如，单个阶段的本发明体外扩增可以指在同一个阶 段的本发明的体外扩增，例如，可以同在本发明的第一阶段体外扩增、同在本发明的第二阶段体外扩增、或同在本发明的第三阶段体外扩增等。
例如，本发明的第一阶段体外扩增中，可以使本发明TIL与一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本发明饲养细胞共培养。例如，本发明的在本发明第二阶段体外扩增中，可以使本发明TIL与本发明一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本发明饲养细胞共培养。例如，本发明的在本发明第三阶段体外扩增中，可以使本发明TIL与本发明一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本发明饲养细胞共培养。
例如，在单个阶段的本发明体外扩增中，可以使本发明细胞与本发明一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本发明饲养细胞共培养。例如，在本发明第一阶段体外扩增中，可以使本发明TIL与本发明一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本发明饲养细胞共培养。例如，在本发明第二阶段体外扩增中，可以使本发明TIL与本发明一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本发明饲养细胞共培养。例如，在本发明第三阶段体外扩增中，可以使本发明TIL与本发明一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本发明饲养细胞共培养。
例如，在单个阶段的本发明体外扩增中，可以使本发明细胞与本发明一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本发明饲养细胞共培养。例如，本发明的一定时间可以为至少约1小时。例如，本发明的一定时间可以为至少约1-72小时，例如至少约1小时、至少约2小时、至少约3小时、至少约4小时、至少约5小时、至少约6小时、至少约7小时、至少约8小时、至少约9小时、至少约10小时、至少约11小时、至少约12小时、至少约 13小时、至少约14小时、至少约15小时、至少约16小时、至少约17小时、至少约18小时、至少约19小时、至少约20小时、至少约21小时、至少约22小时、至少约23小时、至少约24小时、至少约36小时、至少约48小时、至少约60小时或至少约72小时。例如，本发明的一定时间可以为约2小时至约72小时。例如，本发明的一定时间可以为约6小时到约7小时、约6小时到约8小时、约6小时到约9小时、约6小时到约10小时、约6小时到约11小时、约6小时到约12小时、约6小时到约13小时、约6小时到约14小时、约6小时到约15小时、约6小时到约16小时、约6小时到约17小时、约6小时到约18小时、约6小时到约19小时、约6小时到约20小时、约6小时到约21小时、约6小时到约22小时、约6小时到约23小时、约6小时到约24小时、约6小时到约36小时、约6小时到约48小时、约6小时到约60小时或约6小时到约72小时。例如，本发明的一定时间可以为约12小时到约13小时、约12小时到约14小时、约12小时到约15小时、约12小时到约16小时、约12小时到约17小时、约12小时到约18小时、约12小时到约19小时、约12小时到约20小时、约12小时到约21小时、约12小时到约22小时、约12小时到约23小时、约12小时到约24小时、约12小时到约36小时、约12小时到约48小时、约12小时到约60小时或约12小时到约72小时。例如，本发明的一定时间可以为约1小时、约2小时、约3小时、约4小时、约5小时、约6小时、约7小时、约8小时、约9小时、约10小时、约11小时、约12小时、约13小时、约14小时、约15小时、约16小时、约17小时、约18小时、约19小时、约20小时、约21小时、约22小时、约23小时、约24小时、约36小时、约48小时、约60小时或约72小时。
例如，本发明的饲养细胞可以包含抗原呈递细胞。例如，本发明的饲养细胞可以包含选自以下组的一种或多种：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。例如，本发明的饲养细胞可以为外周单个核细胞。例如，本发明的饲养细胞可以为经过辐照的饲养细胞。 例如，本发明的饲养细胞可以为分离的人工抗原呈递细胞(aAPC)，本发明的人工抗原呈递细胞可以包含表达HLA-A/B/C、CD64、CD80、ICOS-L和/或CD58的细胞，并可以被修饰以表达一种以上本发明的细胞激活剂。例如，本发明的饲养细胞可以经过辐照，例如，可以经过伽马射线辐照，或可以经过X射线辐照。
例如，本发明的细胞与本发明的饲养细胞共培养可以包含使本发明的饲养细胞的表面与本发明的细胞的表面相接触。例如，本发明的细胞与本发明的饲养细胞共培养包含将本发明的饲养细胞添加至本发明细胞的细胞培养基中。
例如，本发明可以以约40:1-约400:1的本发明饲养细胞与本发明细胞的比例，将本发明饲养细胞添加至本发明细胞的细胞培养基中。例如，本发明可以以约40:1-约400:1、以约40:1-约300:1、以约40:1-约200:1、以约40:1-约100:1、以约40:1-约90:1、以约40:1-约80:1、以约40:1-约70:1、以约40:1-约60:1、以约40:1-约50:1、以约50:1-约400:1、以约60:1-约400:1、以约70:1-约400:1、以约80:1-约400:1、以约90:1-约400:1、以约100:1-约400:1、以约200:1-约400:1、或以约300:1-约400:1的本发明饲养细胞与本发明细胞的比例，将本发明饲养细胞添加至本发明细胞的细胞培养基中。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：(A)使源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱，且使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；(C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
在一种实施方式中，本发明的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中，本发明的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中，本发明的经第一阶段体外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中，本发明的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中，如有需要，本发明的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中，如有需要，本发明的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种细胞生长因子接触、与多种细胞激活剂接触、使选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中 的至少2个家族的成员的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与细胞生长因子接触、与细胞激活剂接触、使选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱且使所述TIL与饲养细胞共培养，选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员可以分别包含TNFAIP3、ZC3H12A、SOCS1、CBLB；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本发明提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织、肿瘤相关淋巴结伴有或不伴有肿瘤转移、肿瘤转移病灶、癌旁组织的碎片、胸腔积液和/或腹腔积液在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液内约2-8℃运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入细胞无血清培养基和浓度为300-9000IU/mL(例如1000-9000IU/mL，例如6000IU/mL)的IL-2培养约3-14天。收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，细胞无血清培养基可以添加本发明的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如1000-9000IU/mL，例如6000IU/mL)的IL-2以及使选自肽酶C64家族、ZC3H12家族、STAT诱导 的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的表达降低和/或活性减弱(选自肽酶C64家族成员可以包含TNFAIP3、ZC3H12家族成员可以包含ZC3H12A、STAT诱导的STAT抑制剂(SSI)家族成员可以包含SOCS1和CBL家族成员可以包含CBLB，例如可以通过用携带包含本发明的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)，或者包含gRNA与Cas蛋白的LNP，或者包含编码gRNA与Cas蛋白的核酸的LNP进行转导使所述TIL中编码选自肽酶C64家族、ZC3H12家族、STAT诱导的STAT抑制剂(SSI)家族和CBL家族中的至少2个家族的成员的基因的细胞比例为约95％以下)，活化本发明的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞存活率可以大于50％，大于80％的细胞可以为记忆效应细胞和效应细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化细胞比例上调的特征。
一方面，本发明提供一种细胞，本发明的细胞可以根据本发明的培养方法培养得到。在一种实施方式中，本发明提供的细胞可以包含一种或一个批次的本发明的培养方法培养得到细胞。在一种实施方式中，本发明提供的细胞可以包含多种或多个批次的本发明的培养方法培养得到并以任意比例组合的细胞。
在一些实施方式中，可以将使用本发明方法扩增的细胞作为药物组合物施用于患者。在一些实施方式中，药物组合物可以是细胞在无菌缓冲液中的悬液。使用本发明的PBMC扩增的细胞可以通过本领域已知的任何合适途径施用。在一些实施方式中，细胞可以以单次动脉内或静脉内输注施用，输注可以持续约30至60分钟。其他合适的施用途径可以包括腹膜内、鞘内和淋巴管内施用。
在一些实施方式中，可以施用任何合适剂量的细胞。在一些实施 方式中，例如当肿瘤是黑色素瘤时，可以施用约1×109至约13.7×1010，优选约2.3×109至约13.7×1010个细胞。在一些实施方式中，可以施用约1×109至约12×1010个细胞。在一些实施方式中，可以施用约1.2×1010至约4.3×1010个细胞。在一些实施方式中，可以施用约3×1010至约12×1010个细胞。在一些实施方式中，可以施用约4×1010至约10×1010个细胞。在一些实施方式中，可以施用约5×1010至约8×1010个细胞。在一些实施方式中，可以施用约6×1010至约8×1010个细胞。在一些实施方式中，可以施用约7×1010至约8×1010个细胞。在一些实施方式中，治疗有效剂量可以为约1×109至约13.7×1010，优选约2.3×109至约13.7×1010。在一些实施方式中，治疗有效剂量可以为约1×109至约12×1010个细胞。在一些实施方式中，治疗有效剂量可以为约1.2×1010至约4.3×1010个细胞。在一些实施方式中，治疗有效剂量可以为约3×1010至约12×1010个细胞。在一些实施方式中，治疗有效剂量可以为约4×1010至约10×1010个细胞。在一些实施方式中，治疗有效剂量可以为约5×1010至约8×1010个细胞。在一些实施方式中，治疗有效剂量可以为约6×1010至约8×1010个细胞。在一些实施方式中，治疗有效剂量可以为约7×1010至约8×1010个细胞。
在一些实施方式中，本发明的组合物中提供的细胞数量可以为约1×106-9×1013，例如约1×106、约2×106、约3×106、约4×106、约5×106、约6×106、约7×106、约8×106、约9×106、约1×107、约2×107、约3×107、约4×107、约5×107、约6×107、约7×107、约8×107、约9×107、约1×108、约2×108、约3×108、约4×108、约5×108、约6×108、约7×108、约8×108、约9×108、约1×109、约2×109、约3×109、约4×109、约5×109、约6×109、约7×109、约8×109、约9×109、约1×1010、约2×1010、约3×1010、约4×1010、约5×1010、约6×1010、约7×1010、约8×1010、约9×1010、约1×1011、约2×1011、约3×1011、约4×1011、约5×1011、约6×1011、约7×1011、约8×1011、约9×1011、约1×1012、约2×1012、约3×1012、约4×1012、约5×1012、约6×1012、约7×1012、约8×1012、约9×1012、约1×1013、约2×1013、约3×1013、约4×1013、约5×1013、约6×1013、约7×1013、约8×1013，或约9×1013。 在一些实施方式中，本发明的组合物中提供的细胞数量的范围可以为约1×106至5×106、约5×106至1×107、约1×107至5×107、约5×107至1×108、约1×108至5×108、约5×108至1×109、约1×109至5×109、约5×109至1×1010、约1×1010至5×1010、约5×1010至1×1011、约5×1011至1×1012、约1×1012至5×1012，约5×1012至1×1013，约1×1013至5×1013、或约5×1013至9×1013。
在一些实施方式中，本发明的组合物中提供的细胞浓度可以小于组合物的约100-0.0001％w/w、w/v或者v/v，例如约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.9％、约0.8％、约0.7％、约0.6％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约0.0002％，或约0.0001％w/w、w/v或者v/v。
在一些实施方式中，本发明的组合物中提供的细胞浓度可以大于组合物的约90-0.0001％w/w、w/v或v/v，例如约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19.75％、约19.50％、约19.25％、约19％、约18.75％、约18.50％、约18.25％、约18％、约17.75％、约17.50％、约17.25％、约17％、约16.75％、约16.50％、约16.25％、约16％、约15.75％、约15.50％、约15.25％、约15％、约14.75％、约14.50％、约14.25％、约14％、约13.75％、约13.50％、约13.25％、约13％、约12.75％、约12.50％、约12.25％、约12％、约11.75％、约11.50％、约11.25％、约11％、约10.75％、约10.50％、约10.25％、约10％、约9.75％、约9.50％、约9.25 ％、约9％、约8.75％、约8.50％、约8.25％、约8％、约7.75％、约7.50％、约7.25％、约7％、约6.75％、约6.50％、约6.25％、约6％、约5.75％、约5.50％、约5.25％、约5％、约4.75％、约4.50％、约4.25％、约4％、约3.75％、约3.50％、约3.25％、约3％、约2.75％、约2.50％、约2.25％、约2％、约1.75％、约1.50％、约1.25％、约1％、约0.9％、约0.8％、约0.7％、约0.6％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约或0.0002％，或者约0.0001％w/w、w/v或v/v。
在一些实施方式中，本发明的组合物中提供的细胞浓度范围可以为组合物的约0.0001％至约50％、约0.001％至约40％、约0.01％至约30％、约0.02％至约29％、约0.03％至约28％、约0.04％至约27％、约0.05％至约26％、约0.06％至约25％、约0.07％至约24％、约0.08％至约23％、约0.09％至约22％、约0.1％至约21％、约0.2％至约20％、约0.3％至约19％、约0.4％至约18％、约0.5％至约17％、约0.6％至约16％、约0.7％至约15％、约0.8％至约14％、约0.9％至约12％，或约1％至约10％w/w、w/v或者v/v。
在一些实施方式中，本发明的组合物中提供的细胞浓度范围可以为组合物的约0.001％至约10％、约0.01％至约5％、约0.02％至约4.5％、约0.03％至约4％、约0.04％至约3.5％、约0.05％至约3％、约0.06％至约2.5％、约0.07％至约2％、约0.08％至约1.5％、约0.09％至约1％、或约0.1％至约0.9％w/w、w/v或者v/v。
在一些实施方式中，本发明的组合物中提供的细胞量可以等于或小于约10-0.0001g，例如约10g、约9.5g、约9.0g、约8.5g、约8.0g、 约7.5g、约7.0g、约6.5g、约6.0g、约5.5g、约5.0g、约4.5g、约4.0g、约3.5g、约3.0g、约2.5g、约2.0g、约1.5g、约1.0g、约0.95g、约0.9g、约0.85g、约0.8g、约0.75g、约0.7g、约0.65g、约0.6g、约0.55g、约0.5g、约0.45g、约0.4g、约0.35g、约0.3g、约0.25g、约0.2g、约0.15g、约0.1g、约0.09g、约0.08g、约0.07g、约0.06g、约0.05g、约0.04g、约0.03g、约0.02g、约0.01g、约0.009g、约0.008g、约0.007g、约0.006g、约0.005g、约0.004g、约0.003g、约0.002g、约0.001g、约0.0009g、约0.0008g、约0.0007g、约0.0006g、约0.0005g、约0.0004g、约0.0003g、约0.0002g，或者约0.0001g。
在一些实施方式中，本发明的组合物中提供的细胞量可以大于约0.0001-10g，例如约0.0001g、约0.0002g、约0.0003g、约0.0004g、约0.0005g、约0.0006g、约0.0007g、约0.0008g、约0.0009g、约0.001g、约0.0015g、约0.002g、约0.0025g、约0.003g、约0.0035g、约0.004g、约0.0045g、约0.005g、约0.0055g、约0.006g、约0.0065g、约0.007g、约0.0075g、约0.008g、约0.0085g、约0.009g、约0.0095g、约0.01g、约0.015g、约0.02g、约0.025g、约0.03g、约0.035g、约0.04g、约0.045g、约0.05g、约0.055g、约0.06g、约0.065g、约0.07g、约0.075g、约0.08g、约0.085g、约0.09g、约0.095g、约0.1g、约0.15g、约0.2g、约0.25g、约0.3g、约0.35g、约0.4g、约0.45g、约0.5g、约0.55g、约0.6g、约0.65g、约0.7g、约0.75g、约0.8g、约0.85g、约0.9g、约0.95g、约1g、约1.5g、约2g、约2.5g、约3g、约3.5g、约4g、约4.5g、约5g、约5.5g、约6g、约6.5g、约7g、约7.5g、约8g、约8.5g、约9g、约9.5g，或者约10g。
在一些实施方式中，细胞可以单剂量施用。此种施用可以通过注射，例如可以静脉内注射。在一些实施方式中，细胞可以多剂量施用。剂量可以是每年一次、两次、三次、四次、五次、六次或超过六次。剂量可以是每月一次、每两周一次、每周一次或每2天一次。在一些实施方式中，细胞的施用可以连续施用。
一方面，本发明提供一种药物组合物。在一些实施方式中，其可以包含本发明的细胞，与药学上可接受的载体。
一方面，本发明提供一种试剂盒，本发明的试剂盒可以包含本发明培养细胞方法的细胞激活剂、细胞生长因子和/或饲养细胞与记载本发明培养细胞方法的步骤的说明书。一方面，本发明提供一种试剂盒，本发明试剂盒可以包含本发明的细胞和/或本发明的药物组合物。
一方面，本发明提供一种影响细胞，例如肿瘤细胞，生长的方法，可以包括向受试者施用本发明的细胞和/或本发明的药物组合物。在一些实施方式中，影响肿瘤生长可以包含肿瘤的体积减少到施用前的约99-0.1％，例如约99％、约95％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.9％、约0.8％、约0.7％、约0.6％、约0.5％、约0.4％、约0.3％、约0.2％或约0.1％。
一方面，本发明提供本发明的细胞和/或本发明的药物组合物在制备药物中的应用，本发明的药物可以用于预防和/或治疗疾病和/或症状。例如，本发明的疾病和/或症状可以包含肿瘤。在一些实施方式中，本发明的肿瘤选自实体瘤。在一些实施方式中，本发明的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
一方面，本发明提供一种预防和/或治疗疾病和/或症状的方法，可以包括向受试者施用本发明的细胞和/或本发明的药物组合物。例如，本发明的疾病和/或症状可以包含肿瘤。在一些实施方式中，本发明的肿瘤选自实体瘤。在一些实施方式中，本发明的肿瘤可以选自以下组 的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
一方面，本发明提供一种本发明的TIL和/或本发明的药物组合物，其可以用于预防和/或治疗疾病和/或症状。例如，本发明的疾病和/或症状可以包含肿瘤。在一些实施方式中，本发明的肿瘤选自实体瘤。在一些实施方式中，本发明的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
实施例
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本发明的方法和用途等，而不用于限制本发明的范围。
实施例1
(一)TIL细胞培养
1.1肿瘤组织接收及处理
1.1.1组织接收
接收供者的肿瘤组织及血样，核对样品信息并记录，打印相应样品标签。
1.1.2组织处理及培养
使用75％酒精消毒样品管及采血管，转移至生物安全柜内。根据上述PBMC手动分离及冻存操作程序分离血样中PBMC细胞并进行冻存。取一种具有透气表面的培养瓶或培养袋，例如培养袋(Origen)，加入300mL已复温的完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度为300-9000IU/mL(例如1000-9000IU/mL，例如6000IU/mL)的IL-2。取数个10厘米培养皿，加入适量培养基，使用无菌眼科镊从 样品管中取出肿瘤组织于10厘米培养皿中，洗涤组织并更换培养皿。使用眼科剪及眼科镊将进行初步剪切，去除脂肪组织及坏死组织，每块组织块继续剪碎至约27立方毫米大小。取非悬浮肿瘤组织块，使用20mL注射器去除内部活塞后，与培养袋连接，使用移液管将约1g组织块通过注射器转入培养袋内。将培养袋放入二氧化碳培养箱内进行培养。清理剪刀及镊子，并用75％酒精进行初步消毒后，超声清洗后进行灭菌，得到第一TIL群。
1.2步骤(A)体外扩增及收获
1.2.1步骤(A)体外扩增
根据细胞生长状态，每3-7天补液或半量换液，保证细胞营养。使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度为300-9000IU/mL(例如1000-9000IU/mL，例如6000IU/mL)的IL-2(双鹭和/或四环)。步骤(A)的3-14天，例如可以第13或14天时取样计数，若细胞数目处于5×105至5×108之间时进入步骤(A)的收获步骤。
1.2.2步骤(A)的收获
收集步骤(A)体外扩增结束细胞，离心，弃去培养基，使用PBS或生理盐水洗涤细胞一次，获得经步骤(A)体外扩增的TIL(第二TIL群)，并取样计数留取约5×105至2×108个细胞进入后续体外扩增步骤；取约5×105个细胞可以进行质量控制检测；其余细胞加入冻存液冻存，作为冻存的preREP TIL体外细胞。
1.3步骤(B)TIL活化
继续培养经步骤(A)体外扩增的TIL(第二TIL群)，或者对冻存的preREP TIL体外细胞进行细胞复苏，进行步骤(B)的TIL活化。
使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，调整细胞密度为5×105至2×106个细胞/mL，于悬浮24孔培养板内，1mL/孔，添加浓度为300-9000IU/mL(例如1000-9000IU/mL，例如6000IU/mL)的IL-2。各TIL细胞群的培养基中同时可以添加T细胞激活剂，例如添加CD3激动剂和/或CD28激动剂，例如，约30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)、约30ng/mL的CD28抗体(Merck，15E8)、以约1:2-2:1的磁珠与TIL的比例加入磁珠(直径约1至10μm Dynabeads，Thermo Fisher)和/或以约1:100-1:2000的transACT(直径约100至500nm，Miltenyi)与TIL的比例加入transACT。培养约0-4天，获得第三TIL群。
1.4步骤(C)TIL细胞基因编辑
将选自本发明的靶向各个靶点的sgRNA合成，解冻并加入无核酸酶水，配至浓度为约100μM。将约2μL的gRNA(50μM)在95℃孵育2分钟退火后加入到P3缓冲液，并加入0.3-1μL的Cas9(例如恺佧、克睿或Acro，10mg/mL)，25℃孵育10分钟以形成核糖核蛋白复合物(RNP)。在P3缓冲液(Lonza)中，通过Lonza电转仪将上述RNP与第三TIL群的约1×106个细胞进行电转。例如，电转程序可以是human T cell stim(EO115)。电转的基因编辑后培养约0-4天，获得第四TIL群。
1.5步骤(D)TIL细胞基因编辑后培养
在第四TIL细胞群中加入饲养细胞(经辐照的健康供者PBMC T细胞)进行培养。TIL与饲养细胞接触的时间需要在步骤(B)的TIL与IL-2以及T细胞激活剂接触后的若干时间Tn以后(例如，Tn可以取0小时到12天，例如24小时或48小时)。首先复苏1-5名供者混合的饲养细胞；将活化的TIL细胞、饲养细胞按照TIL细胞:饲养细胞约为1:200的比例混合，转入G-Rex100培养瓶或者透气袋内，补充完全 培养基，每1-3天取样计数，并根据细胞状态补液或半量换液直至细胞总数大于1×109或步骤(D)体外扩增培养约5天至约14天，终止步骤(D)体外扩增的培养。
1.6肿瘤浸润淋巴细胞的收获
取步骤(D)扩增的细胞，离心后弃去培养基上清，并使用PBS或生理盐水或复方电解质溶液清洗三次，获得经步骤(D)扩增的TIL(第五TIL群)，第三次清洗时取样计数，根据计数结果，最后一次离心后弃上清，取3×106细胞送质量控制检测；其余全部细胞加入冻存液，调整细胞密度1-3×108个细胞/mL冻存。
(二)TCR-T细胞培养
T细胞活化：将冻存于液氮中的T细胞复苏培养，使用T细胞培养基RPMI 1640(Gibco)+10％FBS(Bovogen)离心重悬至5E5/ml，1:100加入T cell TransAct(Miltenyi)，添加浓度为30IU/ml的重组人IL-2，培养约72hr。
TCR转导：转导前1天使用终浓度为15μg/mL的重组人纤维蛋白片段(Retronectin，Takara)包被24孔悬浮培养板，24孔板每孔250μL。避光，4℃过夜备用。取出包被好的24孔板，吸弃包被液，加入含2％BSA封闭液500μL室温封闭30分钟。吸弃封闭液，用含2.5％HEPES的洗板液500μL/孔洗板2次，吸弃洗板液。实验组用携带NY-ESO-1抗原肽的特异性TCR核酸片段的逆转录病毒进行转导。每孔加0.1～1mL逆转录病毒液，32℃，2000g，离心2小时。弃去24孔板上清液，24孔板每孔加入复苏活化后的T细胞，体积500-1000μL，细胞浓度约为5×105个/mL。30-32℃，1000g，离心10分钟。离心完毕后，将培养板置于37℃，5％CO2培养箱中培养，得到转导后细胞。转导后培养约0-4天，获得TCR-T细胞群。转导后细胞根据细胞每1～3天细胞计数密度及活率，根据计数结果添加T细胞培养基，添加浓度为30～100IU/ml的重组人IL-2，调整初始培养细胞密度为0.5～2×106个/ml，继 续培养。
TCR-T基因编辑：将选自本发明的靶向各个靶点的sgRNA合成，解冻并加入无核酸酶水，配至浓度为约100μM。将约2μL的gRNA(50μM)在95℃孵育2分钟退火后加入到P3缓冲液，并加入0.3-1μL的Cas9(例如恺佧、克睿、Acro，10mg/mL)，25℃孵育10分钟以形成核糖核蛋白复合物(RNP)。在P3缓冲液(Lonza)中，通过Lonza电转仪将上述RNP与约1×106个TCR-T细胞进行电转。例如，电转程序可以是human T cell stim(EO115)。电转后的T细胞添加浓度为100～300IU/ml的重组人IL-2，继续培养，获得本发明的目标基因编辑后的TCR-T细胞。
(三)敲除效率检测
用于检测细胞的敲除效率，试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在T细胞敲除(参照实施例1(一)的步骤1.4的基因编辑方法、或实施例1(二)的TCR-T基因编辑的方法)后的约2-7天，取约1×105个至约2×105个细胞，用PBS清洗1次，再用44μL PBS重悬基因编辑后细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDropTM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
并按照以下PCR程序扩增：
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本发明相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
(四)扩增情况检测
试验准备
在实施例1(一)基因编辑后第7-10天检测TIL细胞的增殖情况(撤去IL-2)，收获各组TIL细胞；或者采用实施例1(二)得到的TCR转导的T细胞。
用PBS清洗细胞1次，再用T细胞培养基(无IL-2)重悬，计数后调整细胞密度至1e5至2e6/mL，并按照100μL/孔加入到平底96孔板。无刺激培养基组，在培养基中不加入细胞激活物质；CD3抗体刺激组加入CD3抗体(OKT3)30ng/ml刺激；TransACT刺激组加入 transACT(直径约100至500nm，Miltenyi)，使transACT工作液浓度为1:1000(v/v)；Medium组，仅加入同等体积的细胞培养基。利用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析T细胞铺板时荧光量，3天后用CTG试剂盒分析T细胞的荧光量，T细胞的扩增效率用第三天荧光量/铺板时荧光量表征。
(五)细胞杀伤能力检测
基因编辑后的第6天开始，将肿瘤靶细胞铺于96孔平底板中，次日，将各组TIL细胞以不同效靶比(T细胞：靶细胞，E：T)与靶细胞共培养。或者采用实施例1(二)得到的TCR转导的T细胞。
靶细胞和T细胞各100μL，每组设置三个复孔，同时设置一组只包含靶细胞的对照组。其中靶细胞可以选自Hey-T30卵巢癌细胞、A375黑色素瘤细胞。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL细胞对靶细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
(六)细胞杀伤能力检测
利用流式细胞仪检测基因编辑后第8天获得的TIL细胞的T细胞耗竭、干性等相关分子的表达情况；或者对实施例1(二)得到的TCR转导的T细胞进行检测。
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052；流式抗体购自BD或Biolegend。
细胞表面分子检测：将每组1×105至5×105个细胞样品，加入流式 管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
细胞内分子检测：配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8，抗体浓度为1:100，细胞活率检测染料浓度(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。每孔加入100μL固定破膜液(BD，Fixation/Permeabilization)，2-8℃避光孵育20-40分钟，固定破膜后使用1×Perm/Wash Buffer清洗2次，(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。使用1×Perm/Wash Buffer配制细胞内分子抗体(例如TCF1)，并重悬TIL细胞(96孔板50μL/孔，流式管100μL/管染色)，2-8℃避光孵育30分钟；细胞内分子染色后使用1×Perm/Wash Buffer清洗1-2次，(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
(七)细胞因子表达流式检测
对于各个试验组基因编辑后的第7天或第8天获得的TIL细胞群进行流式细胞仪检测细胞因子表达情况；或者对实施例1(二)得到的TCR转导的T细胞进行检测。
配制胞内因子表达检测所需培养基：取TIL细胞培养基，按照体积比添加：Golgistop 0.7:1000，Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的T细胞离心后，使用上述胞内因子表达检测所需培养基重悬各组T细胞，计数后调整细胞密度为1×106个细胞/mL，加入96孔板内，200μL/孔。CD3抗体刺激组加入CD3抗体(OKT3)30ng/ml刺激；TransACT刺激组加入transACT(直径约100至500nm，Miltenyi)，使transACT工作液浓度为1:1000(v/v)；Medium组，仅加入同等体积的细胞培养基。置于37℃培养箱孵育过夜。
细胞因子流式检测试验主要试剂及材料的来源：
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052；流式抗体购自BD或Biolegend。
孵育结束后，用200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8，抗体浓度为1:100，细胞活率检测染料浓度(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。用PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。每孔加入100μL固定破膜液(BD，Fixation/Permeabilization)，2-8℃避光孵育20-40分钟，固定破膜后使用1×Perm/Wash Buffer清洗2次，(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。使用1×Perm/Wash Buffer配制细胞因子检测抗体(例如GZMB,TNF-α,IFN-γ)，并重悬TIL细胞(96孔板50μL/孔，流式管100μL/管染色)，2-8℃避光孵育30分钟；细胞因子染色后使用1×Perm/Wash Buffer清洗1-2次，(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
(八)细胞凋亡检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的 TIL群进行凋亡检测；或者对实施例1(二)得到的TCR转导的T细胞进行检测。
将基因敲除组或对照组(NT，no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
(九)PDO模型培养
肿瘤组织自手术取得，离体后2～8℃运输及保存，24小时内机械解离，将组织剪碎至约为0.5mm3大小，后使用专用组织消化液(bioGenous(伯桢生物))于37℃消化处理后使用10％FBS终止反应，该组织悬液使用100μm滤网过滤后洗涤，置于冰上与Martrigel充分混匀，之后将混合液点涂至细胞培养板底部，将培养板放于37℃培养箱，待Martrigel充分凝固后小心加入完全培养基进行培养。待类器官生长到足够大小和密度时，进行传代或者冻存，得到PDO(肿瘤类器官，Patient-Derived Organoid)模型。
用移液器吸头尖端刮下Matrigel和类器官混合物，转移至含基础培养基的离心管中，吹打使类器官和Matrigel分离，离心后加入传代用组织消化液，混匀后置于37℃培养箱中消化，加入基础培养基稀释消化液后离心洗涤，取少量细胞悬液加入8倍体积消化液消化使之变为单细胞，使用FBS终止反应后进行计数，依据计数结果吸取所需体积类器官离心后使用T细胞培养基重悬；对待测试的细胞(例如TIL细胞或TCR-T细胞)进行计数，按不同效靶比重悬待测试的细胞至所需密度；于平底96孔板中加入PDO靶细胞和待测试的细胞各100μl，每组设置三个复孔，用于PDO共培养检测。设置几个不同效靶比，同时，设置一组只包含PDO靶细胞的组，设置一组对照组(NT)用于仅在PDO靶细胞中共培养未编辑的待测试的细胞。
PDO模型中杀伤能力检测
参照本发明实施例制备PDO模型并与TIL细胞或者TCR-T细胞共培养，加入Caspase3/7底物进行凋亡信号标记后将实验板放于Incucyte中进行观察记录。
(十)细胞多轮杀伤能力检测
用IncuCyte监测多轮杀伤中靶细胞A375-GFP的荧光强度变化，用A375-GFP细胞均匀铺在多孔板中，在37℃培养4小时后，取未编辑或敲除本发明靶点的细胞，加入对应孔后开始用IncuCyte记录GFP荧光信号，每组各3个复孔。杀伤进行24小时后取共培养上清(20μL/孔)进行CBA检测；杀伤进行3天后，用新的A375-GFP均匀平铺的多孔板中，37℃培养4小时后，将上一轮杀伤的未编辑或敲除本发明靶点的细胞及A375-GFP细胞混合物转移到新的96孔板中，用IncuCyte记录新一轮杀伤中GFP荧光信号的变化曲线。
(十一)CBA法检测细胞因子释放
对于基因编辑后的细胞与自体肿瘤细胞共培养，收集共培养上清液，采用CBA试剂盒(BD)检测未编辑或敲除本发明靶点的细胞的细胞因子释放情况。
(十二)基因编辑细胞的体内药效检测
手术获得肿瘤组织样本，无菌条件下转移至实验室，在无菌超净台内切除肿瘤坏死组织，并将肿瘤组织切割至1-2mm3大小，将切割的肿瘤组织滴加少许Matrigel胶在SPF级动物房内皮下接种于NOG小鼠(维通利华，品系代码408)，每只小鼠接种4-5块组织，该批肿瘤组织为F0代。待F0代肿瘤组织在小鼠皮下长至800mm3，手术切除该F0肿瘤组织，再次将肿瘤组织分割为1-2mm3，部分冻存备用，部分皮下接种于第二批NOG小鼠，在小鼠中继续传代扩增，该批肿瘤组织为F1代。依次类推，直至肿瘤组织在小鼠中传至F3代，定期测量肿瘤组织大小，直至肿瘤体积生长至约100mm3后，对小鼠进行随机分组。NT组小鼠静脉注射未经基因编辑的细胞，试验组小鼠静脉注射本 发明基因编辑的细胞。
靶向各个靶点的sgRNA(single guide RNA，或简称guide)的序列，根据本发明提供的序列进行合成。其中，靶向各个靶点的区域可以选自：该靶点基因的外显子区域、距离该靶点基因的外显子约100bp或约20bp的内含子区域、和该靶点基因的起始密码子之前约1500bp的区域。当靶向各个靶点的区域选自该靶点基因的起始密码子之前约1500bp的区域时，图1A-1D显示了本发明提供的相对于起始密码子的之前的，具有约3个以上的转录因子结合数的一段连续区域(如纵坐标在3以上的、黑色线段显示的区域)。本发明还提供了，靶向各个靶点的外显子区域、距离该靶点基因的外显子约100bp或约20bp的内含子区域，敲除后可增强细胞功能的sgRNA。优选地，在实施例中的sgRNA编号信息如下所示：



实施例2
根据实施例中的试验方法，检测靶向TNFAIP3的sgRNA的敲除效果。
(A)敲除效率

其中，A表示敲除效率大于或等于70％，B表示敲除效率大于或等于50％且小于70％，C表示敲除效率大于或等于10％且小于50％，D表示敲除效率小于10％，N/A表示未检测。
其中，供者045为健康供者，供者053为健康供者，用于分离PBMC细胞后转导TCR作为TCR-T细胞。
(B)增殖能力
图2A显示无刺激培养基组中TNFAIP3基因编辑的TCR-T扩增倍数。
图2B-2C显示TransACT刺激组中TNFAIP3基因编辑的TCR-T扩增倍数。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于未基因编辑的对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的扩增能力。
(C)杀伤能力
图2D-2G显示不同供者来源的TCR-T细胞在TNFAIP3基因编辑 后的靶细胞杀伤能力。图2D、2F展示各时间点杀伤曲线，以及图2E、2G展示试验终点各个试验组的杀伤情况，均高于未编辑的NT组。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的靶细胞杀伤能力和/或连续杀伤能力。
(D)细胞因子释放能力
采用CBA试剂盒检测未编辑或敲除本发明靶点的细胞的细胞因子释放情况。
图2H-2K显示TNFAIP3基因编辑的TCR-T细胞的细胞因子释放能力。
结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的细胞因子释放能力。
实施例3
根据实施例中的试验方法，检测靶向ZC3H12A的sgRNA的敲除效果。
其中，A表示敲除效率大于或等于70％，B表示敲除效率大于或等于50％且小于70％，C表示敲除效率大于或等于10％且小于50％，D表示敲除效率小于10％，N/A表示未检测。
(B)增殖能力
图3A显示无刺激培养基组中ZC3H12A基因编辑的TCR-T扩增倍数。
图3B显示TransACT刺激组中ZC3H12A基因编辑的TCR-T扩增倍数。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于未基因编辑的对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的扩增能力。
(C)杀伤能力
图3C-3F显示ZC3H12A基因编辑的TCR-T细胞的靶细胞杀伤能力。图3C、3E展示各时间点杀伤曲线，以及图3D、3F展示试验终点各个试验组的杀伤情况，均高于未编辑的NT组。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的靶细胞杀伤能力和/或连续杀伤能力。
(D)细胞因子释放能力
采用CBA试剂盒检测未编辑或敲除本发明靶点的细胞的细胞因子释放情况。
图3G-3J显示ZC3H12A基因编辑的TCR-T细胞的细胞因子释放能力。
结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细 胞可以具有显著提升的细胞因子释放能力。
实施例4
根据实施例中的试验方法，检测靶向SOCS1的sgRNA的敲除效果。
其中，A表示敲除效率大于或等于70％，B表示敲除效率大于或等于50％且小于70％，C表示敲除效率大于或等于10％且小于50％，D表示敲除效率小于10％，N/A表示未检测。
其中，供者005为健康供者，供者006为健康供者，供者031为健康供者，用于分离PBMC细胞后转导TCR作为TCR-T细胞。供者105为肺癌供者、供者306为宫颈癌供者，用于分离提供TIL。
(B)增殖能力
图4A显示无刺激培养基组中SOCS1基因编辑的TCR-T扩增倍数。图4B显示TransACT刺激组中SOCS1基因编辑的TCR-T扩增倍数。
图4C显示无刺激培养基组中SOCS1基因编辑的TIL扩增倍数。图4D显示TransACT刺激组中SOCS1基因编辑的TIL扩增倍数。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于未基因编辑的对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的扩增能力。
(C)杀伤能力
图4E-4H显示SOCS1基因编辑的TCR-T细胞的靶细胞杀伤能力。各图展示各时间点杀伤曲线，以及展示具体时间点各个试验组的杀伤情况，均高于未编辑的NT组。
图4I显示SOCS1基因编辑的TIL细胞的靶细胞杀伤能力。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的靶细胞杀伤能力和/或连续杀伤能力。
(D)细胞因子表达和释放
采用流式细胞仪检测未编辑或敲除本发明靶点的细胞的细胞因子表达情况，采用CBA试剂盒检测细胞因子释放能力。
图4J显示未刺激组SOCS1基因编辑的TCR-T细胞的细胞因子表达。图4K显示CD3抗体刺激组SOCS1基因编辑的TCR-T细胞的细胞因子表达。图4L显示与A375靶细胞共培养的SOCS1基因编辑的TCR-T细胞的细胞因子释放能力。
图4M显示未刺激组SOCS1基因编辑的TIL细胞的细胞因子表达。图4N显示TransACT刺激组SOCS1基因编辑的TIL细胞的细胞因子表达。
结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的细胞因子表达和/或释放能力。
(E)流式细胞检测
图4O显示SOCS1基因编辑后的TIL细胞具有更高的干性细胞比例。
图4P显示SOCS1基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
例如，干性细胞可以是CD39阴性CD69阴性细胞。例如，耗竭T细胞可以是PD-1阳性、LAG-3阳性、TIM-3阳性、CD38阳性和/或CD101阳性细胞。结果表明，相对于未基因编辑的对照组(NT)，本发明的基因编辑的TIL细胞可以具有更为有利的细胞表型特征。
实施例5
根据实施例中的试验方法，检测靶向CBLB的sgRNA的敲除效果。
其中，A表示敲除效率大于或等于70％，B表示敲除效率大于或等于50％且小于70％，C表示敲除效率大于或等于10％且小于50％，D表示敲除效率小于10％，N/A表示未检测。
(B)增殖能力
图5A-5B显示无刺激培养基组中CBLB基因编辑的TCR-T扩增倍数。图5C-5D显示TransACT刺激组中CBLB基因编辑的TCR-T扩增倍数。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于未基因编辑的对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的扩增能力。
(C)杀伤能力
图5E-5H显示CBLB基因编辑的TCR-T细胞的靶细胞杀伤能力。图5E、5G展示各时间点杀伤曲线，以及图5F、5H展示试验终点各个试验组的杀伤情况，均高于未编辑的NT组。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的靶细胞杀伤能力和/或连续杀伤能力。
(D)细胞因子释放能力
采用CBA试剂盒检测未编辑或敲除本发明靶点的细胞的细胞因子释放情况。
图5I-5L显示CBLB基因编辑的TCR-T细胞的细胞因子释放能力。
结果表明，相对于对照组(NT)，本发明的靶点基因编辑的T细胞可以具有显著提升的细胞因子释放能力。
实施例6
根据实施例中的试验方法，检测靶向选自TNFAIP3、ZC3H12A、SOCS1和CBLB中两个或更多个的sgRNA的敲除效果。

其中，A表示敲除效率大于或等于70％，B表示敲除效率大于或等于50％且小于70％，C表示敲除效率大于或等于10％且小于50％，D表示敲除效率小于10％，N/A表示未检测。
其中供者713为宫颈癌患者，供者316为恶性黑色素瘤患者，供者504、607为卵巢癌患者。供者801为宫颈癌患者、供者312为宫颈癌患者。
(B)扩增情况
图6A、6B、6C、6E、6G、6H、6J显示无刺激培养基组中组合基因编辑的TIL扩增倍数。
图6D、6F、6I显示CD3抗体刺激组中组合基因编辑的TIL扩增倍数。
图6K显示TransACT抗体刺激组中组合基因编辑的TIL扩增倍数。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于未基因编辑的对照组(NT)，本发明的靶点组合基因编辑的TIL细胞可以具有显著提升的扩增能力。
(C)杀伤情况
图7A至7S显示组合基因编辑的TIL细胞的靶细胞杀伤能力。
图7T显示组合基因编辑的TIL细胞的对自体肿瘤类器官的杀伤能力。
其中显著性差异为相对于未编辑的NT组，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.0001。结果表明，相对于对照组(NT)，本发明的靶点组合基因编辑的TIL细胞可以具有显著提升的靶细胞杀伤能力。
(D)细胞流式检测
图8A、8C、8E、8G、8H、8I、8L、8M、8O显示组合基因编辑后的TIL细胞具有更低的耗竭T细胞比例。
图8B、8D、8F、8K显示组合基因编辑后的TIL细胞具有更高的中心记忆T细胞比例。
图8J显示TNFAIP3和ZC3H12A组合基因编辑后的TIL细胞具有更高的幼稚T细胞比例。
图8N显示TNFAIP3和SOCS1组合基因编辑后的TIL细胞具有更高的干性细胞比例。
例如，耗竭T细胞可以是PD-1阳性、LAG-3阳性、TIM-3阳性、CD38阳性和/或CD101阳性细胞。例如，中心记忆T细胞可以是CD45RO阳性CD62L阳性细胞。例如，干性细胞可以是CD39阴性CD69阴性细胞。结果表明，相对于未基因编辑的对照组(NT)，本发明的靶点组合基因编辑的TIL细胞可以具有更为有利的细胞表型特征。
(E)细胞因子表达和释放
图9A、9C、9E、9G、9I、9L显示CD3抗体刺激组的各种组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9B、9D、9F、9H、9J、9K显示无刺激Medium组的各种各种组合基因编辑后的TIL细胞具有更高的细胞因子表达比例。
图9M显示与自体肿瘤类器官共培养的TNFAIP3和SOCS1组合基因编辑后的TIL细胞具有更高的细胞因子释放能力。
例如，细胞因子表达能力包含更高的IFN-γ表达能力、更高的TNF-α表达能力、更高的CD107a表达能力或更高的GZMB表达能力。
结果表明，本发明的靶点组合基因编辑的TIL细胞具有更高的功能性细胞因子表达能力。
(F)细胞凋亡检测
图10显示对于来源于供者504的TIL细胞的凋亡检测结果。
结果表明，本发明的靶点组合基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
实施例7基因编辑细胞的体内药效检测
(一)本发明基因编辑的TCRT的体内效果
对免疫缺陷鼠(NOG小鼠，维通利华，品系代码408)进行皮下接种A375细胞，接种数量约为1×106个细胞/只。接种7天后，在肿瘤体积到达50-80mm3左右，根据肿瘤体积大小对小鼠，按照以下方案进行随机分组。

各组尾静脉注射本发明提供的sgRNA编号进行基因编辑的TCR-T细胞当日记为第0天。同时进行腹腔注射IL-2，每12小时一次，连续6次，IL-2的注射剂量为NT组3E5IU/次，各实验组0.6E5IU/次。每周测量肿瘤体积两次，计算第28天肿瘤生长抑制率，如下所示。
数据使用T-test分析。其中，A表示TGI大于或等于50％。
(二)本发明基因编辑的TIL的体内效果
对免疫缺陷鼠(NOG小鼠，维通利华，品系代码408)进行皮下接种HeyT-30细胞，接种数量约为1×106个细胞/只。接种7天后，在肿瘤体积到达50-80mm3左右，根据肿瘤体积大小对小鼠，按照以下方案进行随机分组。
各组尾静脉注射本发明提供的sgRNA编号进行基因编辑的TIL细胞当日记为第0天。同时进行腹腔注射IL-2，每12小时一次，连续6次，IL-2的注射剂量为NT组3E5IU/次，各实验组0.6E5IU/次。每周测量肿瘤体积两次，计算肿瘤生长抑制率，如下所示。
数据使用T-test分析。其中，A表示TGI大于或等于80％。
结果显示，实验组的小鼠相比于NT组产生了更好的肿瘤控制效果和/或体内功能性细胞因子释放，证明本发明的提供的sgRNA敲除的细胞抑瘤效果明显强于NT组。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本发明所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。
表1A人TNFAIP3优选靶向亚区域的基因组坐标

表1B人ZC3H12A优选靶向亚区域的基因组坐标
表1C人SOCS1优选靶向亚区域的基因组坐标
表1D人CBLB优选靶向亚区域的基因组坐标

表2A人TNFAIP3用于多敲基因组坐标


表2B人ZC3H12A用于多敲基因组坐标

表2C人SOCS1用于多敲基因组坐标
表2D人CBLB用于多敲基因组坐标

表3A本发明人TNFAIP3基因组坐标












表3B本发明人ZC3H12A基因组坐标







表3C本发明人SOCS1基因组坐标









表3D本发明人CBLB基因组坐标













Claims (41)

  1. 一种培养细胞的方法，所述方法包含：使所述细胞的肽酶C64家族成员和/或其功能活性片段的表达降低和/或活性减弱。
  2. 根据权利要求1所述的方法，其中所述细胞包含免疫细胞。
  3. 根据权利要求2所述的方法，其中所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
  4. 根据权利要求2-3中任一项所述的方法，其中所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
  5. 根据权利要求2-4中任一项所述的方法，其中所述免疫细胞来源于干细胞分化的免疫细胞。
  6. 根据权利要求5所述的方法，其中所述干细胞包含诱导的多能干细胞(iPSC)。
  7. 根据权利要求2-6中任一项所述的方法，其中所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。
  8. 根据权利要求2-7中任一项所述的方法，其中所述免疫细胞包含αβT细胞和/或γδT细胞。
  9. 根据权利要求2-8中任一项所述的方法，其中所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
  10. 根据权利要求9所述的方法，其中所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的 TIL。
  11. 根据权利要求10所述的方法，其中所述碎片的体积为约1立方毫米至约27立方毫米。
  12. 根据权利要求2-11中任一项所述的方法，其中所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
  13. 根据权利要求12所述的方法，其中所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
  14. 根据权利要求2-13中任一项所述的方法，其中所述免疫细胞包含嵌合抗原受体和/或T细胞受体。
  15. 根据权利要求1-14中任一项所述的方法，其中使所述细胞的肽酶C64家族成员的表达降低和/或活性减弱包含抑制去泛素化酶和/或锌指核酸酶的功能。
  16. 根据权利要求1-15中任一项所述的方法，其中与肽酶C64家族成员的表达和/或活性未改变的细胞相比，使所述肽酶C64家族成员的表达降低和/或活性减弱获得的细胞显示出改善的细胞特性。
  17. 根据权利要求16所述的方法，其中所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
  18. 根据权利要求17所述的方法，其中所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细 胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
  19. 根据权利要求1-18中任一项所述的方法，其中所述肽酶C64家族成员包含泛素结合域。
  20. 根据权利要求1-19中任一项所述的方法，其中所述肽酶C64家族成员包含TNFAIP3。
  21. 根据权利要求1-20中任一项所述的方法，其中使所述细胞的肽酶C64家族成员的表达降低和/或活性减弱包含将基因调控系统引入所述细胞中。
  22. 根据权利要求21所述的方法，其中所述基因调控系统能够在DNA水平破坏所述肽酶C64家族成员。
  23. 根据权利要求21-22中任一项所述的方法，其中所述基因调控系统包含指导核酸分子和酶蛋白。
  24. 根据权利要求23所述的方法，其中使所述肽酶C64家族成员的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)、包含gRNA与Cas蛋白的LNP，或者包含编码gRNA与编码Cas蛋白的核酸的LNP引入所述细胞中。
  25. 根据权利要求23-24中任一项所述的方法，其中所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
  26. 根据权利要求23-25中任一项所述的方法，其中所述指导核酸分子包含指导RNA(gRNA)。
  27. 根据权利要求23-26中任一项所述的方法，其中所述指导核酸 分子能够与所述肽酶C64家族成员的序列结合。
  28. 根据权利要求23-27中任一项所述的方法，其中所述指导核酸分子能够与选自表1A所示的基因组坐标定义的区域或其片段结合。
  29. 根据权利要求23-28中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：107-212、1562-2532。
  30. 根据权利要求23-29中任一项所述的方法，其中所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、CGG和GGG。
  31. 根据权利要求23-30中任一项所述的方法，其中所述指导核酸分子包括靶向结构域，所述靶向结构域包含如SEQ ID NO：1-106、591-1561、7267-7324、7419、7420中任一项所示的序列。
  32. 根据权利要求1-31中任一项所述的方法，其中与肽酶C64家族成员的表达和/或活性未改变的细胞相比，使所述肽酶C64家族成员的表达降低和/或活性减弱获得的细胞中表达所述目的基因的产物的细胞比例降低和/或单个细胞中所述目的基因的表达量下降。
  33. 根据权利要求1-32中任一项所述的方法，其中所述使所述肽酶C64家族成员的表达降低和/或活性减弱获得的细胞中，表达所述目的基因的细胞比例为约95％或以下。
  34. 一种细胞，所述细胞经过权利要求1-33中任一项所述的方法获得。
  35. 一种组合物，其包含权利要求34所述的细胞。
  36. 一种药物组合物，其包含权利要求34所述的细胞和/或权利要求35所述的组合物，以及任选的药学上可接受的载体。
  37. 一种影响细胞生长的方法，其包含施用权利要求34所述的细胞、权利要求35所述的组合物和/或权利要求36所述的药物组合物。
  38. 权利要求34所述的细胞、权利要求35所述的组合物和/或权利要求36所述的药物组合物在制备药物中的应用，其中所述药物用于预防和/或治疗疾病和/或症状。
  39. 根据权利要求38所述的应用，其中所述疾病和/或症状包含肿瘤。
  40. 根据权利要求38-39中任一项所述的应用，其中所述疾病和/或症状包含实体瘤。
  41. 根据权利要求38-40中任一项所述的应用，其中所述疾病和/或症状包含选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
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