WO2025157155A1 - Cellule modifiée et son utilisation - Google Patents

Cellule modifiée et son utilisation

Info

Publication number
WO2025157155A1
WO2025157155A1 PCT/CN2025/073804 CN2025073804W WO2025157155A1 WO 2025157155 A1 WO2025157155 A1 WO 2025157155A1 CN 2025073804 W CN2025073804 W CN 2025073804W WO 2025157155 A1 WO2025157155 A1 WO 2025157155A1
Authority
WO
WIPO (PCT)
Prior art keywords
family
cells
present
protein
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/073804
Other languages
English (en)
Chinese (zh)
Inventor
刘雅容
孙静玮
盛耀
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Grit Biotechnology Co Ltd
Shanghai Grit Biotechnology Co Ltd
Suzhou Grit Biotechnology Co Ltd
Zhuhai Tuoyu Biotechnology Co Ltd
Original Assignee
Shenzhen Grit Biotechnology Co Ltd
Shanghai Grit Biotechnology Co Ltd
Suzhou Grit Biotechnology Co Ltd
Zhuhai Tuoyu Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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
Publication of WO2025157155A1 publication Critical patent/WO2025157155A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a modified cell and its use.
  • 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-like cells.
  • the present invention provides a method for culturing cells, comprising: reducing the expression and/or weakening the activity of family members and/or functionally active fragments thereof selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen permease regulator family, TRAF-related factor family, tumor necrosis factor receptor-related factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family.
  • RBR RING-Between-RING
  • carnitine/choline acetyltransferase family peptidase M1 family
  • nitric oxide synthase interacting protein family nitrogen permease regulator family
  • TRAF-related factor family tumor necros
  • 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 cells of the present invention, and optionally a pharmaceutically acceptable carrier.
  • the present invention provides a method of influencing cell growth comprising administering a cell of the present invention and/or a pharmaceutical composition of the present 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.
  • Figures 1A-1L show the human ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2 gene editing targeting segments relative to the start codon provided by the present invention, for example, can be a continuous region having about 3 or more transcription factor binding numbers; and can be an exon region of the gene or an intron region about 100 bp away from the exon.
  • Figure 2A shows the expansion fold of TILs edited with the gene of the present invention in the non-stimulation culture medium group.
  • FIG2B shows the expansion fold of TILs edited with the gene of the present invention in the TransACT stimulation group.
  • FIGS 3A-3H show the target cell killing ability of the gene-edited TIL cells of the present invention.
  • Figure 3I shows the killing ability of the gene-edited TIL cells of the present invention on the autologous tumor cell PDO model.
  • FIG4 shows the apoptosis ratio of gene-edited TIL cells of the present invention.
  • Figure 5A shows that the gene-edited TIL cells of the present invention have a higher proportion of central memory T cells.
  • Figure 5B shows that the gene-edited TIL cells of the present invention have a higher proportion of stem cells.
  • Figures 5C-5D show that the gene-edited TIL cells of the present invention have a lower proportion of exhausted T cells.
  • Figures 6A-6G show the cytokine expression of the gene-edited TIL cells of the unstimulated group.
  • Figures 6H-6O show the cytokine expression of the gene-edited TIL cells of the present invention in the TransACT stimulation group.
  • Figure 6P shows the cytokine release after co-culture of the gene-edited TIL cells of the present invention with the autologous tumor cell PDO model.
  • Figure 7A shows the expansion fold of TILs edited with the combination gene of the present invention in the non-stimulation culture medium group.
  • FIG7B shows the expansion fold of TILs edited with the combination gene of the present invention in the TransACT stimulation group.
  • FIGS 8A-8D show the target cell killing ability of TIL cells edited by the combination of genes of the present invention.
  • FIGS 9A-9B show that the TIL cells after gene editing of the combination of the present invention have a higher proportion of central memory T cells.
  • Figures 10A-10C show the cytokine expression of TIL cells edited with the combination gene of the present invention in the unstimulated group.
  • Figures 10D-10F show the cytokine expression of TIL cells edited with the combination gene of the present invention in the TransACT stimulation group.
  • Figure 10G shows the cytokine release after co-culture of TIL cells edited by the combination of genes of the present invention with the autologous tumor cell PDO model.
  • the term "adenylate cyclase family member” generally refers to a family member protein having a cyclase domain or a functionally active fragment thereof.
  • the adenylate cyclase family member may include ADCY7.
  • the UniProt number of the adenylate cyclase family member may be P51828.
  • the adenylate cyclase family member of the present application 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 its processing and/or modification in cells.
  • the adenylate cyclase family member of the present application may include its functionally active fragments and any other domains.
  • the term "RBR (RING-Between-RING) family member” generally refers to a family member protein having a RING-type zinc finger domain or a functionally active fragment thereof.
  • the RBR (RING-Between-RING) family member may include ARIH2.
  • the UniProt number of the RBR (RING-Between-RING) family member may be O95376.
  • the RBR (RING-Between-RING) family member of the present application may 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 RBR (RING-Between-RING) family member of the present application may include its functionally active fragments and any other domains.
  • the term "carnitine/choline acetyltransferase family member” generally refers to a family member protein or its functionally active fragment with an acetyltransferase domain.
  • the carnitine/choline acetyltransferase family member may include CPT2.
  • the UniProt numbering of the peptidase M1 family member may be P23786.
  • the carnitine/choline acetyltransferase family member of the present application may also encompass its functionally active fragments, and is not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, its active fragments, or the materials comprising the functionally active fragments produced after the processing and/or modification thereof occurring in the cell.
  • the carnitine/choline acetyltransferase family member of the present application may include its functionally active fragments and other arbitrary domains.
  • peptidase M1 family member generally refers to a family member protein having an aminopeptidase domain or a functionally active fragment thereof.
  • a peptidase M1 family member may include LNPEP.
  • the UniProt number of a peptidase M1 family member may be Q9UIQ6.
  • the peptidase M1 family member of this application may also encompass 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 thereof in cells.
  • the peptidase M1 family member of this application may include functionally active fragments thereof as well as any other domains.
  • nitric oxide synthase interacting protein family member generally refers to a family member protein or a functionally active fragment thereof having a nitric oxide synthase binding domain.
  • a nitric oxide synthase interacting protein family member may include NOSIP.
  • the UniProt numbering of a nitric oxide synthase interacting protein family member may be Q9Y314.
  • the nitric oxide synthase interacting protein family members of the present application may also encompass 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 thereof in cells.
  • the nitric oxide synthase interacting protein family members of the present application may include functionally active fragments thereof and any other domains.
  • nitrogen permease regulator family member generally refers to a family member protein or a functionally active fragment thereof having a GTPase domain.
  • the nitrogen permease regulator family member may include NPRL3.
  • the UniProt number of the nitrogen permease regulator family member may be Q12980.
  • the nitrogen permease regulator family member of the present application 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 comprising the functionally active fragments produced after processing and/or modification thereof in cells.
  • the nitrogen permease regulator family member of the present application may include its functionally active fragments and other arbitrary domains.
  • the term "TRAF-associated factor family member” generally refers to a family member protein having a TRAF binding domain or a functionally active fragment thereof.
  • a TRAF-associated factor family member may include TANK.
  • the UniProt number of a TRAF-associated factor family member may be Q92844.
  • the TRAF-associated factor family members of this application may also encompass 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 TRAF-associated factor family members of this application may include functionally active fragments thereof as well as any other domains.
  • tumor necrosis factor receptor-associated factor family member generally refers to a family member protein having a receptor cytoplasmic binding domain or a functionally active fragment thereof.
  • a tumor necrosis factor receptor-associated factor family member may include TRAF3.
  • the UniProt number of a tumor necrosis factor receptor-associated factor family member may be Q13114.
  • the tumor necrosis factor receptor-associated factor family member of this application may also encompass 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 tumor necrosis factor receptor-associated factor family member of this application may include functionally active fragments thereof as well as any other domains.
  • the term "TSC protein family member” generally refers to a family member protein having a helical domain or a functionally active fragment thereof.
  • the TSC protein family member may include TSC1.
  • the UniProt number of the TSC protein family member may be Q92574.
  • the TSC protein family members of the present application may also cover 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 TSC protein family members of the present application may include functionally active fragments thereof and any other structural domains.
  • BTB Broad-Complex, Tramtrack and Bric-a-brac protein family member
  • a BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may include ZBTB7B.
  • the UniProt number of a BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may be O15156.
  • the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member in this application may also include functionally active fragments thereof, including but 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 BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family members of the present application may include their functionally active fragments and any other structural domains.
  • ZC3H12 protein family member generally refers to a family member protein having a C3H1-type zinc finger domain or a functionally active fragment thereof.
  • a ZC3H12 protein family member may include ZC3H12D.
  • the UniProt number of a ZC3H12 protein family member may be A2A288.
  • the ZC3H12 protein family members of this application may also encompass 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 protein family members of this application may include functionally active fragments thereof as well as any other domains.
  • the term "RC3H family member” generally refers to a family member protein having an ROQ domain or a functionally active fragment thereof.
  • the RC3H family member may include RC3H2.
  • the UniProt number of the RC3H family member may be Q9HBD1.
  • the RC3H family members of this application 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 RC3H family members of this application may include functionally active fragments thereof as well as any other domains.
  • AF4 family member generally refers to a family member protein having a transcriptional activation domain or a functionally active fragment thereof.
  • the AF4 family member may include AFF3.
  • the UniProt number of the AF4 family member may be P51826.
  • the AF4 family members of this application may also encompass 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 AF4 family members of this application may include functionally active fragments thereof as well as any other domains.
  • tyrosine protein kinase family member generally refers to a family member protein having a phosphotransferase domain or a functionally active fragment thereof.
  • a tyrosine protein kinase family member may include AXL.
  • the UniProt number of a tyrosine protein kinase family member may be P30530.
  • the tyrosine protein kinase family members of this application 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 such functionally active fragments produced after processing and/or modification thereof in cells.
  • the tyrosine protein kinase family members of this application may include functionally active fragments thereof as well as any other domains.
  • bZIP family member generally refers to a family member protein or a functionally active fragment thereof having a bZIP-related DNA binding domain.
  • a bZIP family member may include NFE2L1.
  • the UniProt number of a bZIP family member may be Q14494.
  • the bZIP family members of this application may also encompass 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 bZIP family members of this application may include functionally active fragments thereof and any other domains.
  • nuclear receptor family member generally refers to a family member protein or a functionally active fragment thereof having a nuclear receptor-associated DNA binding domain.
  • a nuclear receptor family member may include RARG.
  • the UniProt number of a nuclear receptor family member may be P13631.
  • the nuclear receptor family members of the present application may also encompass 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 nuclear receptor family members of the present application may include functionally active fragments thereof and any other domains.
  • the term "ubiquitin family member” generally refers to a family member protein having a ubiquitin-like domain or a functionally active fragment thereof.
  • the ubiquitin family member may include UBFD1.
  • the UniProt number of the ubiquitin family member may be O14562.
  • the ubiquitin family members of this application may also encompass 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 ubiquitin family members of this application may include functionally active fragments thereof and any other domains.
  • Bcl-2 family member generally refers to a family member protein having a Bcl-2 homology domain 3 domain or a functionally active fragment thereof.
  • a Bcl-2 family member may include BCL2L11.
  • the UniProt number of a Bcl-2 family member may be O43521.
  • the Bcl-2 family members of this application may also encompass functionally active fragments thereof, not limited to 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 Bcl-2 family members of this application may include functionally active fragments thereof as well as any other domains.
  • protein tyrosine phosphatase family member generally refers to a family member protein or a functionally active fragment thereof having a tyrosine phosphatase domain.
  • the protein tyrosine phosphatase family member may include PTPN2.
  • the UniProt number of the protein tyrosine phosphatase family member may be P17706.
  • the protein tyrosine phosphatase family member of the present application may also encompass functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances comprising the functionally active fragments thereof produced after processing and/or modification thereof in cells.
  • the protein tyrosine phosphatase family member of the present application may include functionally active fragments thereof and any other domains.
  • 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 herein may also encompass functionally active fragments thereof, including but not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing such functionally active fragments produced by cellular processing and/or modification thereof.
  • the CBL family members herein may include functionally active fragments thereof as well as 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 a STAT-induced STAT inhibitor (SSI) family member may be O15524.
  • the STAT-induced STAT inhibitor (SSI) family member of the present application may also encompass 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 STAT-induced STAT inhibitor (SSI) family member of the present application may include functionally active fragments thereof and any other domains.
  • the term "peptidase C64 family member” generally refers to a family member protein having a ubiquitin binding domain or a functionally active fragment thereof.
  • 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 this application 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 this application may include its functionally active fragments and any other domains.
  • ZC3H12 family member generally refers to a family member protein having a C3H1-type zinc finger domain or a functionally active fragment thereof.
  • a ZC3H12 family member may include ZC3H12A.
  • the UniProt number of a ZC3H12 family member may be Q5D1E8.
  • the ZC3H12 family members of this application may also encompass 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 this application may include functionally active fragments thereof as well as any other domains.
  • IKAROS zinc finger protein family member generally refers to a family member protein having a zinc finger domain or a functionally active fragment thereof.
  • an IKAROS zinc finger protein family member may include IKZF1.
  • the UniProt number of an IKAROS zinc finger protein family member may be Q13422.
  • the IKAROS zinc finger protein family members of the present invention may also include functionally active fragments thereof, including but not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing such functionally active fragments produced after processing and/or modification thereof in cells.
  • the IKAROS zinc finger protein family members of the present invention may include functionally active fragments thereof as well as any other domains.
  • TNFAIP3 tumor necrosis factor alpha-induced protein 3
  • TNFAIP3 can ubiquitinate signal transduction substances of the NF- ⁇ B pathway.
  • the UniProt accession number of TNFAIP3 can be P21580.
  • TNFAIP3 can include unprocessed TNFAIP3, any form of processed TNFAIP3, variants of TNFAIP3, or substances comprising functionally active fragments 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 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 GTPase activating protein 1 family member of the present invention may include functionally active fragments thereof and any other domains.
  • FGF-binding protein family member generally refers to a family member protein having an FGF-binding domain, or a functionally active fragment thereof.
  • an FGF-binding protein family member may include FIBP.
  • the UniProt number for an FGF-binding protein family member may be O43427.
  • the FGF-binding protein family members of the present invention may also encompass functionally active fragments thereof, including but not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing such functionally active fragments produced by cellular processing and/or modification thereof.
  • the FGF-binding protein family members of the present invention may include functionally active fragments thereof as well as any other domains.
  • the term "Mediator (MED) family member” generally refers to a family member protein having a CDK8 binding domain or a functionally active fragment thereof.
  • 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 encompass 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 Mediator (MED) family members of the present invention may include functionally active fragments thereof and any other structural 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 CTLs), 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, thereby leading to activation and proliferation of the cell.
  • the extracellular domain of CAR can recognize a tag fused to an antibody or its antigen-binding fragment. For example, a single CAR construct can be made to target a variety of different antigens by replacing another antibody with one antibody.
  • the extracellular domain of CAR may include an antigen-binding fragment derived from an antibody.
  • the antigen-binding domains used in the present invention may include, for example, scFv, antibodies, antigen-binding regions of antibodies, variable regions of heavy chains/light chains, and/or single-chain antibodies.
  • T cell receptor generally refers to an engineered antigen receptor.
  • a TCR may comprise TCR ⁇ and/or TCR ⁇ chains that have been isolated and cloned from a T cell population that recognizes a specific target antigen.
  • TCR ⁇ and/or TCR ⁇ genes i.e., TRAC and TRBC
  • TRAC and TRBC can 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 leading to activation and proliferation of TCR engineered cells.
  • MHC major histocompatibility complex
  • a gene regulatory system generally refers to a system that regulates the expression or activity of a target gene.
  • a gene regulatory system may comprise a gene regulatory molecule.
  • a gene regulatory system may regulate the expression or activity of a gene, such as by inactivating or activating the gene, increasing or decreasing the amount of the gene, increasing or decreasing the amount of transcription of the gene, and/or inactivating or activating the transcription product of the gene; for example, a gene regulatory system may regulate the expression or activity of a gene, such as by 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 location within the target DNA.
  • hybridization between the gRNA and the DNA targeting sequence promotes the formation of a CRISPR complex
  • complete complementarity may not be required, for example, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex.
  • 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 that can 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. Cutting 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 encompass 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 cleave a target sequence under the guidance of the nucleic acid therein.
  • the ribonucleoprotein complex can be a complex formed by a Cas protein and a guide RNA.
  • lipid nanoparticle generally refers to a lipid-nucleic acid particle or 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, self
  • 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 can also include lipid derivatives, such as lipids covalently or non-covalently bound to proteins or polypeptides.
  • the components in LNP may further include a polypeptide component, wherein the polypeptide component may 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.
  • cleaving an exon sequence of a target gene can reduce the activity or function of the target gene.
  • the term "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 are sheared off from the endogenous RNA before being translated into protein. For example, targeting the position of the intron for editing can reduce the activity or function of the target gene. For example, targeting the junction of introns and exons, such as editing an intron region 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 to 1500 bp upstream of the start codon, preferably 0 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 following a target sequence.
  • PAM sequence can be used to determine the location of the cleavage. For example, by determining the PAM region, those skilled in the art can easily determine the appropriate target sequence location and can easily design the guide RNA 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%).
  • the amount of the product expressed by the gene in the cell is reduced or the proportion of cells comprising the product expressed by 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 can be detected in a cell population 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 transcriptional and/or translational status of the gene.
  • a reduction in gene activity e.g., at least about 5-100% may mean that the transcriptional function of the gene is reduced, the gene cannot be transcribed normally, or the function of the gene transcription product is inhibited.
  • CD80 generally refers to a cell-stimulatory molecule.
  • CD80 can be a ligand for CD28.
  • CD80 can be found in GenBank Accession No. P33681.
  • the CD80 protein of the present invention also encompasses functionally active fragments thereof, and is not limited to substances comprising 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 as well as any other domains.
  • CD86 generally refers to a cell stimulatory molecule.
  • CD86 can be a ligand for CD28.
  • CD86 can be found in GenBank Accession No. P42081.
  • the CD86 protein of the present invention can also include functionally active fragments thereof, and is 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 as well as any other domains.
  • secreted generally refers to a substance that can be located outside of a cell.
  • a secreted substance can be synthesized within a cell and then transported to the extracellular space of the cell.
  • whether a substance is secreted can be detected using an 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.
  • TCR can be responsible for recognizing antigens that are bound to major histocompatibility complex molecules.
  • TCR can be composed of a heterodimer of alpha ( ⁇ ) and beta ( ⁇ ) chains, or composed of gamma and delta ( ⁇ / ⁇ ) chains.
  • TCR can exist in ⁇ / ⁇ and ⁇ / ⁇ forms, which are structurally similar but have unique anatomical locations and functions.
  • TCR can be a TCR that is modified on any cell that expresses TCR.
  • the type of TCR can be analyzed by TCR subtyping reagents.
  • clonal diversity generally refers to the presence of multiple clonal types in a given substance.
  • TCR clonal diversity can refer to TCRs having different sequence structures and/or antigen recognition capabilities.
  • TCR diversity is often distinguished by ⁇ -chain subtypes, which can 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 to have higher clonal diversity.
  • CD4 + cells generally refer to CD4-positive cells, such as T cells.
  • CD4 + cells and “CD4-positive cells” can be used interchangeably. 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. For example, 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. For example, see Tay, RE, Richardson, EK et al. (2020). Cancer Gene Therapy, 1-13. However, 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 CD8-positive, such as T cells.
  • CD8 + cells and CD8-positive cells are used interchangeably. These cells can be identified by methods known in the art, such as staining the cells with fluorescently labeled antibodies against CD8 and using fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • IC50 value or “IC50 value” generally refers to the concentration of the target substance required to achieve 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 a BIAcore system (Pharmacia Biosensor, Piscataway, NJ) to measure the real-time binding interaction between a ligand (a substance immobilized on a biosensor matrix) and an analyte (a substance in solution) by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • SPR surface plasmon resonance
  • SPR surface plasmon resonance
  • the term "encode” generally refers to the ability to directly or indirectly infer, based on essentially defined rules, the structure or composition of one molecule from the structure or composition of another related class of molecules.
  • the nucleotide sequence can be inferred from the amino acid sequence, or from the properties of a deoxyribonucleic acid that transcribes complementary nucleic acids, including nucleic acids that can be translated into polypeptides.
  • a deoxyribonucleic acid can encode an RNA transcribed from the deoxyribonucleic acid.
  • a deoxyribonucleic acid can encode a polypeptide translated from the RNA transcribed from the 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 also known as “natural killer cell,” generally refers to a cell with large granules in its cytoplasm. NK cells develop from bone marrow lymphoid stem cells and can differentiate and develop in the bone marrow or thymic microenvironment. In the present invention, the proportion of NK cells in TIL cells can be altered using the methods of the present invention.
  • the term "antibody” generally refers to an immunoglobulin or its fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybridized, mutated and transplanted antibodies.
  • the term “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). Typically, 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 five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites.
  • IgA antibodies consist of two to five basic four-chain units that can combine with the J chain to form multivalent combinations.
  • the four-chain unit is generally approximately 150,000 daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are interconnected by one or more disulfide bonds that depend on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the ⁇ and ⁇ chains and four CH domains for the ⁇ and ⁇ isotypes.
  • VH variable domain
  • CH constant domains
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end.
  • the VL corresponds to the VH
  • the CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains.
  • the VH and VL pairs together to form a single antigen-binding site.
  • the L chains from any vertebrate species can be divided into one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • Immunoglobulins can be divided into different classes or isotypes based on the amino acid sequence of the heavy chain (CH) constant domain. There are currently 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 the gene encoding the target polypeptide in the cell.
  • the transcription level of the gene encoding the target polypeptide in the host cell can be determined by measuring the amount of the corresponding mRNA present in the cell.
  • the mRNA transcribed from 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 various methods, such as by ELISA, by a polypeptide biological activity test, or by Western blotting or radioimmunoassay.
  • the term "expression” generally also refers to the transcription and/or translation process of the product.
  • the expression of a cytokine can be the process by which the cell transcribes and/or translates 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 in “a stage of in vitro expansion”, “single stage of in vitro expansion”, or “first stage of in vitro expansion” generally refers to a stage of expansion process that TIL undergoes 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.
  • the TIL cells when T cell activators and/or T cell growth factors are added or supplemented to the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion. In one embodiment, when the TIL cells are centrifuged and/or washed, the TIL cells can be considered to 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.
  • the 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, the 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 primary TILs are obtained from tissues.
  • 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 a patient with metastatic cancer.
  • the amplification of the present invention can be in vivo amplification performed by autologous or allogeneic means, 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.
  • preREP pre-rapid amplification
  • TILs derived from tumor tissue and not amplified in vitro can be referred to as the first TIL group.
  • the TILs obtained through the first stage in vitro amplification in the culture method of the present invention divided into two steps can be referred to as the second TIL group.
  • the term "second stage in vitro expansion” generally refers to the stage in which the tissue removed from the subject is expanded and then expanded again.
  • the number of TIL cells expanded in vitro in the second stage of the present invention is increased compared to the TIL expanded in vitro in the first stage, for example, it can be increased by at least about 10 times (or at least about 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.
  • the culture conditions of the second stage in vitro expansion can be different from those of the first stage in vitro expansion, for example, the culture substances added can be different.
  • the second stage in vitro expansion can also be called the REP (rapid expansion) stage.
  • the TIL obtained by the second stage in vitro expansion can be called the third TIL population.
  • the term "in vivo" generally refers to events that occur 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 an event 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 can be returned to the subject's body through surgery or treatment.
  • 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 TILs of the present invention's T cell activators and/or T cell growth factors.
  • the term "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 can, in one embodiment, be direct contact, i.e., cells from one population are in direct physical contact with cells from another population.
  • the contact can be indirect contact mediated by a shared culture medium.
  • the shared culture medium of the present invention can contain metabolites produced and released by at least one population of co-cultured cells and be used to culture cells from the other population.
  • the term "contact” generally refers to the contact of two or more different types of substances in any order, in any manner, and for any duration.
  • direct contact can be used, for example, one or more feeder cells, T cell activators, and/or T cell growth factors can 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 can 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 can be used to culture TIL cells; in one embodiment, indirect contact can be used, for example, metabolites produced and released by feeder cells can be used to culture TIL cells.
  • the terms “contacting simultaneously,” “contacting together,” “contacting simultaneously with,” “contacting simultaneously with,” and “concurrently” generally refer to administering two or more substances to a subject and/or cell such that the substances are present simultaneously in the subject and/or cell culture environment. Concurrent contacting can include administering different compositions simultaneously, administering different compositions at different times, or administering a composition in which two or more active pharmaceutical ingredients are present. For example, “contacting simultaneously” as used herein generally refers to contacting substantially simultaneously.
  • the term “expanded” generally refers to an increase in the number of cells by several folds over a period of time. In one embodiment, the number of cells can be increased by at least about 3 fold (or 4, 5, 6, 7, 8, or 9 fold), in one embodiment, the number of cells can be increased by at least about 10 fold (or 20, 30, 40, 50, 60, 70, 80, or 90 fold), or in one embodiment, the number of cells can be increased by at least about 100 fold. As used herein, the term “expanded” generally refers to cells of the invention undergoing one or more of the above-described amplifications.
  • the term “polymer” generally refers to a molecule consisting of separate chemical moieties linked together, which can be the same or different.
  • the term “polymer” can refer to separate chemical moieties linked end-to-end to form a linear molecule, as well as separate chemical moieties linked together in a branched (e.g., "multi-arm” or "star") structure.
  • a polymer can include, for example, a polysaccharide, a dextran, a hydrogel, a polyethylene glycol, or a poloxamer.
  • a poloxamer is a nonionic triblock copolymer having a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).
  • the substances encompassed by the present invention can be formulated with or administered with any polymer described herein or known in the art.
  • the term "chimeric antibody” generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can mitigate the immune response induced by the murine antibody.
  • a hybridoma secreting a murine-specific monoclonal antibody can be established.
  • the variable region genes can then be cloned from the murine hybridoma cells.
  • the constant region genes of a human antibody can be cloned as needed.
  • the murine variable region genes and human constant region genes can be linked to form a chimeric gene, which can then be inserted into an expression vector.
  • the chimeric antibody molecule can then be expressed in either eukaryotic or prokaryotic systems.
  • humanized antibody also known as CDR-grafted antibody
  • CDR-grafted antibody generally refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibody framework sequences. This can overcome the heterologous reaction induced by chimeric antibodies due to the large amount of mouse protein components they carry.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy 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.
  • Related 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 the T cell growth factor known as interleukin 2 and includes all forms of IL-2, including, 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 in complex 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).
  • TCRs T cell receptors
  • APCs can process antigens and present them to T cells.
  • antigen presenting cells can 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 can include: increased TIL cell number, increased proportion of viable cells, 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 tissues, or any combination thereof.
  • the changes of the present invention can be either increases or decreases.
  • an increase in the persistence of TIL cells may refer to an increase in the duration of TIL cell survival in vivo.
  • an increase in persistence may refer to an increase in the duration of cell survival within a subject's tissues, such as a tumor, spleen, bone marrow, lung tissue, or blood.
  • an increase in persistence may refer to 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 comprising a complex of the exogenous antigen and the major histocompatibility complex (MHC) on the surface of the artificial antigen-presenting cell.
  • MHC major histocompatibility complex
  • isolated artificial antigen-presenting cells can be included, which can include cells expressing HLA-A/B/C (the gene encoding the gene may be 3105, 3106, or 3107), CD64 (the gene encoding the gene may be 2209), CD80 (the gene encoding the gene may be 941), ICOS-L (the gene encoding the gene may be 23308), and CD58 (the gene encoding the gene may be 965), and can be modified to express one or more T cell activators.
  • HLA-A/B/C the gene encoding the gene may be 3105, 3106, or 3107
  • CD64 the gene encoding the gene may be 2209
  • CD80 the gene encoding the gene may be 941
  • ICOS-L the gene encoding the gene may be 23308
  • CD58 the gene encoding the gene may be 965
  • 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 generally a part of the first polypeptide or protein or a fragment, analog or derivative thereof).
  • a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide.
  • the heterologous protein, polypeptide or peptide of the present invention may or may not be a different type of prophylactic or therapeutic drug.
  • two different proteins, polypeptides or peptides having immunomodulatory activity can be fused together to form a fusion protein.
  • the fusion protein may retain or increase the activity compared to the activity of the original polypeptide or protein before 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. 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.
  • administer generally refer 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.
  • kit generally refers to two or more components packaged together in a container, receptacle or other container, one of which corresponds to a substance of the present invention, for example, comprising TIL cells of the present invention.
  • the term "subject” generally refers to a cell or an animal, which can 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 cell of interest.
  • the feeder cell can be grown in vitro and secrete at least one factor into the culture medium.
  • the feeder cell can include an antigen presenting cell.
  • the term "specific binding” generally refers to recognizing a specific target substance but not substantially recognizing or binding to other molecules in the sample.
  • the binding substance of the present invention may also specifically bind to target substances of the present invention or homologous target substances 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 may 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 expansions, 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.
  • a cell culture medium comprises a buffer, salts, carbohydrates, amino acids, vitamins, and essential trace elements.
  • a cell culture medium may or may not contain serum, peptone, and/or protein.
  • a cell culture medium may be supplemented with additional components or increased concentrations of components, such as amino 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 does not contain additional components that are unacceptably toxic to the subject to whom the preparation is administered. Such preparations are sterile. "Pharmaceutically acceptable” excipients (carriers, additives) are those 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 white blood cells, which 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 Th17 CD4 + 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 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 possess long-term memory and are capable of antigen restimulation.
  • Central memory T cells may have a CD45RO + CD62L + phenotype, and can be identified, for example, by their CD45RO + and CD62L + characteristics.
  • Central memory T cells may possess a stronger ability to fight tumor growth than standard T cells.
  • regulatory T cells generally refers to a subpopulation of T cells that regulate autoimmune reactivity in the body. These cells can have a CD4 + CD25 + Foxp3 + phenotype, for example, by being identified by their CD4 + , CD25 + , and Foxp3 + phenotypes. These cells can also 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 fight tumor growth.
  • Activated T cells may have a phenotype of PD-1 + (PD1 + ), LAG-3 + (LAG3 + ), or CD28 + .
  • activated T cells can be identified by PD-1 + , LAG-3 + , or CD28 + .
  • Activated T cells may have the ability to fight tumor growth.
  • tumor-specific T cells generally refers to T cells that can specifically fight tumor growth.
  • Tumor-specific T cells can have a CD103 + CD39 + phenotype.
  • tumor-specific T cells can be identified by their CD103 + and CD39 + characteristics.
  • Tumor-specific T cells can have more specific anti-tumor activity than standard T cells.
  • stem cell-like T cells generally refers to a type of T cell 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- and CD62L + .
  • stem cell-like T cells can be identified by CD39- and 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 anti-tumor growth ability 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 component such as a carrier, stabilizer, diluent, dispersant, suspending agent, thickener and/or excipient.
  • the term "pharmaceutically acceptable carrier” generally refers to one or more non-toxic materials that do not interfere with the active ingredient.
  • a pharmaceutically acceptable carrier may not interfere with the biological activity of the active ingredient; for example, a pharmaceutically acceptable carrier may not interfere with the effectiveness of the biological activity possessed by the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable carriers may also contain compatible solid or liquid fillers, diluents, or encapsulating materials suitable for human administration.
  • contemplated carriers, excipients, and/or additives may include, for example, flavorings, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (e.g., serum albumin, gelatin, casein), salt-forming counterions (e.g., sodium), and the like.
  • pharmaceutically acceptable carrier may be understood to mean a vector that does not include nucleic acid forms used in genetic engineering.
  • the term "functionally active fragment” generally refers to a fragment that has a partial region of a full-length protein or nucleic acid but retains or partially retains the biological activity or function of the full-length protein or nucleic acid.
  • a functionally active fragment may 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, homolog, or 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 activating 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 958), CDS, ICAM-1 (the gene encoding it may be 3383), LFA-1 (CD11a/CD18) (the gene encoding it may be 3689), 4-1BB (CD137) (the gene encoding it may be 3604), B7-H3 (the gene
  • the gene encoding the gene may be 3682), ITGAL (the gene encoding the gene may be 3683), CD11a (the gene encoding the gene may be 3683), LFA-1 (the gene encoding the gene may be 3683), ITGAM (the gene encoding the gene may be 3684), CD11b (the gene encoding the gene may be 3684), ITGAX (the gene encoding the gene may be 3687), CD11c (the gene encoding the gene may be 3687), ITGB1 (the gene encoding the gene may be 3688), CD29 (the gene encoding the gene may be 3688), ITGB2 (the gene encoding the gene may be 3689), CD18 (the gene encoding the gene may be 3690), and CD29 (the gene encoding the gene may be 3691).
  • LFA-1 the gene encoding it may be 3689
  • ITGB7 the gene encoding it may be 3695
  • NKG2D the gene encoding it may be 22914)
  • NKG2C the gene encoding it may be 3822
  • TNFR2 the gene encoding it may be 7133
  • TRANCE/RANKL the gene encoding it may be 8600
  • DNAM1 CD226)
  • SLAMF4 CD244, 2B4
  • CD84 the gene encoding it may be 8832
  • CD96 Tactile
  • the co-stimulatory intracellular signaling domain can refer to the intracellular portion of a T cell activator.
  • the intracellular signaling domain can include the 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 its variants, homologs, or any substance containing its functionally active fragments.
  • the T cell growth factor can be selected from one or more of the following groups: IL-2 (the gene encoding it may be 3558), IL-4 (the gene encoding it may be 3565), IL-6 (the gene encoding it may be 3569), IL-7 (the gene encoding it may be 3574), IL-10 (the gene encoding it may be 3586), IL-12 (the gene encoding it may be 3592 or 3593), IL-15 (the gene encoding it may be 3600), IL-21 (the gene encoding it may be 59067), TNF- ⁇ (the gene encoding it may be 100137091), gamma interferon (the gene encoding it may be 3458), GZMB (the gene encoding it may be 3002), CD107a (the gene encoding it may be 6499), and the like.
  • IL-2 the gene encoding it may be 3558
  • IL-4 the gene encoding it may be 3565
  • IL-6 the gene encoding it may
  • substantially simultaneously generally refers to a period of time during which the TIL can be in contact with two or more substances simultaneously, but is not limited to always being in contact with two or more substances simultaneously during the entire contact process.
  • substantially simultaneously can 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%, or 95% of each of the two or more substances simultaneously 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 that can be derived from a hematopoietic stem cell or a monocyte.
  • Dendritic cells and their precursors can be isolated from various lymphoid organs such as the 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 proportion of cells, a change in secretory capacity, a change in killing capacity, or a change in expression capacity, 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; in order to detect the function of TIL cells, such as detecting the ability of TIL cells to release cytokines, the operation steps performed on TIL cells (such as adding one or more substances to the culture medium of TIL cells to detect the ability of TIL cells to release cytokines) may not belong to the in vitro expansion of the present invention.
  • peripheral mononuclear cells or “peripheral blood mononuclear cells” generally refers to cells with a single nucleus in peripheral blood.
  • the peripheral blood mononuclear cells 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 monokine, 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 a 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 diameter of a substance can be determined by methods commonly used in the art, such as transmission electron microscopy.
  • tumor generally refers to any new pathological tissue proliferation.
  • the tumors of the present invention may be benign or malignant.
  • the tumors of the present invention may be solid or hematologic.
  • 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 that is not a solid tumor in a 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 immune activities and/or differentiation capabilities.
  • the T cell subsets of the present invention can be distinguished based on 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 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, for example, including but not limited to manual cell counting with a cell counting plate and/or counting with an automatic cell counter.
  • 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 variations encompassed by the terms “about” or “approximately” may depend on the specific system under study and can be readily understood by those of ordinary skill in the art.
  • the present invention provides a method for culturing cells, thereby reducing the expression and/or attenuating the activity of members and/or functionally active fragments thereof selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family.
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the nitric oxide synthase interacting protein family the nitrogen per
  • the cell can further comprise reduced expression and/or decreased activity of a gene optionally selected from the group consisting of BRD4, FAS, TNFAIP3, ZC3H12A, SOCS1, CBLB, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6, TNIP1, BCL2L11, and PTPN2.
  • a gene optionally selected from the group consisting of BRD4, FAS, TNFAIP3, ZC3H12A, SOCS1, CBLB, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6, TNIP1, BCL2L11, and PTPN2.
  • the adenylate cyclase family member may comprise a cyclase domain.
  • the adenylate cyclase family member may comprise ADCY7.
  • the RBR (RING-Between-RING) family member may comprise a RING-type zinc finger domain.
  • the RBR (RING-Between-RING) family member may comprise ARIH2.
  • the carnitine/choline acetyltransferase family member may comprise an acetyltransferase domain.
  • the carnitine/choline acetyltransferase family member may comprise CPT2.
  • the peptidase M1 family member may comprise an aminopeptidase domain.
  • the peptidase M1 family member may comprise LNPEP.
  • the nitric oxide synthase interacting protein family member may comprise a nitric oxide synthase binding domain.
  • the nitric oxide synthase interacting protein family member may comprise NOSIP.
  • the nitrogen permease regulator family member may comprise a GTPase domain.
  • the nitrogen permease regulator family member may comprise NPRL3.
  • the TRAF-associated factor family member may comprise a TRAF binding domain.
  • the TRAF-associated factor family member may comprise a TANK.
  • the tumor necrosis factor receptor-associated factor family member may comprise a receptor cytoplasmic binding domain.
  • the tumor necrosis factor receptor-associated factor family member may comprise TRAF3.
  • the TSC protein family member may comprise a helical domain.
  • the TSC protein family member may comprise TSC1.
  • the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may comprise a BTB domain.
  • the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may comprise ZBTB7B.
  • the ZC3H12 protein family member may comprise a C3H1-type zinc finger domain.
  • the ZC3H12 protein family member may comprise ZC3H12D.
  • the RC3H family member may comprise a ROQ domain.
  • the RC3H family member may comprise RC3H2.
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the nitric oxide synthase interacting protein family the nitrogen permease regulator family
  • the TRAF-related factor family the tumor necrosis factor receptor-related factor
  • ADCY7 ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2 are reduced.
  • the target gene of the present invention can be a gene encoding a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and/or its functionally active fragment.
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the nitric oxide synthase interacting protein family the nitrogen permease regulator family
  • the TRAF-related factor family the
  • cells obtained by reducing the expression and/or attenuating the activity of the target gene of the cell can exhibit improved cell characteristics.
  • cells in which the expression and/or activity of the target gene is not altered can refer to cells derived from the same donor and in which the expression and/or activity of the target gene of the cell has not been reduced and/or attenuated.
  • cells in which the expression and/or activity of the target gene is not altered 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 cells have not been reduced (for example, knocking out the other gene has substantially no effect on the function of the cell).
  • the corresponding cells that have not reduced the expression and/or weakened the activity of the target gene of the cell may refer to cells isolated in the same manner from the same donor and that have not reduced the expression and/or weakened the activity of the target gene of the cell. In one embodiment, the corresponding cells that have not reduced the expression and/or weakened the activity of the target gene of the cell may refer to cells from the same tumor origin of the same donor and that have not reduced the expression and/or weakened the activity of the target gene of the cell.
  • the corresponding cells that have not reduced the expression and/or weakened the activity of the target gene of the cell may refer to cells from the same tumor origin of the same donor being divided into two groups, wherein one group of cells that have not reduced the expression and/or weakened the activity of the target gene of the cell may be the corresponding cells that have not reduced the expression and/or weakened the activity of the target gene of the cell.
  • reduced expression and/or weakened activity of a target gene may refer to a state in which the target gene in a natural cell is expressed to a certain extent, and after the treatment of the present invention, the expression level of the target gene in the cell may be reduced, that is, the reduction in the expression level of the target gene may be such that the natural cell changes from expressing the target gene to substantially not expressing the target gene or expressing a reduced amount of the target gene.
  • the cell comprises an immune cell.
  • the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and/or a basophil.
  • the cell comprises a monocyte, a macrophage and/or a dendritic cell.
  • 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 through 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/or peripheral blood stem cells.
  • stem cells also includes 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 fetal reproductive tissue.
  • 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, non-targeted gRNA, 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 ascites and/or TIL recovered after cryopreservation.
  • the TILs of the present invention can be derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue fragments, pleural effusions, and/or peritoneal effusions, and/or TILs revived after cryopreservation.
  • the TILs of the present invention can 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.
  • tumor fragments of the invention can have a volume of 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 immunoreceptor displayed on the cell surface.
  • the engineered immunoreceptor 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 include: reducing the expression and/or activity of members and/or functionally active fragments thereof selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen permease regulator family, TRAF-related factor family, tumor necrosis factor receptor-related factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family 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 adjacent cancerous 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 selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and/or their functional
  • the TILs of the present invention that are derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and/or ascites and have 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 members and/or functionally active fragments thereof selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack
  • the TILs of the present invention that are derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and/or ascites and have 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 members and/or functionally active fragments thereof selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family
  • the TILs of the present invention derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and/or peritoneal effusion and not 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 TILs can be selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein
  • the expression and/or activity of members of the TIL family
  • the TILs of the present invention that are derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and/or ascites and have 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 members and/or functionally active fragments thereof selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bri
  • the TILs of the present invention that are derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and/or ascites and have 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 members and/or functionally active fragments thereof selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bri
  • the TILs of the present invention that are derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and/or ascites and have 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 members and/or functionally active fragments thereof selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bri
  • 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 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 TILs can be selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-br ac) protein
  • 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 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 TILs can be selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-br ac) protein
  • 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 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 TILs can be selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-br ac) protein
  • 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 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 TILs can be selected from the group consisting of adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen
  • RBR RING-Between-RING
  • each stage of in vitro expansion can be divided by the change in the number of TIL cells, for example, when the number of TIL cells increases by at least about 1 times, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • the number of TIL cells increases by at least about 1-1000 times, for example, 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 the TIL cells have entered the next stage of in vitro expansion.
  • each stage of in vitro expansion can also be divided by the change in the conditions of TIL cell culture.
  • the TIL cells when cell activators and/or cell growth factors are added or supplemented to the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • the TIL cells when IL-2 is added or supplemented to the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • feeder cells are added or supplemented to the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • each stage can also be divided by the number of days the TIL cells are cultured.
  • 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 adenylate cyclase family member in the cell comprises inhibiting the function of adenylate cyclization.
  • the reducing the expression and/or weakening the activity of RBR (RING-Between-RING) family members in the cell comprises inhibiting ubiquitination.
  • the reducing the expression and/or weakening the activity of the carnitine/choline acetyltransferase family member in the cell comprises inhibiting the function of acetyl transfer.
  • said reducing the expression and/or weakening the activity of a peptidase M1 family member in said cell comprises inhibiting the function of degrading polypeptides.
  • the reducing the expression and/or weakening the activity of a member of the nitric oxide synthase interacting protein family in the cell comprises inhibiting the function of regulating nitric oxide production.
  • the reducing the expression and/or weakening the activity of the nitrogen permease regulator family member in the cell comprises inhibiting the function of negatively regulating the TOR signaling pathway.
  • the reducing the expression and/or weakening the activity of TRAF-related factor family members in the cells comprises inhibiting the function of regulating NF-kappa-B.
  • the reducing the expression and/or weakening the activity of tumor necrosis factor receptor-associated factor family members in the cells comprises inhibiting the signal transduction function of TNFR.
  • the reducing the expression and/or weakening the activity of TSC protein family members in the cell comprises inhibiting the function of negatively regulating the mTORC1 signaling pathway.
  • the reducing the expression and/or weakening the activity of BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family members in the cell comprises inhibiting transcriptional regulatory function.
  • the reducing the expression and/or weakening the activity of the ZC3H12 protein family member in the cell comprises inhibiting the activity of endonuclease.
  • the reducing the expression and/or weakening the activity of the RC3H family member in the cell comprises inhibiting the function of ubiquitin protein ligase.
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is not altered
  • 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 ratio, 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 refers to the expression and/or activity of a member selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen permease regulator family, TRAF-related factor family, tumor necrosis factor receptor-related factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family in at least one in vitro expansion stage compared to cells in which the expression and/or activity of a member selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase
  • the cell number of the cells of the present invention in which the expression and/or activity of a member of the nitrogen synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced 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 30 times, at least about 40 times, or at least about 50 times.
  • an increased proportion of live cells can be manifested as an increase in cell survival.
  • the increased proportion of live cells in the present invention can refer to the increase in the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family in at least one in vitro expansion stage compared to cells in which the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family,
  • the proportion of viable cells of the cells of the present invention in which the expression and/or activity of a member of the lex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced can be increased by at least about 100-0.1%, for example, 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 20%, at least about 3 ...
  • the improved cytokine secretion capacity of the present invention may refer to the improvement of the cytokine secretion capacity of the cell selected from the following groups: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention may refer to the improvement of the cytokine secretion capacity of the cell selected from the following groups: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention may refer to the improvement of the cytokine secretion capacity of the cell selected from the following groups: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the improved cytokine secretion capacity of the present invention may refer to the improvement of the cytokine secretion capacity of the cell selected from the following groups: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • RBR RING-Between-RING
  • the proportion of cells secreting cytokines 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 30 times, at least about 40 times, or at least about 50 times.
  • the improved cytokine secretion capacity of the present invention can refer to the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family, compared to cells in which the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1
  • the proportion of cells secreting cytokines can be increased by at least about 100-0.1%, for example, 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 20%, at least about 30%, at least about 40%, 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 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19 ...
  • a member of the protein family e.g., Tramtrack and Bric-a-brac
  • 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 can refer to the expression and/or activity of a member selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen permease regulator family, TRAF-related factor family, tumor necrosis factor receptor-related factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family in at least one in vitro expansion stage compared to cells in which the expression and/or activity of a member selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family is unchanged.
  • the tumor cell killing rate of the cells of the present invention in which the expression of a member of the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced and/or the activity is reduced 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 30 times, at least about 40 times, or at
  • the improved in vitro tumor cell killing ability and/or improved in vivo tumor killing ability of the present invention can refer to the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family, compared to cells in which the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase
  • the tumor cell killing rate of the cells of the present invention in which the expression and/or activity of a member of the road-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced 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%, or more.
  • 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 of the cells of the present invention can refer to the ability of the cells to kill solid tumor cells.
  • the improved cell subpopulation ratios of the present invention may include one or more selected from the following groups: an increased proportion of CD8 + cells, an increased proportion of central memory cells and/or naive cells, a decreased proportion of regulatory cells, an increased proportion of activated cells, an increased proportion of tumor-specific cells (having a phenotype of CD103 + CD39 + ), an increased proportion of stem-like cells, a decreased proportion of exhausted cells, and a decreased proportion of apoptotic cells.
  • the increased proportion of CD8 + cells in the present invention may be an increase in the proportion of CD8-positive cells in cells.
  • the proportion of CD8 + cells in a cell 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%, at least about 0.4%, at least about 3%
  • 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 cell.
  • the proportion of activated cells in the cell 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
  • the amount of the active ingredient in the active ingredient in the present invention may be increased by at least about 1 fold, at least about
  • 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 2%, at least about 1 In some embodiments, the present invention may reduce the amount of the active ingredient
  • the ratio of regulatory cells decreased in the present invention may be a decrease in the ratio 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 that is reduced in the present invention may be a reduction in the ratio of Annexin V + 7-AAD + cells and/or Annexin V + 7-AAD ⁇ cells among 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.
  • 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 a cell 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 least about
  • the increased proportion of naive T cells in the present invention may be an increase in the proportion of CD45RO ⁇ CD62L + cells among cells.
  • the proportion of immature cells in a cell 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%, at least about
  • the method of the present invention can include editing the target gene in vivo, in vitro and/or in vitro.
  • the gene regulatory system can be delivered and edited in vivo to reduce the expression level of the target gene in vivo in cells in vivo.
  • the target gene in vivo can be edited.
  • the composition and/or ratio of the LNP components, or introducing components with targeting capabilities the in vivo editing efficiency of the present invention can be improved.
  • the culture method of the present invention may include a gene editing step for the cells.
  • a gene editing step for the cells includes: subjecting the cells to at least one stage of in vitro expansion, wherein a gene regulatory system may be introduced into the cells during at least one stage of in vitro expansion.
  • the gene regulatory system can destroy the target gene at the DNA level.
  • the gene regulatory system can destroy the region or fragment thereof of the target gene in the genome of the cell.
  • the DNA region or fragment thereof 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 regulatory system on the target gene can be long-term and continuous.
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the nitric oxide synthase interacting protein family the nitrogen permease regulator family
  • the TRAF-related factor family the tumor necrosis factor receptor-related factor family
  • the TSC protein family
  • the genomic region of the present invention is determined based on the human reference genome hg38 version.
  • the gene regulation system can include a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein can have a nucleic acid shearing enzyme activity, and the guide nucleic acid molecule can guide the enzyme protein to specifically shear the region where the target gene is located or its fragment.
  • 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.
  • the polynucleotide encoding gRNA and Cas protein can be introduced or each independently introduced into the target cell.
  • the present invention can reduce the expression and/or weaken the activity of the target gene of the cell by introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell.
  • RNP ribonucleoprotein complex
  • the enzyme protein can comprise a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.
  • the guide nucleic acid molecule can comprise a guide RNA (gRNA).
  • 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 fully complementary, partially complementary, or hybridize 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 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 affinity.
  • 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 subregions shown in Tables 1A to 1L.
  • the guiding nucleic acid molecule targeting ADCY7 described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 4146-8290.
  • the guiding nucleic acid molecule targeting ARIH2 described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 10494-12696.
  • the guiding nucleic acid molecule targeting CPT2 described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 13414-14130.
  • the guiding nucleic acid molecule targeting LNPEP described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 15950-17768.
  • the guiding nucleic acid molecule targeting NOSIP described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 19561-21352.
  • the guiding nucleic acid molecule targeting NPRL3 described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 22857-24360.
  • the guiding nucleic acid molecule targeting TANK described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 25134-25906.
  • the guiding nucleic acid molecule targeting TRAF3 described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 29216-32524.
  • the guiding nucleic acid molecule targeting TSC1 described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 34537-36548.
  • the guiding nucleic acid molecule targeting ZBTB7B described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 39784-43018.
  • the guiding nucleic acid molecule targeting ZC3H12D described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 44428-45836.
  • the guiding nucleic acid molecule targeting RC3H2 described in the present invention can bind to a region or a fragment thereof selected from the following group: SEQ ID NO: 47259-48680.
  • 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, 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 upstream of the 5' 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 upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG and CGG.
  • PAM protospacer adjacent motif
  • the region targeted by the guide nucleic acid molecule of the present invention can have a protospacer adjacent motif (PAM) upstream
  • the protospacer adjacent motif (PAM) can be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, or TTTN, ATTN, GTTN, CTTN, TTC, TTG, TTA, TTT, TAN, TGN, or TCN, 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.
  • the protospacer adjacent motif (PAM) can be TTTN.
  • the protospacer adjacent motif can be TTN.
  • the PAM region of the target gene is determined, one 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 3' downstream of the PAM of the target gene, and can also 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 3' downstream of the protospacer adjacent motif (PAM) selected from the group consisting of NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, or TTTN, ATTN, GTTN, CTTN, TTC, TTG, TTA, TTT, TAN, TGN, or TCN, 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.
  • the protospacer adjacent motif (PAM) may be TTTN.
  • the protospacer adjacent motif (PAM) may be TTN.
  • 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 can be A, T, C or G.
  • 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 region of the guide nucleic acid molecule targeting 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 can be A, T, C or G, Y can be T or C, V can be A, C or G, and R can 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 upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTTV (TTTA, TTTC, or TTTG), wherein V can be A, C or G.
  • TTTV TTTA, TTTC, or TTTG
  • 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 region targeted by the guide nucleic acid molecule of the present invention 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 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 can be A, T, C or G.
  • 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: TTN, wherein N can be A, T, C or G.
  • TTN TTN
  • N can be A, T, C or G.
  • 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.
  • 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: TTN or TTTN, wherein N may be A, T, C or G.
  • TTN or TTTN a PAM sequence selected from the following: TTN or TTTN, wherein N may be A, T, C or G.
  • N may be A, T, C or G.
  • 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.
  • the guide nucleic acid molecule can 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 gene selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and/or its functionally active fragments.
  • a gene selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carn
  • the guide nucleic acid molecule can comprise an AGG, T sequence in the DNA encoding a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and/or its functionally active fragment.
  • adenylate cyclase family the RBR (RING-Between-RING) family
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the target sequence can be a region defined by the genomic coordinates shown in Tables 2A-2L, or a fragment thereof.
  • the guide nucleic acid molecule may comprise an sgRNA targeting ADCY7 as shown in any one of SEQ ID NOs: 1-4145, an sgRNA targeting ARIH2 as shown in any one of SEQ ID NOs: 8291-10493, an sgRNA targeting CPT2 as shown in any one of SEQ ID NOs: 12697-13413, an sgRNA targeting LNPEP as shown in any one of SEQ ID NOs: 14131-15949, an sgRNA targeting NOSIP as shown in any one of SEQ ID NOs: 17769-19560, an sgRNA targeting NPRL3 as shown in any one of SEQ ID NOs: 21353-22856.
  • ADCY7 as shown in any one of SEQ ID NOs: 1-4145
  • an sgRNA targeting ARIH2 as shown in any one of SEQ ID NOs: 8291-10493
  • an sgRNA targeting CPT2 as shown in any one of SEQ ID NOs: 12697-13413
  • RNA sgRNA targeting TANK as shown in any one of SEQ ID NOs: 24361-25133
  • sgRNA targeting TRAF3 as shown in any one of SEQ ID NOs: 25907-29215
  • sgRNA targeting TSC1 as shown in any one of SEQ ID NOs: 32525-34536
  • sgRNA targeting ZBTB7B as shown in any one of SEQ ID NOs: 36549-39783
  • sgRNA targeting ZC3H12D as shown in any one of SEQ ID NOs: 43019-44427
  • sgRNA targeting RC3H2 as shown in any one of SEQ ID NOs: 45837-47258.
  • 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 cells obtained by reducing the expression and/or attenuating the activity 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 changed.
  • the protein encoding the product of the target gene is expressed in a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and/or its
  • the cellular proportion of the product of a gene of the functionally active fragment is reduced by at least about 100-5%, for example, 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%
  • the proportion of cells that are blasted can be reduced to at least about 100-1%, for example, 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
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the nitric oxide synthase interacting protein family the nitrogen permease regulator family
  • the cellular proportion of the product of the gene of the functionally active fragment can be at most about 95-5%, e.g., 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 least
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the nitric oxide synthase interacting protein family the nitrogen permease regulator family
  • the expression and/or activity of the target gene of the cell is reduced and/or the activity is attenuated, and the expression of the target gene in the cell can be reduced by at least about 5% compared to cells in which the expression and/or activity of the target gene is not altered.
  • 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 reduced from the observed expression level 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 can be at most about 95% of that in cells where the expression and/or activity of the target gene is not changed.
  • the expression level of the gene encoding a member selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen permease regulator family, TRAF-related factor family, tumor necrosis factor receptor-related factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family and/or its functionally active fragment (e.g., gene encoding ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, RC3H2) in a single cell can be the gene encoding a member selected from the adenylate cyclase family, RBR (RING
  • the method of the present invention comprises: subjecting the cells 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 a member of the cell selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced.
  • a member of the cell selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acety
  • the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and/or ascites 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 a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family
  • 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 cells can be contacted with the one or more cell activators and the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and/or its functionally active fragments can be reduced in the cells.
  • the cell activator can include an agonist of one or more targets selected from the group consisting of CD3, CD28, HVEM, CD40L, OX40, and 4-1BB.
  • the expression and/or activity of a member of the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family of the cells of the present invention is reduced and the cells are contacted with one or more cell activators of the present invention.
  • a member of the TIL of the present invention selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the
  • a member of the TIL of the present invention selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the
  • a member of the TIL of the present invention selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the
  • the cells of the present invention essentially simultaneously reduce the expression and/or attenuate the activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H 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 a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H 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.
  • 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, and then the expression and/or activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced.
  • 2-48 hours in advance such as 2 hours in advance, 4 hours in advance, 8 hours in advance, 12
  • the TILs of the present invention substantially simultaneously reduce the expression and/or weaken the activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and contact with one or more cell activators of the present invention.
  • the TILs of the present invention substantially simultaneously reduce the expression and/or weaken the activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and contact with one or more cell activators of the present invention.
  • the TILs of the present invention substantially simultaneously reduce the expression and/or weaken the activity of a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and contact with one or more cell activators of the present invention.
  • the second stage of in vitro expansion of the present invention is carried out for at least about 9 days.
  • the second stage of in vitro expansion of the present invention can be carried out for 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 of 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 of in vitro expansion of the present invention can be considered the REP (rapid expansion protocol) stage.
  • the first stage of in vitro expansion of the present invention can be considered the preREP stage.
  • 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 second stage in vitro amplification starts, 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 comprise one or more selected from the following groups: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and functionally active fragments thereof.
  • the cell activator of the present invention may comprise 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 comprise an antibody selected from the following groups: CD3, CD28, HVEM, CD40L, OX40, and 4-1BB, and an antigen-binding fragment thereof.
  • the cell activator of the present invention may comprise a CD3 agonist.
  • the cell activator of the present invention may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, such as OKT3 from Miltenyi Biotech, or SP34 from 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, such as Merck's 15E8.
  • 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 Miltenyi Biotech's OKT3, or it may comprise the light chain VL and heavy chain VH of BD's SP34.
  • 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 Merck's 15E8.
  • 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 Miltenyi Biotech's OKT3, or it may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of BD's SP34.
  • 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 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, the light chain LCDR1-3 and heavy chain HCDR1-3 of Merck's 15E8.
  • the anti-CD28 antibody and/or antigen-binding fragment thereof of the present invention may have CD28 binding ability.
  • the antibody or antigen-binding protein thereof of the present invention comprises at least one CDR in the antibody heavy chain variable region VH and/or at least one CDR in the antibody light chain variable region VL.
  • the CDRs of the present invention may be defined according to the IMGT nomenclature, the CDRs of the present invention may be defined according to Chothia, or the CDRs 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 comprise one or more methods selected from the following group: (1) adding the cell activator of the present invention to the cell culture medium of the cells of the present invention; (2) adding engineered cells expressing the cell activator 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 activator 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 comprise adding a solid phase medium containing the cell activator 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 comprise 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 selected from about 30 ng/mL to about 300 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 arbitrarily selected from about 30 ng/mL to about 300 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 arbitrarily selected from about 30 ng/mL to about 300 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 arbitrarily selected from about 30 ng/mL to about 300 ng/mL
  • the initial concentration of the CD28 antibody of the present invention can be selected independently 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.
  • a solid phase medium containing a cell activator of the present invention is added to a 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.
  • a solid phase medium containing a cell activator of the present invention is added to a 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 activating agent of the present invention can be 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 about 2:1 to about 1:2.
  • the solid phase medium containing the cell activating agent of the present invention can be 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 about 2:1 to about 1:2, about 2:1 to about 1:1, or 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.
  • 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 1:2000, about 1:100 to about 1:2000, about 1:2000 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 1:2000
  • a solid phase medium comprising a CD28 agonist and a CD3 agonist of the present invention can be added to a cell culture
  • 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 TILs 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 TILs 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 TILs 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 activators of the present invention and one or more cell growth factors of the present invention at substantially the same time.
  • the cells of the present invention can be contacted with the one or more cell growth factors of the present invention and one or more cell activators of the present invention at substantially the same time.
  • the cells of the present invention can be contacted with the 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, and then contacted with the one or more cell activators of the present invention.
  • the cells of the present invention can be contacted with the 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, and then contacted with the one or more cell growth factors of the present invention.
  • 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 TILs 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 TILs 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 functionally active fragments thereof.
  • the cell growth factor of the present invention can comprise IL-2 and/or functionally active fragments thereof.
  • the functionally active fragments of IL-2 can comprise fragments of IL-2 known in the art that can bind to the IL-2 receptor of a cell.
  • the cell growth factor of the present invention can comprise IL-2 and/or functionally active fragments thereof, IL-7 and/or functionally active fragments thereof, and IL-15 and/or functionally active fragments thereof.
  • contacting the cells of the invention with one or more cell growth factors of the invention can 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 can be at least about 300 IU/mL.
  • the initial concentration of IL-2 of the invention in the cell culture medium of the cells of the 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, at least about 2000 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL.
  • the cells of the present invention can be contacted with IL-2, IL-7, and IL-15 to reduce the amount of cytokines used compared to contact with IL-2 alone.
  • the amount of IL-2 added can be reduced under the conditions of adding IL-7 and IL-15.
  • the concentration of IL-7 can be about 1 to 1000 ng/mL, preferably about 1-100 ng/mL.
  • the concentration of IL-15 can be about 1 to 1000 ng/mL, preferably about 1-100 ng/mL.
  • the amount of IL-2 added can be reduced to the range commonly used in the art for various immune cells, for example, to 50-10% of the range commonly used in the art, such as 50%, 20%, or 10%.
  • the amount of IL-2 added for TCR-T cells can be in the range commonly used in the art of 30-300 IU/mL.
  • the amount of IL-2 added for TIL cells can be in the range commonly used in the art of 300-9000 IU/mL (e.g., 1000-9000 IU/mL).
  • the method of the present invention may further comprise: during 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 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.
  • a single stage of in vitro expansion of the present invention can refer to in vitro expansion of the present invention at the same stage, for example, the cells can be expanded 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 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 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 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 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours.
  • the certain period of time of the present invention can be from about 2 hours to about 72 hours.
  • the certain time period 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 period 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 period 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 comprise antigen-presenting cells.
  • the feeder cells of the present invention may comprise one or more selected from the following groups: 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 may be isolated artificial antigen-presenting cells (aAPCs), which may comprise cells expressing HLA-A/B/C, CD64, CD80, ICOS-L and/or CD58, and may be modified to express one or more cell activators of the present invention.
  • the feeder cells of the present invention may be irradiated, for example, by gamma ray irradiation, or by X-ray irradiation.
  • co-culturing the cells of the present invention with the feeder cells of the present invention can comprise contacting the surface of the feeder cells of the present invention with the surface of the cells of the present invention.
  • co-culturing the cells of the present invention with the feeder cells of the present invention comprises adding the feeder cells of the present invention to the cell culture medium of the cells of the present 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 feeder cells of the invention to cells of the invention of about 60:1 to about 400:1, about 70:1 to about 400:1, about 80:1 to about 400:1, about 90:1 to about 400:1, about 100:1 to about 400:1, about 200:1 to about 400:1, or about 300:1 to about
  • the present invention provides a method for culturing tumor-infiltrating lymphocytes (TILs), 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 adjacent cancerous 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 activity of a member of the second TIL population selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family,
  • the present invention provides a method for culturing tumor-infiltrating lymphocytes (TILs), comprising: (A) contacting a first TIL population 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 and not expanded in vitro with one or more T cell growth factors, wherein a second TIL population is obtained by step (A); (B) the second TIL population is selected from the group consisting of adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein
  • the method comprises the steps of: (a) reducing the expression and/or weakening the activity of members of the TIL family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necros
  • 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 TILs of the present invention that have undergone 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 TILs of the present invention that have undergone 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 TILs of the present invention that have undergone 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 (TILs), 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, paracancerous tissue fragments, 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 cytokine selected from the adenylate cyclase family, RBR (RING-Betwee
  • the method comprises the steps of: reducing the expression and/or weakening the activity of members of the TIL family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting
  • the present invention provides a method for culturing tumor infiltrating lymphocytes (TILs), 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 adjacent cancerous 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, a cell activator, and a protein selected from the group consisting of adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen permease regulator family, TRAF-related factor family, tumor necrosis factor receptor-related factor family, TSC protein family,
  • 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, which can weigh 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 adjacent 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°C and processed within 48 hours.
  • the 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 the addition of cell 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 in the culture medium are collected and transferred into a breathable culture bag, or Grex, or Xuri device.
  • the serum-free culture medium of the cells can be supplemented with CD28 antibodies of the present invention, CD3 antibodies comprising CD3 antibodies and CD28 antibodies, magnetic beads (e.g., Dynabeads) and/or nanomatrices (e.g., transACT) comprising CD3 antibodies and CD28 antibodies, IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, e.g., 6000 IU/mL), and IL-2 selected from the adenylate cyclase family, RBR (RING-Between-RING) family, carnitine/choline acetyltransferase family, peptidase M1 family, nitric oxide synthase interacting protein family, nitrogen permease regulator family, TRAF-related factors.
  • CD28 antibodies of the present invention CD3 antibodies comprising CD3 antibodies and CD28 antibodies, magnetic beads (e.g., Dynabeads) and/or nanomatrices (e
  • the adenylate cyclase family member may include ADCY7
  • the RBR (RING-Between-RING) family member may include ARIH2
  • the carnitine/choline acetyltransferase family member may include CPT2
  • the peptidase M1 family member may include LNPEP
  • the nitric oxide synthase interacting protein family member may include NOSIP
  • the nitrogen permease regulator family member may include NPRL3
  • the T The RAF-related factor family member may include TANK
  • the tumor necrosis factor receptor-related factor family member may include TRAF3
  • the TSC protein family member may include TSC1
  • the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may include ZBTB7B
  • the ZBTB7B the ZBTB7B
  • the transduction may be performed using a ribonucleoprotein complex (RNP) containing the gRNA and Cas protein of the present invention, or an LNP containing the gRNA and Cas protein, or an LNP containing a nucleic acid encoding the gRNA and Cas protein.
  • RNP ribonucleoprotein complex
  • the TILs contain a gene encoding a member selected from the adenylate cyclase family, the RBR (RING-Between-RING) family, the carnitine/choline acetyltransferase family, the peptidase M1 family, the nitric oxide synthase interacting protein family, the nitrogen permease regulator family, the TRAF-related factor family, the tumor necrosis factor receptor-related factor family, the TSC protein family, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family (the ratio is about 95% or less).
  • the RBR RING-Between-RING
  • the carnitine/choline acetyltransferase family the peptidase M1 family
  • the nitric oxide synthase interacting protein family the nitrogen permease regulator family
  • the TRAF-related factor family the tumor necros
  • TIL to PBMC ratio is about 1:40 to about 1:400
  • 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 more than 80% of the cells can be memory effector cells and effector cells.
  • IFN- ⁇ can be secreted and/or the ratio of activated cells can be increased.
  • the present invention provides a cell, which can be cultured according to the culture method of the present invention.
  • the cell provided by the present invention can include one or a batch of cells cultured according to the culture method of the present invention.
  • the cell provided by the present invention can include multiple or multiple batches of cells cultured according to the culture method of the present invention and combined in any proportion.
  • the cells expanded using the methods 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 expanded using the PBMCs of the present invention can be administered by any suitable route known in the art.
  • the cells can be administered as 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 may 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, a therapeutically effective dose can be from 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 , 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, for example, 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%, about 1%.
  • % about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, about 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 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, for example, 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 16%, about 17.75%, about 17.75%, about 17.50%, about 17.25%, about 17%.
  • the cells provided in the compositions of the present invention may be present at 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 26%, about 0.09% to about 27%, about 0.10% to about 28%, about 0.11% to about 29%, about 0.12% to about 29%, about 0.13% to about 29%, about 0.14% to about 29%, about 0.15% to about 29%, about 0.16% to about 29%, about 0.17% to about 29%, about 0.18% to about 29%, about 0.19% to about 29%, about 0.19% to about 29%, about 0.11% to about 29%, From about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 23%, from about
  • cells are provided in the compositions of the invention at 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, for example, about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g, about 6.5 g, about 6.0 g, about 5.5 g, about 5.0 g, about 4.5 g, about 4.0 g, about 3.5 g, about 3.0 g, about 2.5 g, about 2.0 g, about 1.5 g, about 1.0 g, about 0.95 g, about 0.9 g, about 0.85 g, about 0.8 g, about 0.75 g, about 0.7 g, about 0.65 g, about 0.6 g, about 0.55 g, about 0.5 g, about 0.45 g, about 0.4 g, about 0.35 g, about 0.3 g, about 0.001 g, about 0.0009 g, about 0.0008 g, about 0.0007 g, about 0.3
  • 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 0.0030 g, about 0.0031 g, about 0.0032 g, about 0.0033 g, about 0.0034 g, about 0.0035 g, about 0.0036 g, about 0.0037 g, about 0.0038 g, about 0.0039 g, about 0.010 g, about 0.0115 g, about 0.012 g, about 0.0137 g, about 0.0140 g, about 0.0157 g, about 0.0160 g, about 0.0175 g, about 0.0180 g,
  • 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 two days. In some embodiments, the cells can be administered continuously.
  • the present invention provides a pharmaceutical composition, which in some embodiments may comprise the cells of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a kit that may include a cell activator, a cell growth factor, and/or feeder cells for the cell culture method of the present invention, and instructions describing the steps of the cell culture method of the present invention.
  • the present invention provides a kit that may include the cells of the present invention and/or the pharmaceutical composition of the present invention.
  • the present invention provides a method of affecting the growth of cells, such as tumor cells, which can include administering to a subject a cell of the present invention and/or a pharmaceutical composition of the present invention.
  • affecting tumor growth can 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 cells of the present invention and/or the pharmaceutical compositions of the present invention in the preparation of a medicament, which can be used to prevent and/or treat a disease and/or symptom.
  • the disease and/or symptom of the present invention may comprise a tumor.
  • the disease and/or symptom of the present invention may comprise an immune-related disease, such as an autoimmune disease.
  • the tumor of the present invention is selected from a solid tumor.
  • the tumor of the present invention 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.
  • the present invention provides a method for preventing and/or treating a disease and/or symptom, which may comprise 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 comprise a tumor.
  • the disease and/or symptom of the present invention may comprise an immune-related disease, such as an autoimmune disease.
  • the tumor of the present invention is selected from a solid tumor.
  • the tumor of the present invention 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.
  • 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 may include a tumor.
  • the disease and/or symptom of the present invention may include an immune-related disease, such as an autoimmune disease.
  • the tumor of the present invention is selected from a solid tumor.
  • the tumor of the present invention 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.
  • PBMC cells Use 75% alcohol to disinfect the sample tubes and blood collection tubes and transfer them to a biosafety cabinet. Isolate PBMC cells from the blood sample and freeze them according to the above-mentioned PBMC manual isolation and freezing procedures. Take a culture flask or culture bag with a breathable surface, such as a culture bag (Origen), and add 300 mL of rewarmed complete culture medium.
  • a culture flask or culture bag with a breathable surface such as a culture bag (Origen)
  • the complete culture medium can be arbitrarily selected from X-vivo15 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 can be supplemented with essential amino acids and antibiotics, and add IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, such as 6000 IU/mL). Take several 10 cm culture dishes, add an appropriate amount of culture medium, use sterile ophthalmic forceps to remove the tumor tissue from the sample tube and place it in a 10 cm culture dish, wash the tissue and replace the culture dish.
  • T cell culture medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium
  • IL-2 add a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, such as 6000 IU/mL).
  • tissue block is then minced to approximately 27 cubic millimeters.
  • a non-suspended tumor tissue block is obtained.
  • a 20 mL syringe is used to remove the internal stopcock and connect to a culture bag.
  • approximately 1 g of tissue is transferred from the syringe into the culture bag.
  • the culture bag is placed in a CO2 incubator for incubation.
  • the scissors and forceps are cleaned and initially disinfected with 75% alcohol. After ultrasonic cleaning, they are sterilized to obtain the first TIL population.
  • the medium is replenished or half-volume is replaced every 3-7 days to ensure cell nutrition.
  • Complete culture medium is used.
  • 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, samples can be taken and counted on the 13th or 14th day. If the cell number is between 5 ⁇ 10 5 and 5 ⁇ 10 8 , the harvesting step of step (A) is entered.
  • step (A) Collect the cells after in vitro expansion in step (A), centrifuge, discard the culture medium, wash the cells once with PBS or normal saline, obtain TIL (second TIL population) expanded in vitro in step (A), and take samples and count to retain approximately 5 ⁇ 10 5 to 2 ⁇ 10 8 cells for the subsequent in vitro expansion step; take approximately 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).
  • X-vivo 15 medium or other commercial T cell culture medium can be arbitrarily selected, such as T cell culture medium from brands such as Stem Cell, Lonza, Thermo, and Miltenyi Biotec. Essential amino acids and antibiotics can be added. Adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL and suspend the cells in a 24-well culture plate at 1 mL/well. Add IL-2 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, such as a CD3 agonist and/or a CD28 agonist, for example, approximately 30 ng/mL of CD3 antibody (Miltenyi Biotech, OKT3), approximately 30 ng/mL of CD28 antibody (Merck, 15E8), magnetic beads (Dynabeads, approximately 1 to 10 ⁇ m in diameter, Thermo Fisher) at a ratio of approximately 1:2-2:1 to TIL, and/or transACT (approximately 100 to 500 nm in diameter, Miltenyi) at a ratio of approximately 1:100-1:2000 to TIL.
  • the cells are cultured for approximately 0-4 days to obtain a third TIL population.
  • 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 incubated at 95 ° C for 2 minutes to anneal and then added to P3 buffer, and 0.3-1 ⁇ L of Cas9 (such as Kaixia, 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 Kaixia, Ke Rui or Acro, 10 mg / mL
  • P3 buffer Lionza
  • the above RNP is electroporated with about 1 ⁇ 10 6 cells of the third TIL population by a Lonza electroporator.
  • the electroporation procedure can be human T cell stim (EO115).
  • the gene editing is cultured for about 0-4 days to obtain a fourth
  • Feeder cells irradiated healthy donor PBMC T cells
  • the time for contact between TIL and feeder cells needs to be after a certain time Tn after the TIL is contacted with IL-2 and T cell activator in step (B) (for example, Tn can be taken from 0 hours to 12 days, such as 24 hours or 48 hours).
  • the activated TIL cells and feeder cells are mixed according to the ratio of TIL cells: feeder cells of about 1:200, transferred to G-Rex100 culture flask or breathable bag, supplemented with complete culture medium, sampled and counted every 1-3 days, and replenished or half-replaced according to the cell status until the total number of cells is greater than 1 ⁇ 10 9 or the step (D) in vitro expansion culture is about 5 days to about 14 days, and the step (D) in vitro expansion culture 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 wash. According to the counting results, discard the supernatant after the last centrifugation and take 3 ⁇ 10 6 cells for quality control testing; add all the remaining cells to the freezing solution and adjust the cell density to 1-3 ⁇ 10 8 cells/mL for freezing.
  • T cell activation T cells frozen in liquid nitrogen were revived and cultured, and resuspended to 5E5/ml by centrifugation in T cell culture medium RPMI 1640 (Gibco) + 10% FBS (Bovogen). T cell TransAct (Miltenyi) was added at a ratio of 1:100, and recombinant human IL-2 was added at a concentration of 30 IU/ml, and cultured for about 72 hours.
  • TCR transduction One day prior to transduction, coat a 24-well suspension culture plate with recombinant human fibrin fragment (Retronectin, Takara) at a final concentration of 15 ⁇ g/mL, adding 250 ⁇ L per well. Protect from light and incubate at 4°C overnight. Remove the coated 24-well plate, discard the coating solution, and add 500 ⁇ L of blocking solution containing 2% BSA for 30 minutes at room temperature. Discard the blocking solution and wash the plate twice with 500 ⁇ L/well of a 2.5% HEPES-containing plate wash solution, then discard the wash solution. The experimental group was transduced with a retrovirus carrying a specific TCR nucleic acid fragment targeting the NY-ESO-1 antigen peptide.
  • TCR-T gene editing Synthesize the sgRNA targeting each target selected from the present invention, thaw and add nuclease-free water to a concentration of about 100 ⁇ M. About 2 ⁇ L of gRNA (50 ⁇ M) is incubated at 95 ° C for 2 minutes to anneal and then added to P3 buffer, and 0.3-1 ⁇ L of Cas9 (such as Kaixia, Ke Rui, Acro, 10 mg / mL) is added and incubated at 25 ° C for 10 minutes to form a ribonucleoprotein complex (RNP). In P3 buffer (Lonza), the above RNP is electroporated with about 1 ⁇ 10 6 TCR-T cells using a Lonza electroporator.
  • Cas9 such as Kaixia, Ke Rui, Acro, 10 mg / mL
  • the electroporation procedure can be human T cell stim (EO115).
  • the electroporated T cells are added with recombinant human IL-2 at a concentration of 100 to 300 IU / ml and cultured to obtain TCR-T cells after target gene editing of the present invention.
  • DNA extraction solution QuickExtract DNA extraction solution, Lucigen, QE09050
  • RNase/DNase free water Tiangen
  • EDTA Ellenggong, 0.5M
  • Recombinant DNase I RNase-free, TAKARA
  • Extract genomic DNA Approximately 2-7 days after T cell knockout (see the gene editing method in step 1.4 of Example 1 (I) or the TCR-T gene editing method in Example 1 (II)), extract approximately 1 ⁇ 10 5 to approximately 2 ⁇ 10 5 cells, wash once with PBS, and resuspend the gene-edited cells in 44 ⁇ L of PBS. Add 6 ⁇ L of the prepared nuclease mix (containing 1 ⁇ L of DNase I and 5 ⁇ L of 10 ⁇ DNase I Buffer) and incubate at 37°C for 5 minutes. Add 2.5 ⁇ L of 0.5 M EDTA to the sample and incubate at 80°C for 10 minutes.
  • DNA sample concentration can be measured using a spectrophotometer (NanoDrop TM ).
  • PCR primers can be designed in the region approximately 100 to 200 nucleotides upstream and downstream of the PAM site. Design the PCR reaction system as follows:
  • the PCR products were analyzed by Sanger sequencing.
  • TIL-2 The proliferation of TIL cells was detected 7-10 days after gene editing in Example 1 (I) (IL-2 was withdrawn), and TIL cells in each group were harvested; or T cells transduced with TCR obtained in Example 1 (II) were used.
  • Cells were washed once with PBS and resuspended in T cell culture medium (without IL-2). After counting, the cells were adjusted to a density of 1e5 to 2e6 cells/mL and plated into flat-bottom 96-well plates at 100 ⁇ L/well.
  • the non-stimulation medium group no cell activating substances were added to the culture medium;
  • the CD3 antibody stimulation group 30 ng/mL of CD3 antibody (OKT3) was added for stimulation;
  • TransACT stimulation group transACT (approximately 100 to 500 nm in diameter, Miltenyi) was added to a transACT working solution concentration of 1:1000 (v/v); for the Medium group, only an equal volume of cell culture medium was added.
  • tumor target cells were plated in 96-well flat-bottom plates.
  • TIL cells from each group were co-cultured with target cells at different effector-to-target ratios (T cells: target cells, E:T).
  • T cells transduced with the TCR obtained in Example 1 (II) were used.
  • target cells 100 ⁇ L of target cells and T cells are added, and three replicates are set up for each group. A control group containing only target cells is also set up.
  • the target cells can be selected from Caski cervical cancer cells, Hey-T30 ovarian cancer cells, and A375 melanoma cells.
  • apoptosis detection reagent Incucyte Caspase-3/7 Green Dye for Apoptosis, Sartorius
  • 0.2 ⁇ L of the apoptosis detection reagent was added per well, and 25 ⁇ L of culture medium was added per well to dilute the Caspase 3/7 Green Dye.
  • Caspase 3/7 activity was recorded using an Incucyte recorder (Sartorius) every 3 hours for approximately 5 days to analyze the cytotoxicity of TIL cells against target cells.
  • T cell exhaustion, stemness and other related molecules of TIL cells obtained on the 8th day after gene editing was detected by flow cytometry; or the TCR-transduced T cells obtained in Example 1 (ii) were detected.
  • V-bottom 96-well plates manufacturer Corning, product number 3894; flow cytometry tubes, manufacturer Corning, product number 352052; flow cytometry antibodies were purchased from BD or Biolegend.
  • Intracellular molecule detection Prepare a mixed antibody working solution for cell surface staining of CD3/CD4/CD8 at an antibody concentration of 1:100 and a cell viability detection dye concentration of 1:10,000. 50 ⁇ L/well of a 96-well plate and 100 ⁇ L/tube of a flow cytometry tube were added for staining and incubated at 2-8°C in the dark for 30 minutes. Wash the cells once with PBS (200 ⁇ L/well for a 96-well plate and 1 mL/well for a flow cytometry tube), centrifuge at 600 g for 3 minutes at room temperature, and discard the supernatant.
  • PBS 200 ⁇ L/well for a 96-well plate and 1 mL/well for a flow cytometry tube
  • fixation/permeabilization buffer (BD, Fixation/Permeabilization)
  • BD Fixation/Permeabilization
  • wash twice with 1 ⁇ Perm/Wash Buffer 200 ⁇ L/well for a 96-well plate and 1 mL/well for a flow cytometry tube
  • centrifuge at 600 g for 3 minutes and discard the supernatant.
  • Prepare intracellular molecule antibodies e.g., TCF1 in 1 ⁇ Perm/Wash Buffer and resuspend TIL cells (50 ⁇ L/well for 96-well plates, 100 ⁇ L/tube for flow cytometry staining).
  • TIL cell populations obtained on the 7th or 8th day after gene editing in each experimental group were tested by flow cytometry for cytokine expression; or the TCR-transduced T cells obtained in Example 1 (ii) were tested.
  • T cells from each experimental group were resuspended in the culture medium described above for intracellular factor expression assays. After counting, the cells were adjusted to a density of 1 ⁇ 106 cells/mL and plated into a 96-well plate at 200 ⁇ L/well.
  • CD3 antibody stimulation 30 ng/mL of CD3 antibody (OKT3) was added to the CD3 antibody stimulation group.
  • TransACT stimulation transACT (approximately 100 to 500 nm in diameter, Miltenyi) was added to a transACT working solution concentration of 1:1000 (v/v).
  • For the Medium group an equal volume of cell culture medium was added. The plates were incubated overnight at 37°C.
  • V-bottom 96-well plates manufacturer Corning, product number 3894; flow cytometry tubes, manufacturer Corning, product number 352052; flow cytometry antibodies were purchased from BD or Biolegend.
  • cytokine detection antibodies e.g., GZMB, TNF- ⁇ , IFN- ⁇
  • cytokine detection antibodies e.g., GZMB, TNF- ⁇ , IFN- ⁇
  • Apoptosis detection was performed on the TIL population obtained on the 7th or 8th day after gene editing in each experimental group in Example 1; or the TCR-transduced T cells obtained in Example 1 (ii) were detected.
  • T cell apoptosis levels of TILs in the gene knockout group or control group were detected using a cell apoptosis detection kit (BD 559763 Annexin V PE Apoptosis kit).
  • Tumor tissue was obtained surgically, transported and stored at 2-8°C after ex vivo treatment. Mechanically dissociated within 24 hours, the tissue was minced to approximately 0.5 mm3 . The tissue was then digested at 37°C using a dedicated tissue digestion solution (bioGenous) and terminated with 10% FBS. The tissue suspension was filtered through a 100 ⁇ m filter, washed, and thoroughly mixed with Martrigel on ice. The mixture was then applied to the bottom of a cell culture plate. The plate was placed in a 37°C incubator. After the Martrigel had fully solidified, complete culture medium was carefully added for incubation. Once the organoids reached sufficient size and density, they were passaged or frozen to obtain a PDO (Patient-Derived Organoid) model.
  • PDO Principal-Derived Organoid
  • Count the cells to be tested e.g., TIL cells or TCR-T cells
  • Count the cells to be tested e.g., TIL cells or TCR-T cells
  • NT control group
  • a PDO model was prepared according to the examples of the present invention and co-cultured with TIL cells or TCR-T cells. After adding Caspase3/7 substrate for apoptosis signal marking, the experimental plate was placed in Incucyte for observation and recording.
  • IncuCyte was used to monitor the changes in the fluorescence intensity of target cell A375-GFP during multiple rounds of killing.
  • A375-GFP cells were evenly plated in a multi-well plate. After culturing at 37°C for 4 hours, cells that had not been edited or knocked out of the target of the present invention were taken and added to the corresponding wells. The GFP fluorescence signal was then recorded using IncuCyte, with 3 replicates per group. After 24 hours of killing, the co-culture supernatant (20 ⁇ L/well) was taken for CBA detection; after 3 days of killing, a new A375-GFP was evenly plated in a multi-well plate.
  • the mixture of cells that had not been edited or knocked out of the target of the present invention and A375-GFP cells from the previous round of killing was transferred to a new 96-well plate, and the change curve of the GFP fluorescence signal in the new round of killing was recorded using IncuCyte.
  • the gene-edited cells were co-cultured with autologous tumor cells, and the co-culture supernatant was collected.
  • the CBA kit (BD) was used to detect the cytokine release of cells that were not edited or knocked out the target of the present invention.
  • Tumor tissue samples were obtained surgically and aseptically transferred to the laboratory. Necrotic tumor tissue was excised in a sterile cleanroom and cut into 1-2 mm3 pieces. A small amount of Matrigel was added to the cut tumor tissue and subcutaneously inoculated into NOG mice (Vitamin B, strain code 408) in an SPF animal room. Four to five pieces of tissue were inoculated per mouse. This batch of tumor tissue was designated the F0 generation. Once the F0 generation tumor tissue had grown to 800 mm3 subcutaneously, it was surgically excised and again cut into 1-2 mm3 pieces. Part of this was frozen for future use, and part was subcutaneously inoculated into a second batch of NOG mice for further passage and expansion.
  • mice in the NT group were intravenously injected with non-gene-edited cells, while mice in the experimental group were intravenously injected with the gene-edited cells of the present invention.
  • the sequence of the sgRNA (single guide RNA, or guide for short) targeting each target is synthesized according to the sequence provided by the present invention.
  • the region targeting each target can be selected from: the exon region of the target gene, the intron region about 100bp or about 20bp away from the exon of the target gene, and the region about 1500bp before the start codon of the target gene.
  • Figures 1A-1L show a continuous region with about 3 or more transcription factor binding numbers before the start codon provided by the present invention (such as the region with a vertical coordinate of more than 3 and a horizontal line segment).
  • the present invention also provides, targeting the exon region of each target, the intron region about 100bp or about 20bp away from the exon of the target gene, sgRNA that can enhance cell function after knockout.
  • sgRNA numbering information in the embodiment is as follows:
  • the knockout effect of sgRNA targeting ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2 was detected.
  • A means the knockout efficiency is greater than or equal to 70%, and N/A means not detected.
  • donors 711, 105, and 505 are lung cancer donors
  • donors 905, 306, and 312 are cervical cancer donors
  • donors 603 and 804 are melanoma donors, and are used to isolate and provide TILs.
  • Figure 2A shows the expansion fold of TILs edited with the gene of the present invention in the non-stimulation culture medium group.
  • FIG2B shows the expansion fold of TILs edited with the gene of the present invention in the TransACT stimulation group.
  • FIGS 3A-3H show the target cell killing ability of the gene-edited TIL cells of the present invention.
  • Figure 3I shows the killing ability of the gene-edited TIL cells of the present invention on the autologous tumor cell PDO model.
  • Each figure shows the killing curve at each time point, as well as the killing status of each experimental group at a specific time point, which is higher than that of the unedited NT group.
  • FIG4 shows the apoptosis ratio of gene-edited TIL cells of the present invention.
  • the target gene-edited T cells of the present invention can have a significantly reduced apoptosis ratio.
  • Figure 5A shows that the gene-edited TIL cells of the present invention have a higher proportion of central memory T cells.
  • Figure 5B shows that the gene-edited TIL cells of the present invention have a higher proportion of stem cells.
  • Figures 5C-5D show that the gene-edited TIL cells of the present invention have a lower proportion of exhausted T cells.
  • central memory T cells can be CD45RO positive CD62L positive cells.
  • stem cells can be CD39 negative CD69 negative cells.
  • exhausted T cells can be PD-1 positive, LAG-3 positive, TIM-3 positive, CD38 positive and/or CD101 positive cells.
  • Flow cytometry was used to detect the cytokine expression of cells that were not edited or knocked out the target of the present invention, and a CBA kit was used to detect the cytokine release ability.
  • Figures 6A-6G show the cytokine expression of the gene-edited TIL cells of the unstimulated group.
  • Figures 6H-6O show the cytokine expression of the gene-edited TIL cells of the present invention in the TransACT stimulation group.
  • Figure 6P shows the cytokine release after co-culture of the gene-edited TIL cells of the present invention with the autologous tumor cell PDO model.
  • the target gene-edited T cells of the present invention can have significantly improved cytokine expression and/or release capacity.
  • the knockout effect of the combination of sgRNA targeting ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2 and sgRNA targeting TNFAIP3, SOCS1, IKZF1, ZC3H12A, and PTPN2 was detected.
  • the sgRNA targeting TNFAIP3, SOCS1, IKZF1, ZC3H12A, and PTPN2 was selected from the guide sequences targeting the genes known in the art.
  • A indicates that the knockout efficiency is greater than or equal to 70%
  • B indicates that the knockout efficiency is greater than or equal to 50% and less than 70%
  • C indicates that the knockout efficiency is greater than or equal to 10% and less than 50%
  • N/A indicates not detected.
  • donors 505 and 105 are lung cancer donors
  • donors 804 and 812 are melanoma donors
  • donor 306 is a cervical cancer donor, and they are used to isolate and provide TILs.
  • Figure 7A shows the expansion fold of TILs edited with the combination gene of the present invention in the non-stimulation culture medium group.
  • FIG7B shows the expansion fold of TILs edited with the combination gene of the present invention in the TransACT stimulation group.
  • FIGS 8A-8D show the target cell killing ability of TIL cells edited by the combination of genes of the present invention.
  • Each figure shows the killing curve at each time point, as well as the killing status of each experimental group at a specific time point, which is higher than that of the unedited NT group.
  • FIGS 9A-9B show that the TIL cells after gene editing of the combination of the present invention have a higher proportion of central memory T cells.
  • central memory T cells can be CD45RO positive CD62L positive cells.
  • NT non-gene-edited control group
  • Flow cytometry was used to detect the cytokine expression of cells that were not edited or knocked out of the target combination of the present invention, and a CBA kit was used to detect the cytokine release ability.
  • Figures 10A-10C show the cytokine expression of TIL cells edited with the combination gene of the present invention in the unstimulated group.
  • Figures 10D-10F show the cytokine expression of TIL cells edited with the combination gene of the present invention in the TransACT stimulation group.
  • Figure 10G shows the cytokine release after co-culture of TIL cells edited by the combination of genes of the present invention with the autologous tumor cell PDO model.
  • T cells edited by the target gene combination of the present invention can have significantly improved cytokine expression and/or release capacity.
  • TIL cells edited by the target combination gene of the present invention can have more significant anti-apoptosis ability.

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Abstract

L'invention concerne une cellule modifiée et son utilisation. La présente invention concerne spécifiquement un procédé de culture d'une cellule, ledit procédé comprenant la réduction de l'expression et/ou l'affaiblissement de l'activité d'un gène cible de la cellule. La présente invention concerne en outre un procédé de prévention et/ou de traitement d'une tumeur à l'aide de la cellule cultivée.
PCT/CN2025/073804 2024-01-23 2025-01-22 Cellule modifiée et son utilisation Pending WO2025157155A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040170A2 (fr) * 1996-04-23 1997-10-30 Cadus Pharmaceutical Corporation Expression fonctionnelle d'adenylcyclase heterologue
WO2003038083A1 (fr) * 2001-10-29 2003-05-08 Bayer Healthcare Ag Regulation de l'adenylate cyclase humaine de type i
CN1617716A (zh) * 2000-06-08 2005-05-18 德克萨斯系统大学评议会 杂环衍生物与使用方法
CN1708582A (zh) * 2002-10-31 2005-12-14 独立行政法人理化学研究所 多能干细胞培养用的组合物及其使用
CN101171022A (zh) * 2005-03-07 2008-04-30 罗彻斯特大学 抑制g蛋白信号传导的组合物和方法
CN116406421A (zh) * 2021-07-13 2023-07-07 苏州沙砾生物科技有限公司 一种免疫细胞的培养方法及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040170A2 (fr) * 1996-04-23 1997-10-30 Cadus Pharmaceutical Corporation Expression fonctionnelle d'adenylcyclase heterologue
CN1617716A (zh) * 2000-06-08 2005-05-18 德克萨斯系统大学评议会 杂环衍生物与使用方法
WO2003038083A1 (fr) * 2001-10-29 2003-05-08 Bayer Healthcare Ag Regulation de l'adenylate cyclase humaine de type i
CN1708582A (zh) * 2002-10-31 2005-12-14 独立行政法人理化学研究所 多能干细胞培养用的组合物及其使用
CN101171022A (zh) * 2005-03-07 2008-04-30 罗彻斯特大学 抑制g蛋白信号传导的组合物和方法
CN116406421A (zh) * 2021-07-13 2023-07-07 苏州沙砾生物科技有限公司 一种免疫细胞的培养方法及其用途

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