Modified cell and use thereof

Abstract

Provided are a modified cell and the use thereof. The present invention specifically relates to a method for culturing a cell, which method comprises reducing the expression and / or weakening the activity of a target gene of the cell. The present invention further relates to a method for preventing and / or treating a tumor by using the cultured cell.

Description

A modified cell and its use Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a modified cell and its use. Background Art

[0002] Currently, immunotherapy is an effective treatment for patients with poor prognosis. However, the immune cells used in immunotherapy suffer from poor function, proliferation, and survival after infusion. Therefore, developing modified immune cells and robust and reliable immune cell culture methods are urgent challenges. Summary of the Invention

[0003] 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.

[0004] On the one hand, the present invention provides a method for culturing cells, comprising: reducing the expression and / or weakening the activity of a family member 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 cell.

[0005] In another aspect, the present invention provides a cell obtained by the method of the present invention.

[0006] In another aspect, the present invention provides a pharmaceutical composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0007] In another aspect, 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.

[0008] In another aspect, 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.

[0009] Those skilled in the art will readily appreciate other aspects and advantages of the present invention from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present invention. As will be appreciated by those skilled in the art, the disclosure of the present invention enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the present invention. Accordingly, the descriptions in the drawings and specification of the present invention are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The features and advantages of the present invention can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. The accompanying drawings are briefly described as follows:

[0011] 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.

[0012] Figure 2A shows the expansion fold of TILs edited with the gene of the present invention in the non-stimulation culture medium group.

[0013] FIG2B shows the expansion fold of TILs edited with the gene of the present invention in the TransACT stimulation group.

[0014] Figures 3A-3H show the target cell killing ability of the gene-edited TIL cells of the present invention.

[0015] Figure 3I shows the killing ability of the gene-edited TIL cells of the present invention on the autologous tumor cell PDO model.

[0016] FIG4 shows the apoptosis ratio of gene-edited TIL cells of the present invention.

[0017] Figure 5A shows that the gene-edited TIL cells of the present invention have a higher proportion of central memory T cells.

[0018] Figure 5B shows that the gene-edited TIL cells of the present invention have a higher proportion of stem cells.

[0019] Figures 5C-5D show that the gene-edited TIL cells of the present invention have a lower proportion of exhausted T cells.

[0020] Figures 6A-6G show the cytokine expression of the gene-edited TIL cells of the unstimulated group.

[0021] Figures 6H-6O show the cytokine expression of the gene-edited TIL cells of the present invention in the TransACT stimulation group.

[0022] 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.

[0023] Figure 7A shows the expansion fold of TILs edited with the combination gene of the present invention in the non-stimulation culture medium group.

[0024] FIG7B shows the expansion fold of TILs edited with the combination gene of the present invention in the TransACT stimulation group.

[0025] Figures 8A-8D show the target cell killing ability of TIL cells edited by the combination of genes of the present invention.

[0026] Figures 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.

[0027] Figures 10A-10C show the cytokine expression of TIL cells edited with the combination gene of the present invention in the unstimulated group.

[0028] Figures 10D-10F show the cytokine expression of TIL cells edited with the combination gene of the present invention in the TransACT stimulation group.

[0029] 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. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0031] Definition of terms

[0032] In the present application, the term "adenylate cyclase family member" generally refers to a family member protein having a cyclase domain or a functionally active fragment thereof. For example, the adenylate cyclase family member may include ADCY7. For example, 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. For example, the adenylate cyclase family member of the present application may include its functionally active fragments and any other domains.

[0033] In the present application, 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. For example, the RBR (RING-Between-RING) family member may include ARIH2. For example, 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. For example, the RBR (RING-Between-RING) family member of the present application may include its functionally active fragments and any other domains.

[0034] In the present application, the term "carnitine / choline acetyltransferase family member" generally refers to a family member protein or its functionally active fragment with an acetyltransferase domain. For example, the carnitine / choline acetyltransferase family member may include CPT2. For example, 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. For example, the carnitine / choline acetyltransferase family member of the present application may include its functionally active fragments and other arbitrary domains.

[0035] In this application, the term "peptidase M1 family member" generally refers to a family member protein having an aminopeptidase domain or a functionally active fragment thereof. For example, a peptidase M1 family member may include LNPEP. For example, 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. For example, the peptidase M1 family member of this application may include functionally active fragments thereof as well as any other domains.

[0036] In the present application, the term "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. For example, a nitric oxide synthase interacting protein family member may include NOSIP. For example, 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. For example, the nitric oxide synthase interacting protein family members of the present application may include functionally active fragments thereof and any other domains.

[0037] In the present application, the term "nitrogen permease regulator family member" generally refers to a family member protein or a functionally active fragment thereof having a GTPase domain. For example, the nitrogen permease regulator family member may include NPRL3. For example, 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. For example, the nitrogen permease regulator family member of the present application may include its functionally active fragments and other arbitrary domains.

[0038] In this application, 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. For example, a TRAF-associated factor family member may include TANK. For example, 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. For example, the TRAF-associated factor family members of this application may include functionally active fragments thereof as well as any other domains.

[0039] In this application, the term "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. For example, a tumor necrosis factor receptor-associated factor family member may include TRAF3. For example, 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. For example, the tumor necrosis factor receptor-associated factor family member of this application may include functionally active fragments thereof as well as any other domains.

[0040] In the present application, the term "TSC protein family member" generally refers to a family member protein having a helical domain or a functionally active fragment thereof. For example, the TSC protein family member may include TSC1. For example, 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. For example, the TSC protein family members of the present application may include functionally active fragments thereof and any other structural domains.

[0041] In this application, the term "BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member" generally refers to a family member protein having a BTB domain or a functionally active fragment thereof. For example, a BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may include ZBTB7B. For example, 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 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. For example, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member of this application may include functionally active fragments thereof as well as any other domains.

[0042] In this application, the term "ZC3H12 protein family member" generally refers to a family member protein having a C3H1-type zinc finger domain or a functionally active fragment thereof. For example, a ZC3H12 protein family member may include ZC3H12D. For example, 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. For example, the ZC3H12 protein family members of this application may include functionally active fragments thereof as well as any other domains.

[0043] In this application, the term "RC3H family member" generally refers to a family member protein having an ROQ domain or a functionally active fragment thereof. For example, the RC3H family member may include RC3H2. For example, 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. For example, the RC3H family members of this application may include functionally active fragments thereof as well as any other domains.

[0044] In this application, the term "AF4 family member" generally refers to a family member protein having a transcriptional activation domain or a functionally active fragment thereof. For example, the AF4 family member may include AFF3. For example, 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. For example, the AF4 family members of this application may include functionally active fragments thereof as well as any other domains.

[0045] In this application, the term "tyrosine protein kinase family member" generally refers to a family member protein having a phosphotransferase domain or a functionally active fragment thereof. For example, a tyrosine protein kinase family member may include AXL. For example, 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. For example, the tyrosine protein kinase family members of this application may include functionally active fragments thereof as well as any other domains.

[0046] In this application, the term "bZIP family member" generally refers to a family member protein or a functionally active fragment thereof having a bZIP-related DNA binding domain. For example, a bZIP family member may include NFE2L1. For example, 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. For example, the bZIP family members of this application may include functionally active fragments thereof and any other domains.

[0047] In the present application, the term "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. For example, a nuclear receptor family member may include RARG. For example, 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. For example, the nuclear receptor family members of the present application may include functionally active fragments thereof and any other domains.

[0048] In this application, the term "ubiquitin family member" generally refers to a family member protein having a ubiquitin-like domain or a functionally active fragment thereof. For example, the ubiquitin family member may include UBFD1. For example, 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. For example, the ubiquitin family members of this application may include functionally active fragments thereof and any other domains.

[0049] In this application, the term "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. For example, a Bcl-2 family member may include BCL2L11. For example, 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. For example, the Bcl-2 family members of this application may include functionally active fragments thereof as well as any other domains.

[0050] In the present application, the term "protein tyrosine phosphatase family member" generally refers to a family member protein or a functionally active fragment thereof having a tyrosine phosphatase domain. For example, the protein tyrosine phosphatase family member may include PTPN2. For example, 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. For example, the protein tyrosine phosphatase family member of the present application may include functionally active fragments thereof and any other domains.

[0051] In this application, the term "CBL family member" generally refers to a family member protein having an SH3 domain or a functionally active fragment thereof. For example, a CBL family member may include CBLB. For example, 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. For example, the CBL family members herein may include functionally active fragments thereof as well as any other domains.

[0052] In the present application, the term "STAT-induced STAT inhibitor (SSI) family member" generally refers to a family member protein having an SH2 domain or a functionally active fragment thereof. For example, a STAT-induced STAT inhibitor (SSI) family member may include SOCS1. For example, 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. For example, the STAT-induced STAT inhibitor (SSI) family member of the present application may include functionally active fragments thereof and any other domains.

[0053] In this application, the term "peptidase C64 family member" generally refers to a family member protein having a ubiquitin binding domain or a functionally active fragment thereof. For example, the peptidase C64 family member may include TNFAIP3. For example, 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. For example, the peptidase C64 family member of this application may include its functionally active fragments and any other domains.

[0054] In this application, the term "ZC3H12 family member" generally refers to a family member protein having a C3H1-type zinc finger domain or a functionally active fragment thereof. For example, a ZC3H12 family member may include ZC3H12A. For example, 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. For example, the ZC3H12 family members of this application may include functionally active fragments thereof as well as any other domains.

[0055] In the present invention, the term "IKAROS zinc finger protein family member" generally refers to a family member protein having a zinc finger domain or a functionally active fragment thereof. For example, an IKAROS zinc finger protein family member may include IKZF1. For example, 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. For example, the IKAROS zinc finger protein family members of the present invention may include functionally active fragments thereof as well as any other domains.

[0056] In this application, the term "tumor necrosis factor alpha-induced protein 3 (TNFAIP3)" generally refers to an inhibitory molecule of a signaling pathway. For example, TNFAIP3 can ubiquitinate signal transduction substances of the NF-κB pathway. For example, the UniProt accession number of TNFAIP3 can be P21580. In this application, TNFAIP3 can include unprocessed TNFAIP3, any form of processed TNFAIP3, variants of TNFAIP3, or substances comprising functionally active fragments of TNFAIP3.

[0057] In the present invention, the term "GTPase activating protein 1 family member" generally refers to a family member protein having a GTPase activation domain or a functionally active fragment thereof. For example, a GTPase activating protein 1 family member may include RASA2. For example, 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. For example, the GTPase activating protein 1 family member of the present invention may include functionally active fragments thereof and any other domains.

[0058] In the present invention, the term "FGF-binding protein family member" generally refers to a family member protein having an FGF-binding domain, or a functionally active fragment thereof. For example, an FGF-binding protein family member may include FIBP. For example, 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. For example, the FGF-binding protein family members of the present invention may include functionally active fragments thereof as well as any other domains.

[0059] In the present invention, the term "Mediator (MED) family member" generally refers to a family member protein having a CDK8 binding domain or a functionally active fragment thereof. For example, the Mediator (MED) family member may include MED12. For example, 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. For example, the Mediator (MED) family members of the present invention may include functionally active fragments thereof and any other structural domains.

[0060] In the present invention, the term "immune cell" generally refers to cells involved in innate and adaptive immune responses. For example, it may include but is not limited to lymphocytes (such as T cells (including thymocytes) and B cells), natural killer (NK) cells, NKT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells and mast cells. In some embodiments, 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 αβT cells and / or γδT cells. For example, the immune cells of the present invention also include immune cells derived from stem cell differentiation. For example, the immune cells of the present invention also include immune cells derived from pluripotent stem cell differentiation. For example, obtaining the stem cells of the present invention can be produced by induction. For example, the above-mentioned stem cells of the present invention can include induced pluripotent stem cells (iPSC).

[0061] In the present invention, the term "chimeric antigen receptor" generally refers to an engineered antigen receptor. For example, CAR may include an extracellular antigen binding domain fused to a cytoplasmic domain comprising a signaling domain via a hinge and a transmembrane domain. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0062] In the present invention, the term "T cell receptor" generally refers to an engineered antigen receptor. For example, a TCR may comprise TCR α and / or TCR β chains that have been isolated and cloned from a T cell population that recognizes a specific target antigen. For example, TCR α and / or TCR β genes (i.e., 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.

[0063] In the present invention, the term "gene regulatory system" generally refers to a system that regulates the expression or activity of a target gene. For example, a gene regulatory system may comprise a gene regulatory molecule. For example, 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.

[0064] In the present invention, the term "guide nucleic acid molecule" generally refers to a nucleic acid molecule that can be used for gene editing. For example, a guide nucleic acid molecule can provide information for nucleotide insertion or deletion to guide the editing process. For example, a guide nucleic acid molecule can be a guide RNA or a guide RNA (gRNA). For example, "gRNA" can refer to an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific position within the target DNA. For example, where hybridization between the gRNA and the DNA targeting sequence promotes the formation of a CRISPR complex, complete complementarity may not necessarily be required, for example, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex.

[0065] In the present invention, the term "enzyme protein" generally refers to a protein with enzymatic activity. For example, the enzyme protein may refer to a Cas protein. For example, 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. In the present invention, 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.

[0066] In the present invention, the term "ribonucleoprotein complex" generally refers to a complex formed by a protein and a nucleic acid. For example, the protein in the ribonucleoprotein complex can have nuclease activity. For example, the ribonucleoprotein complex can cleave a target sequence under the guidance of the nucleic acid therein. For example, the ribonucleoprotein complex can be a complex formed by a Cas protein and a guide RNA.

[0067] In the present invention, the term "lipid nanoparticle (LNP)" generally refers to a lipid-nucleic acid particle or nucleic acid-lipid particle. For example, 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. For example, proteins can be encapsulated in LNPs, for example, Cas proteins known in the art can be encapsulated in LNPs. For example, 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. For example, the lipids in LNPs can also include lipid derivatives, such as lipids covalently or non-covalently bound to proteins or polypeptides. For example, 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.

[0068] In the present invention, the term "exon" generally refers to a portion of a gene that can be expressed as a protein. For example, an exon can refer to a portion of a gene that has the ability to be expressed as a protein during protein biosynthesis. For example, cleaving an exon sequence of a target gene can reduce the activity or function of the target gene.

[0069] In the present invention, 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.

[0070] In the present invention, the term "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.

[0071] In the present invention, the term "protospacer adjacent motif (PAM)" generally refers to a short sequence following a target sequence. For example, when Cas9 performs site-specific cleavage of target DNA, the 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.

[0072] In the present invention, the term "reduced expression" generally refers to a decrease in the amount of expression of a product or its gene and / or a decrease in the proportion of cells capable of expressing the product (e.g., at least about 5-100%). For example, it may be that 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. For example, 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. For example, 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.

[0073] In the present invention, the term "activity" generally refers to the biological function of a substance. For example, the activity of a gene may refer to the transcriptional and / or translational status of the gene. For example, 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.

[0074] In the present invention, the term "CD80" generally refers to a cell-stimulatory molecule. For example, CD80 can be a ligand for CD28. For example, 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. For example, the CD80 of the present invention can include functionally active fragments of CD80 as well as any other domains.

[0075] In the present invention, the term "CD86" generally refers to a cell stimulatory molecule. For example, CD86 can be a ligand for CD28. For example, 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. For example, the CD86 of the present invention can include functionally active fragments of CD86 as well as any other domains.

[0076] As used herein, the term "secreted" generally refers to a substance that can be located outside of a cell. For example, a secreted substance can be synthesized within a cell and then transported to the extracellular space of the cell. For example, whether a substance is secreted can be detected using an enzyme-linked immunosorbent assay or other detection methods.

[0077] In the present invention, the term "T cell receptor" or "TCR" 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 (α) and beta (β) chains, or composed of gamma and delta (γ / δ) chains. TCR can exist in α / β and γ / δ forms, which are structurally similar but have unique anatomical locations and functions. For example, TCR can be a TCR that is modified on any cell that expresses TCR. For example, the type of TCR can be analyzed by TCR subtyping reagents.

[0078] In the present invention, the term "clonal diversity" generally refers to the presence of multiple clonal types in a given substance. For example, TCR clonal diversity can refer to TCRs having different sequence structures and / or antigen recognition capabilities. For example, TCR diversity is often distinguished by β-chain subtypes, which can include Vβ23, Vβ7.2, Vβ5.2, Vβ11, Vβ16, Vβ3, etc. When a T cell population has more β-chain subtypes, it can be considered to have higher clonal diversity.

[0079] In the present invention, "CD4 + Cells" generally refer to CD4-positive cells, such as T cells. The term "CD4 + "CD4 positive cells" can be used interchangeably. These cells can be identified by methods known in the art, such as staining the cells with fluorescently labeled antibodies against CD4 and using fluorescence activated cell sorting. For example, existing data can show that CD4 +The increase in the cell ratio can increase the ability of the cell population to secrete IFN and / or TNF, and can improve 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, there is a lack of a method to increase CD4 + The present invention can provide a method for affecting the CD4 + Cell ratio method.

[0080] In the present invention, "CD8 + Cells” generally refer to CD8-positive cells, such as T cells. The term “CD8 + These cells can be identified by methods known in the art, such as by staining the cells with fluorescently labeled antibodies against CD8 and using fluorescence-activated cell sorting.

[0081] In the present invention, the term "IC 50 The term "IC50 value" or "IC50 value" generally refers to the concentration of the target substance required to inhibit a biological process by 50%. The IC50 value can be converted to an absolute inhibition constant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22: 3099).

[0082] In the present invention, the term "K D A "KD value" or "KD value" generally refers to the dissociation constant, which can be determined by surface plasmon resonance. Typically, 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). Surface plasmon analysis can also be performed with an immobilized analyte (a substance on a biosensor matrix) and a presented ligand.

[0083] In the present invention, 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. For example, 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. For example, a deoxyribonucleic acid can encode an RNA transcribed from the deoxyribonucleic acid. Similarly, a deoxyribonucleic acid can encode a polypeptide translated from the RNA transcribed from the deoxyribonucleic acid.

[0084] In the present invention, the term "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.

[0085] In the present invention, the term "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.

[0086] In 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. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, 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., specifically bind CD3). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting 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, on the other hand, consist of two to five basic four-chain units that can combine with the J chain to form multivalent combinations. For IgG, 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 α and γ chains and four CH domains for the μ and ε isotypes. 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, and 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 α, δ, ε, γ, and μ, respectively.

[0087] In the present invention, the term "antigen-binding fragment" generally refers to one or more polypeptide fragments that have the ability to specifically bind to an antigen. In the present invention, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0088] In the present invention, 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. For example, 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. In the present invention, the term "expression" generally also refers to the transcription and / or translation process of the product. For example, the expression of a cytokine can be the process by which the cell transcribes and / or translates the cytokine. For example, 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.

[0089] In the present invention, the term "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. In one embodiment, each stage can be divided by the change in the number of TIL cells. In one embodiment, 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. In some embodiments, when the number of TIL cells increases by at least about 1-50 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, or at least about 50 times, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the conditions of TIL cell culture. In one embodiment, 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. In one embodiment, 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.

[0090] In the present invention, 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. In one embodiment, 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. In one embodiment, 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. For example, TILs derived from tumor tissue and not amplified in vitro can be referred to as the first TIL group. For example, 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.

[0091] In the present invention, 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. In one embodiment, 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. In one embodiment, 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. For example, in the culture method of the present invention divided by the two-step method, the second stage in vitro expansion can also be called the REP (rapid expansion) stage. For example, in the culture method of the present invention divided by the two-step method, the TIL obtained by the second stage in vitro expansion can be called the third TIL population.

[0092] In the present invention, the term "in vivo" generally refers to events that occur within the body of a subject.

[0093] In the present invention, the term "in vitro" generally refers to events that occur outside the body of a subject.

[0094] In the present invention, the term "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. In one embodiment, the cells, tissues and / or organs can be returned to the subject's body through surgery or treatment.

[0095] In the present invention, the term "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.

[0096] In the present invention, the term "irradiation" generally refers to the treatment of a substance by radiation. For example, in one embodiment, irradiation may refer to irradiating a substance by X-rays, α-rays, β-rays, or γ-rays.

[0097] In the present invention, 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. In one embodiment, the engineered cell can be genetically modified to express TILs of the present invention's T cell activators and / or T cell growth factors.

[0098] In the present invention, 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. Alternatively, in one embodiment, 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.

[0099] In the present invention, 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. In one embodiment, 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.

[0100] As used herein, 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.

[0101] As used herein, 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.

[0102] In the present invention, the term "polymer" generally refers to a molecule consisting of separate chemical moieties linked together, which can be the same or different. In one embodiment, 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. In one embodiment, 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.

[0103] In the present invention, 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. To create a chimeric 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. Alternatively, 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.

[0104] In the present invention, the term "humanized antibody", also known as CDR-grafted antibody, generally refers to an antibody produced by transplanting mouse CDR sequences into the antibody variable region framework of a human, i.e., 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. Such 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.

[0105] In the present invention, the terms "fully human antibody", "fully human antibody" or "completely human antibody" are also called "fully human monoclonal antibody", and the variable region and constant region of the antibody can be both human, eliminating immunogenicity and toxic side effects. 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.

[0106] In the present invention, the term "CDR" generally refers to one of the six hypervariable regions in the variable domains of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs can be provided by Kabat EA et al., Chothia et al., and MacCallum et al. As used in the present invention, the Kabat definition of CDR can be applied to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3, or L1, L2, L3) of a light chain variable domain, and CDR1, CDR2, and CDR3 (CDR H1, CDR H2, CDR H3, or H1, H2, H3) of a heavy chain variable domain.

[0107] In the present invention, the term "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.

[0108] In the present invention, 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. T cells can recognize these complexes using their T cell receptors (TCR). APCs can process antigens and present them to T cells. In one embodiment, antigen presenting cells can include those selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.

[0109] In the present invention, the term "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.

[0110] In the present invention, the term "persistence" generally refers to the presence of cells in vitro and / or in a subject. For example, an increase in the persistence of TIL cells may refer to an increase in the duration of TIL cell survival in vivo. For example, 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. For example, an increase in persistence may refer to an increase in the persistence of TIL cells after IL-2 is removed from the culture medium.

[0111] In the present invention, the term "artificial antigen-presenting cell" generally refers to an artificially constructed immune cell for presenting exogenous antigens. For example, 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. In one embodiment, isolated artificial antigen-presenting cells (aAPCs) 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.

[0112] In the present invention, the term "fusion protein" generally refers to a polypeptide or protein containing the amino acid sequence of a first polypeptide or protein or a fragment, analog or derivative thereof and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein or a fragment, analog or derivative thereof that is different from the first polypeptide or protein or a fragment, analog or derivative thereof, or that is not generally a part of the first polypeptide or protein or a fragment, analog or derivative thereof). In some cases, 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. For example, two different proteins, polypeptides or peptides having immunomodulatory activity can be fused together to form a fusion protein. In some cases, 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.

[0113] In the present invention, 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. In one embodiment, 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.

[0114] As used herein, the terms "administer" or "administering" 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.

[0115] In the present invention, the term "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.

[0116] In 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.

[0117] In the present invention, the term "feeder cell" generally refers to a cultured cell that can be used to support the growth of another cell of interest. For example, the feeder cell can be grown in vitro and secrete at least one factor into the culture medium. In one embodiment, the feeder cell can include an antigen presenting cell.

[0118] In the present invention, the term "specific binding" generally refers to recognizing a specific target substance but not substantially recognizing or binding to other molecules in the sample. For example, if a binding substance can specifically bind to a specific target substance of the present invention from one species, the binding substance of the present invention 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. In some cases, a binding substance that specifically binds to a target substance may also bind to different allelic forms of the target substance.

[0119] In the present invention, 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.

[0120] As used herein, the term "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. Typically, 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.

[0121] In the present invention, 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.

[0122] In the present invention, the term "tumor infiltrating lymphocytes" or "TIL" generally refers to a population of cells originally obtained as leukocytes that have left the bloodstream of a subject and migrated into a 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 can include primary TIL and secondary TIL. "Primary TIL" can be those TIL cells obtained from a subject's tissue sample, and "secondary TIL" can be any TIL population that has been expanded or amplified in the present invention. In some embodiments, the tumor infiltrating lymphocytes of the present invention may not be isolated and purified, or may be infiltrated with tumor cells. In one embodiment, the TIL of the present invention may refer to a TIL population.

[0123] In the present invention, the term "central memory T cells" generally refers to T cells with long-term memory and capable of accepting antigen restimulation. Central memory T cells may have CD45RO + CD62L + The phenotype can be, for example, CD45RO + and CD62L + To identify central memory T cells. Central memory T cells can have stronger anti-tumor growth ability than ordinary T cells.

[0124] In the present invention, the term "regulatory T cells" generally refers to a type of T cell subpopulation that controls autoimmune reactivity in the body. Regulatory T cells may have CD4 + CD25 + Foxp3 + The phenotype can be, for example, CD4 + 、CD25 + and Foxp3 + To identify regulatory T cells, which can suppress the anti-tumor growth ability of T cells.

[0125] In the present invention, the term "activated T cells" generally refers to T cells that have been activated to have the ability to resist tumor growth. Activated T cells may have PD-1 + (PD1 + ), LAG-3 + (LAG3 + ) or CD28 + The phenotype, for example, can be PD-1 + 、LAG-3 + or CD28 + To identify activated T cells. Activated T cells can have the ability to fight tumor growth.

[0126] In the present invention, the term "tumor-specific T cells" generally refers to T cells that can specifically resist tumor growth. Tumor-specific T cells may have CD103 + CD39 + The phenotype, for example, can be determined by CD103+ and CD39 + To identify tumor-specific T cells. Tumor-specific T cells can have more specific anti-tumor growth capabilities than ordinary T cells.

[0127] In the present invention, the term "stem cell-like T cells" generally refers to a type of T cell that has the potential for self-proliferation and / or differentiation. For example, in the present invention, cells with differentiation potential and / or sustained proliferation ability can be considered stem cell-like cells. For example, naive T cells (CD45RO - CD62L + ) can be considered as stem cell-like cells. For example, naive T cells may have CD45RO - CD62L + For example, it can be through CD45RO - and CD62L + To identify stem cell-like T cells. For example, CD39 - and CD69 - To identify stem-like T cells. For example, stem-like T cells can have TCF1 + The phenotype, for example, can be caused by TCF1 + To identify stem cell-like T cells. Stem cell-like T cells may have stronger and / or longer-term anti-tumor growth capabilities than ordinary T cells.

[0128] In the present invention, the term 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.

[0129] In the present invention, the term "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.

[0130] As used herein, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the active ingredient. For example, 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. Other contemplated carriers, excipients, and / or additives that may be used in the formulations described herein 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. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art. As used herein, "pharmaceutically acceptable carrier" may be understood to mean a vector that does not include nucleic acid forms used in genetic engineering.

[0131] In the present invention, 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. For example, a functionally active fragment may retain or partially retain the ability of the full-length protein to bind to another molecule.

[0132] In the present invention, the term "T cell activator" generally refers to a substance that binds to the corresponding binding receptor on the T cell and mediates the T cell co-stimulatory reaction. A T cell activator can be a substance other than an antigen receptor required for T cells to produce an effective immune response. A T cell activator can refer to a T cell co-stimulatory molecule. For example, the T cell activator of the present invention can include its variant, homologue or any substance comprising its functionally active fragment. T cell activators can include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal lymphocyte activation molecules (SLAM proteins), NK cell activation receptors, BTLA (the gene GeneID encoding it can be 151888), Toll ligand receptor, OX40 (the gene GeneID encoding it can be 7293), CD2 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 924), CD27 (the gene GeneID encoding it can be 939), CD28 (the gene GeneID encoding it can be 939), CD29 (the gene GeneID encoding it can be 941), CD30 (the gene GeneID encoding it can be 943), CD31 (the gene GeneID encoding it can be 944), CD32 (the gene GeneID encoding it can be 945), CD33 (the gene GeneID encoding it can be 946), CD34 (the gene GeneID encoding it can be 947), CD35 (the gene GeneID encoding it can be 948), CD36 (the gene GeneID encoding it can be 949), CD37 (the gene GeneID encoding it can be 949), CD38 (the gene GeneID encoding it can be 948), CD39 (the gene GeneID encoding it can be 949), CD40 (the gene GeneID encoding it can be 949), CD41 (the gene GeneID encoding it can be 9 The following are the genes encoding the cytokine: (GeneID 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 encoding it may be 80381), ICOS (CD278) (the gene encoding it may be 29851), GITR (the gene encoding it may be 8784), BAFFR (the gene encoding it may be 115650), LIGHT (the gene encoding it may be 8740), HVEM (LIGHTR) (the gene encoding it may be 8764), KIRDS2 (the gene encoding it may be 100132285), SLAMF7 (the gene encoding it may be 57823), NKp80 (KL RF1) (the gene encoding it may be 51348), NKp44 (the gene encoding it may be 9436), NKp30 (the gene encoding it may be 259197), NKp46 (the gene encoding it may be 9437), CD19 (the gene encoding it may be 930), CD4 (the gene encoding it may be 920), CD8α (the gene encoding it may be 925), CD8β (the gene encoding it may be 926),IL-2Rβ, IL-2Rγ, IL7Rα (the gene encoding it can be GeneID 3575), ITGA4 (the gene encoding it can be GeneID 3676), VLA1 (the gene encoding it can be GeneID 3672), CD49a (the gene encoding it can be GeneID 3672), IA4 (the gene encoding it can be GeneID 3732), CD49D (the gene encoding it can be GeneID 3676), ITGA6 (the gene encoding it can be GeneID 3655), VLA-6 (the gene encoding it can be GeneID 3655), CD49f (the gene encoding it can be GeneID 3672), 3655), ITGAD (the gene encoding it may be 3681), CD11d (the gene encoding it may be 3681), ITGAE (the gene encoding it may be 3682), CD103 (the gene encoding it may be 3682), ITGAL (the gene encoding it may be 3683), CD11a (the gene encoding it may be 3683), LFA-1 (the gene encoding it may be 3683), ITGAM (the gene encoding it may be 3684), CD11b (the gene encoding it may be 3684) , ITGAX (the gene encoding it may be 3687), CD11c (the gene encoding it may be 3687), ITGB1 (the gene encoding it may be 3688), CD29 (the gene encoding it may be 3688), ITGB2 (the gene encoding it may be 3689), CD18 (the gene encoding it may be 3689), 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) (the gene encoding it may be 10666), SLAMF4 (CD244, 2B4) (the gene encoding it may be 51744), CD84 (the gene encoding it may be 8832), CD96 (Tactile) (the gene encoding it may be 10225), CEACAM1 (the gene encoding it may be 634),CRTAM (the gene encoding it may be 56253 in GeneID), Ly9 (CD229) (the gene encoding it may be 4063 in GeneID), CD160 (BY55) (the gene encoding it may be 11126 in GeneID), PSGL1 (the gene encoding it may be 6404 in GeneID), CD100 (SEMA4D) (the gene encoding it may be 10507 in GeneID), CD69 (the gene encoding it may be 969 in GeneID), SLAMF6 (NTB-A, Ly108) (the gene encoding it may be 114836 in GeneID), SLAM (SLAMF1, CD150, IPO-3) (the gene encoding it may be 6504 in GeneID), BLAME (SLAM F8) (the gene encoding it may be 56833), SELPLG (CD162) (the gene encoding it may be 6404), LTBR (the gene encoding it may be 4055), LAT (the gene encoding it may be 27040), GADS (the gene encoding it may be 9402), SLP-76 (the gene encoding it may be 3937), PAG / Cbp (the gene encoding it may be 55824), CD19a, a ligand that specifically binds to CD3, a ligand that specifically binds to CD28, a ligand that specifically binds to HVEM, a ligand that specifically binds to CD40L, a ligand that specifically binds to OX40, and a ligand that specifically binds to 4-1BB. The co-stimulatory intracellular signaling domain may refer to the intracellular portion of a T cell activator. The intracellular signaling domain may comprise the entire intracellular portion of a molecule derived therefrom or an entire native intracellular signaling domain or a functional fragment thereof. ,

[0133] In the present invention, the term "T cell growth factor" generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation. For example, the T cell growth factor of the present invention may include its variants, homologs, or any substance containing its functionally active fragments. In one embodiment, 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-α (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.

[0134] In the present invention, the term "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. In one embodiment, 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.

[0135] In the present invention, 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. Typically, 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.

[0136] In the present invention, the term "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 the present invention, 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.

[0137] In the present invention, the term "peripheral mononuclear cells" or "peripheral blood mononuclear cells" generally refers to cells with a single nucleus in peripheral blood. For example, in the present invention, the peripheral blood mononuclear cells of the present invention may include lymphocytes, monocytes and / or dendritic cells.

[0138] In the present invention, the term "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-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-15, IL-21 and / or IL-12. In the present invention, 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.

[0139] In the present invention, the term "diameter" generally refers to the diameter of a cross-section of the substance of the present invention. For example, when the substance of the present invention is not spherical, 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.

[0140] In the present invention, the term "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.

[0141] In the present invention, the term "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.

[0142] In the present invention, the term "T cell subset ratio" generally refers to the ratio of different T cell subsets to TIL cells or TIL populations. For example, different T cell subsets of the present invention have different immune activities and / or differentiation abilities. For example, the T cell subsets of the present invention can be distinguished based on T cell surface markers. For example, central memory T cells may have CD45RO + CD62L + For example, naive T cells may have a CD45RO - CD62L + For example, regulatory T cells may have a CD4 + CD25 + Foxp3 + For example, activated T cells may have CD25 + 、CD28 + , PD-1 + or 41BB + For example, tumor-specific T cells may have CD103 + CD39 + For example, stem-like T cells can have TCF1 + phenotype.

[0143] In the present invention, the term "TIL cell number" generally refers to the number of cells in the TIL cells of the present invention. In 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. For example, 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. For example, the number of TIL cells may refer to the number of cells of a second TIL population amplified in vitro in the first stage. For example, the number of TIL cells may refer to the number of cells of a third TIL population amplified in vitro in the second stage. For example, 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. In 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.

[0144] In the present invention, 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.

[0145] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0146] Detailed Description of the Invention

[0147] On the one hand, the present invention provides a method for culturing cells, thereby reducing the expression and / or weakening the 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.

[0148] For example, 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.

[0149] For example, the adenylate cyclase family member may comprise a cyclase domain. For example, the adenylate cyclase family member may comprise ADCY7.

[0150] For example, the RBR (RING-Between-RING) family member may comprise a RING-type zinc finger domain. For example, the RBR (RING-Between-RING) family member may comprise ARIH2.

[0151] For example, the carnitine / choline acetyltransferase family member may comprise an acetyltransferase domain. For example, the carnitine / choline acetyltransferase family member may comprise CPT2.

[0152] For example, the peptidase M1 family member may comprise an aminopeptidase domain. For example, the peptidase M1 family member may comprise LNPEP.

[0153] For example, the nitric oxide synthase interacting protein family member may comprise a nitric oxide synthase binding domain. For example, the nitric oxide synthase interacting protein family member may comprise NOSIP.

[0154] For example, the nitrogen permease regulator family member may comprise a GTPase domain. For example, the nitrogen permease regulator family member may comprise NPRL3.

[0155] For example, the TRAF-associated factor family member may comprise a TRAF binding domain. For example, the TRAF-associated factor family member may comprise a TANK.

[0156] For example, the tumor necrosis factor receptor-associated factor family member may comprise a receptor cytoplasmic binding domain. For example, the tumor necrosis factor receptor-associated factor family member may comprise TRAF3.

[0157] For example, the TSC protein family member may comprise a helical domain. For example, the TSC protein family member may comprise TSC1.

[0158] For example, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may comprise a BTB domain. For example, the BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member may comprise ZBTB7B.

[0159] For example, the ZC3H12 protein family member may comprise a C3H1-type zinc finger domain. For example, the ZC3H12 protein family member may comprise ZC3H12D.

[0160] For example, the RC3H family member may comprise a ROQ domain.For example, the RC3H family member may comprise RC3H2.

[0161] For example, in the cells of the present invention, the expression and / or activity of members 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, and the RC3H family and / or their functionally active fragments are reduced. For example, the expression and / or activity of ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2 are reduced.

[0162] For example, the gene of interest 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 associated factor family, the tumor necrosis factor receptor associated 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. For example, compared with cells in which the expression and / or activity of the gene of interest is not changed, the cells obtained by reducing the expression and / or weakening the activity of the gene of interest of the cell can show improved cell characteristics. In one embodiment, the cells in which the expression and / or activity of the gene of interest is not changed can refer to cells derived from the same donor and in which the expression and / or activity of the gene of interest of the cell is not reduced and / or the activity is weakened. In one embodiment, 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).

[0163] 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 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. 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 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. For example, 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.

[0164] For example, the cell comprises an immune cell. For example, the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and / or a basophil. For example, the cell comprises a monocyte, a macrophage and / or a dendritic cell.

[0165] For example, the cells of the present invention also include cells derived from stem cell differentiation. For example, the cells of the present invention also include cells derived from pluripotent stem cell differentiation. For example, obtaining the stem cells of the present invention can be through induction. For example, 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.

[0166] For example, the term "stem cells" of the present invention also includes pluripotent cells, multipotent cells, precursor cells, and progenitor cells. For example, 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. Exemplary 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."

[0167] For example, the cell comprises B cells, T cells, natural killer cells and / or natural killer-like T cells (NKT). For example, "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.). For example, the cell comprises αβT cells and / or γδT cells. For example, the cell comprises tumor infiltrating lymphocytes (TIL). For example, 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.

[0168] For example, 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. For example, the TILs of the present invention can be obtained by processing tumor tissue into tumor fragments. For example, the volume of the tumor fragments of the present invention is about 1-27 cubic millimeters. For example, 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.

[0169] For example, the cell comprises an engineered immunoreceptor displayed on the cell surface. For example, the engineered immunoreceptor specifically binds to an antigen expressed on a target cell. For example, the cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0170] On the one hand, the present invention provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: reducing 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 in the TIL.

[0171] For example, TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and / or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of 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 functionally active fragments can be reduced.

[0172] For example, 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 ascites 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 second stage in vitro expansion of the present invention, 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 functionally active fragments can be reduced. For example, 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 ascites 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 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 functionally active fragments can be reduced.

[0173] For example, 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 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 family, ZC3H12 protein family, or RC3H family members and / or their functionally active fragments are reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of members 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 their functionally active fragments can be reduced.

[0174] For example, 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 ascites and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of members 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 functionally active fragments can be reduced.

[0175] For example, 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 ascites and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of members 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 functionally active fragments can be reduced.

[0176] For example, 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 ascites and not expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of members 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 functionally active fragments can be reduced.

[0177] For example, 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 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, ZC3H12 protein family, or RC3H family members and / or their functionally active fragments are reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of members 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 their functionally active fragments can be reduced.

[0178] For example, 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 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, ZC3H12 protein family, or RC3H family members and / or their functionally active fragments are reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of members 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 their functionally active fragments can be reduced.

[0179] For example, 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 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-brac) protein family, ZC3H12 protein family, or RC3H family members and / or their functionally active fragments are reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of members 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 their functionally active fragments can be reduced.

[0180] For example, 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 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, ZC3H12 protein family, or RC3H family members and / or their functionally active fragments are reduced in expression and / or activity, and in the second stage of in vitro expansion of the present invention, the TIL can be 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 members and / or their functionally active fragments are reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of members 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 their functionally active fragments can be reduced.

[0181] For example, 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. In some embodiments, when 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. For example, each stage of in vitro expansion can also be divided by the change in the conditions of TIL cell culture. For example, 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. For example, 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. For example, when one or more gene regulatory systems 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. For example, when 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. For example, after the TIL cells are centrifuged and / or washed, the TIL cells can be considered to have entered the next stage of in vitro expansion. For example, each stage can also be divided by the number of days the TIL cells are cultured. For example, after 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.

[0182] For example, 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.

[0183] For example, the reducing the expression and / or weakening the activity of RBR (RING-Between-RING) family members in the cell comprises the function of inhibiting ubiquitination.

[0184] For example, 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.

[0185] For example, the reducing the expression and / or weakening the activity of a peptidase M1 family member in the cell comprises inhibiting the function of degrading polypeptides.

[0186] For example, 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.

[0187] For example, 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.

[0188] For example, 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.

[0189] For example, 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.

[0190] For example, 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.

[0191] For example, reducing the expression and / or weakening the activity of a BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family member in the cell comprises inhibiting transcriptional regulatory function.

[0192] For example, the reducing the expression and / or weakening the activity of the ZC3H12 protein family member in the cell comprises inhibiting the activity of endonuclease.

[0193] For example, 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.

[0194] For example, 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 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, Cells obtained by reducing the expression and / or activity of members of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family exhibit improved cell properties.

[0195] For example, 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.

[0196] For example, 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.

[0197] For example, 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, 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 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 is unchanged in at least one in vitro expansion stage. The cell number of cells of the invention having reduced expression and / or reduced activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family can be increased by at least about 1-50 fold, for example, at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold.

[0198] For example, an increased proportion of live cells can be manifested as an increase in cell survival. For example, the increased proportion of viable cells in the present invention may 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 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 unchanged in at least one in vitro expansion stage. The proportion of viable cells of the cells of the present invention in which the expression of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced and / or the activity is attenuated 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 17%, at least about 16%, or more. %, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0199] For example, the increased cytokine secretion capacity of the present invention may refer to an increased cytokine secretion capacity of cells selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF-α, and IFN-γ. For example, 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, 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 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 is unchanged. In cells of the present invention in which the expression and / or activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced, 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.For example, the improved cytokine secretion capacity of the present invention may 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 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 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 unchanged. The proportion of cells secreting cytokines in the cells of the present invention in which the expression of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced and / or the activity is attenuated 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 17%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about At least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%. For example, the cytokine secretion capacity of the cells of the invention is determined by flow cytometry or CBA (Cytometric Bead Array).

[0200] For example, the improved in vitro tumor cell killing ability and / or improved in vivo tumor killing ability of the present invention can refer to 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, BTB (Broad-Complex, Tramtrack and The expression and / or activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is unchanged compared to cells in which the expression and / or activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is unchanged, wherein in at least one in vitro expansion stage, the 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 The tumor cell killing rate of the cells of the present invention with reduced expression and / or reduced activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family can be increased by at least about 1-50 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, or at least about 50 times.For example, the improved in vitro tumor cell killing ability and / or improved in vivo tumor killing ability of the present invention can refer to 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, BTB (Broad-Complex, Tramtrack and The expression and / or activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is unchanged compared to cells in which the expression and / or activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is unchanged, wherein in at least one in vitro expansion stage, the 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 The tumor cell killing rate of the cells of the present invention with reduced expression and / or reduced activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family 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 18%, at least about 19%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 16%, at least about 17%, at least about 18%, at least about 19 ... At least about 6%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%. For example, the tumor cell killing rate of the cells of the invention can be measured by the IncuCyte system or CFSE and DAPI staining. For example, tumor cell killing of the cells of the invention can refer to the ability of the cells to kill solid tumor cells.

[0201] For example, the cell subpopulation ratio improved by the present invention may comprise one or more selected from the following groups: increased CD8 + The proportion of cells, increased proportion of central memory cells and / or naive cells, decreased proportion of regulatory cells, increased proportion of activated cells, increased proportion of tumor-specific cells (with CD103 + CD39 +phenotype), increased proportion of stem-like cells, decreased proportion of exhausted cells, and decreased proportion of apoptotic cells.

[0202] For example, the present invention increases CD8 + The cell ratio can be an increase in the ratio of CD8 positive cells in the cells. + The proportion of cells 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 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0203] For example, the ratio of activated cells increased by the present invention can be CD28 + 、CD25 + and / or 41BB + For example, the proportion of activated 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 In some embodiments, 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 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold.

[0204] For example, the ratio of exhausted cells reduced by the present invention can be PD-1 + 、LAG-3 + 、TIM-3 + 、CD39 + 、CD38 + and / or CD101 + For example, the proportion of depleted 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%, or at least about 1%. In some embodiments, the present invention may reduce the amount of the active ingredient in the pharmaceutical composition to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 600%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 700%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about 770%, about 780%, about 790%, about

[0205] For example, the ratio of regulatory cells reduced by the present invention can be CD4 + CD25 + Foxp3 + A decrease in the proportion of cells. For example, 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%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0206] For example, the ratio of apoptotic cells reduced by the present invention can be + 7-AAD + Cells and / or Annexin V + 7-AAD - A decrease in the proportion of cells. For example, the proportion of apoptotic cells in a cell can be reduced by at least about 100-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.9%, at least about 0.8%, at least about 0.7%, at least about 0.6%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0207] For example, the proportion of cells with stemness increased by the present invention can be CD69 - CD39 - cells and / or TCF1 + The proportion of cells increased. For example, 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 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0208] For example, the increased central memory cell ratio of the present invention can be CD45RA - CCR7 + or CD45RO + CD62L +The proportion of cells increased. For example, 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 about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0209] For example, the ratio of naive T cells increased by the present invention can be CD45RO - CD62L + The proportion of cells increased. For example, 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 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0210] For example, the method of the present invention can include editing the target gene in vivo, in vitro and / or in vitro. For example, 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. For example, by targeting immune cells or their precursor cells, such as bone marrow stem cells, delivering LNPs containing the gene regulatory system or mRNA encoding the gene regulatory system, the target gene in vivo can be edited. By adjusting 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.

[0211] For example, the culture method of the present invention may include a gene editing step for the cells. For example, it 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.

[0212] For example, the gene regulation system can destroy the target gene at the DNA level. For example, the gene regulation system can destroy the region or fragment thereof of the target gene in the genome of the cell. For example, after using the gene regulation system, 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. For example, the editing effect of the gene regulation system on the target gene can be long-term and continuous. For example, in the cells of the present invention, 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 is inhibited.

[0213] The genomic region of the present invention is determined based on the human reference genome version hg38.

[0214] For example, the gene regulation system can include a guide nucleic acid molecule and an enzyme protein. For example, 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. For example, 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. For example, the enzyme protein can include a Cas protein. For example, the polynucleotide encoding gRNA and Cas protein can be introduced or each independently introduced into the target cell.

[0215] For example, 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. For example, the enzyme protein can comprise a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the guide nucleic acid molecule can comprise a guide RNA (gRNA). For example, a complex comprising a polynucleotide encoding a gRNA and a Cas protein can be introduced into the cell. For example, a complex comprising a gRNA and a Cas protein can be introduced into the cell.

[0216] For example, the gRNA can be used to bind to the sequence of the target gene. For example, 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. For example, 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.

[0217] For example, the editing target region of the present invention may be a region before the start codon. For example, the editing target region of the present invention may be a region with high transcription factor binding affinity. For example, the editing target region of the present invention may be a region with a specific number of transcription factor binding numbers. For example, the editing target region of the present invention may be a continuous region with about 3 or more transcription factor binding numbers. For example, 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.

[0218] For example, the guide nucleic acid molecule targeting ADCY7 of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 4146-8290.

[0219] For example, the guide nucleic acid molecule targeting ARIH2 of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 10494-12696.

[0220] For example, the guide nucleic acid molecule targeting CPT2 of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 13414-14130.

[0221] For example, the guide nucleic acid molecule targeting LNPEP of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 15950-17768.

[0222] For example, the guide nucleic acid molecule targeting NOSIP of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 19561-21352.

[0223] For example, the guide nucleic acid molecule targeting NPRL3 of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 22857-24360.

[0224] For example, the guide nucleic acid molecule targeting TANK of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 25134-25906.

[0225] For example, the TRAF3-targeting guide nucleic acid molecule of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 29216-32524.

[0226] For example, the guide nucleic acid molecule targeting TSC1 of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 34537-36548.

[0227] For example, the guide nucleic acid molecule targeting ZBTB7B of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 39784-43018.

[0228] For example, the guide nucleic acid molecule targeting ZC3H12D of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 44428-45836.

[0229] For example, the guide nucleic acid molecule targeting RC3H2 of the present invention can bind to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 47259-48680.

[0230] For example, when the gene editing system includes CRISPR / Cas9, 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. For example, when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides upstream of the 5' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence. For example, 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.

[0231] For example, when the gene editing system comprises CRISPR / Cas12, the region targeted by the guide nucleic acid molecule of the present invention can have a protospacer adjacent motif (PAM) upstream, and 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. For example, the protospacer adjacent motif (PAM) can be TTTN. For example, the protospacer adjacent motif (PAM) can be TTN. For example, once 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. For example, 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. For example, the protospacer adjacent motif (PAM) may be TTTN. For example, the protospacer adjacent motif (PAM) may be TTN.

[0232] For example, when the gene editing system of the present invention comprises wild-type Cas12a (also referred to as Cpf1, such as AsCas12a, FnCas12a, LbCas12a, BbCas12a, CMaCas12a and OsCas12a), 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. For example, 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.

[0233] For example, when the gene editing system of the present invention includes a mutant Cas12a, such as enAsCas12a (mutation sites E174R, S542R and K548R), 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. For example, 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.

[0234] For example, when the gene editing system of the present invention includes a mutant Cas12a, such as opAsCas12a (mutation sites: E174R and S542R), 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 may be A, C or G. For example, 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.

[0235] For example, when the gene editing system of the present invention includes a mutant Cas12a, such as AsCas12aUltra (mutation sites: M537R and F870L), 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. For example, 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.

[0236] For example, when the gene editing system of the present invention includes mutant Cas12a, such as hfCas12Max (mutation site: N243R / E336R / D892R) and Cas12Max (mutation site: N243R), 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. For example, 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.

[0237] For example, when the gene editing system of the present invention comprises wild-type Cas12b or mutant Cas12b (such as AaCas12b protein from Alicyclobacillus acidiphilus), 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. For example, when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.

[0238] For example, when the gene editing system of the present invention comprises wild-type Cas12i or mutant Cas12i (Cas protein with a smaller size), 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 can be A, T, C or G. For example, when the PAM region of the target gene is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the PAM of the target gene, and can design a suitable gRNA for the target sequence.

[0239] For example, 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 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 fragments. For example, the guide nucleic acid molecule can comprise a family of proteins encoding proteins 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, BTB (Broad-Complex, Tramtrack and The target sequence comprises about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 22 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 20, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides preceding the PAM region represented by AGG, TGG, GGG and / or CGG in the DNA where the gene for a member of the Bric-a-brac protein family, the ZC3H12 protein family, or the RC3H family and / or functionally active fragments thereof is located. For example, the target sequence can be selected from a region defined by the genomic coordinates shown in Tables 2A-2L, or a fragment thereof.

[0240] For example, the guide nucleic acid molecule can 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, an sgRNA targeting TANK as shown in any one of SEQ ID NOs: 24361-25133, an sgRNA targeting TRAF3 as shown in any one of SEQ ID NOs: 25907-29215, an sgRNA targeting VEGF as shown in any one of SEQ ID NOs: 2690-27147, an sgRNA targeting VEGF as shown in any one of SEQ ID NOs: 2791-2830 an sgRNA targeting TSC1 as shown in any one of SEQ ID NOs: 32525-34536, an sgRNA targeting ZBTB7B as shown in any one of SEQ ID NOs: 36549-39783, an sgRNA targeting ZC3H12D as shown in any one of SEQ ID NOs: 43019-44427, or an sgRNA targeting RC3H2 as shown in any one of SEQ ID NOs: 45837-47258.

[0241] For example, compared to cells in which the expression and / or activity of the target gene is not altered, the proportion of cells expressing the product of the 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.

[0242] For example, in the method of the present invention, the proportion of cells expressing the product of the target gene in the cells obtained by reducing the expression and / or weakening 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. For example, the expression of the encoding protein is 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 In some embodiments, the cellular proportion of a gene product of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family, and / or functionally active fragments thereof, is reduced by at least about 100-5%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%. For example, the proportion of cells expressing the product of 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 can be from the observable cell proportion to 1%.For example, the expression of the coding member is 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 The cellular proportion of the product of a gene of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family, and / or functionally active fragments thereof, 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 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%. For example, the proportion of cells expressing the product of 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 fragments can be detected by flow cytometry.

[0243] For example, in the method of the present invention, the expression and / or activity of the target gene in the cells obtained by reducing isotope expression and / or attenuating isotope activity, and the proportion of cells expressing the product of the 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 fragments can be up to about 95%. For example, the expression of the coding member is 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 The cellular proportion of the product of a gene of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family, and / or functionally active fragments thereof, can be at most about 95-5%, such as at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%. For example, the proportion of cells expressing the product of 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 fragments can be detected by flow cytometry.

[0244] For example, in the methods of the present invention, 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. For example, 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%. For example, the expression of the target gene in a single cell can be reduced from the observed expression level to 1%. For example, 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%.

[0245] For example, the expression of the target gene in a single cell obtained by reducing the expression and / or weakening the activity of the target gene of the cell by the method of the present invention can be at most about 95% of the expression and / or activity of the target gene in the cell in which the expression and / or activity of the target gene is unchanged. For example, the encoding protein in a single cell is 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 The expression level of a gene encoding a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family and / or a functionally active fragment thereof (e.g., a gene encoding ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, or RC3H2) can be selected from the group consisting 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 In some embodiments, the expression and / or activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family, and / or functionally active fragments thereof, is unchanged in at most about 95-5%, such as at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%.

[0246] For example, the method of the present invention comprises: subjecting the cell to at least one stage of in vitro expansion, wherein, in at least one stage of 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.

[0247] For example, the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and / or 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 of 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 expanded in vitro in the first stage are reduced.

[0248] For example, the first stage in vitro expansion is performed for at least about 7 days.For example, the second stage in vitro expansion is performed for at least about 7 days.

[0249] For example, the cells can be contacted with the one or more cell activators and the expression and / or activity of a member 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, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family and / or its functionally active fragment can be reduced in the in vitro expansion of the present invention at a single stage. For example, the cell activator can include an agonist of one or more targets selected from the following groups: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB. For example, in a single 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 in the cells of the present invention is reduced and / or the activity is attenuated, and the cells are contacted with one or more cell activators of the present invention. For example, in the first stage of in vitro expansion of the present invention, the expression and / or activity of 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 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 can be reduced and the TIL is contacted with one or more cell activators of the present invention.For example, in the second stage in vitro expansion of the present invention, the expression and / or activity of 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 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 can be reduced and the TIL is contacted with one or more cell activators of the present invention. For example, in the third stage in vitro expansion of the present invention, the expression of 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 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 can be reduced and / or the activity is weakened and the TIL is contacted with one or more cell activators of the present invention.

[0250] For example, in a single stage of in vitro expansion, the cells of the present invention substantially 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 one or more cell activators of the present invention. For example, in a single stage of in vitro expansion, the cells of the present invention first 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, 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 the cells with one or more cell activators of the present invention. For example, in a single stage of in vitro expansion, 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.

[0251] For example, in the first stage of in vitro expansion of the present invention, the TILs of the present invention substantially simultaneously reduce 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 contact with one or more cell activators of the present invention. For example, in the second stage of in vitro expansion of the present invention, the TILs of the present invention substantially simultaneously reduce 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 contact with one or more cell activators of the present invention. For example, in the third stage of in vitro expansion of the present invention, the TILs of the present invention substantially simultaneously reduce 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 contact with one or more cell activators of the present invention.

[0252] For example, the second stage of in vitro expansion of the present invention is performed for at least about 9 days. For example, the second stage of in vitro expansion of the present invention can be performed 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. For example, the second stage of in vitro expansion of the present invention can be performed 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. For example, the second stage of in vitro expansion of the present invention can be considered the REP (rapid expansion protocol) stage. For example, the first stage of in vitro expansion of the present invention can be considered the preREP stage.

[0253] For example, 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. For example, when 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. For example, about 24 hours after the start of the second stage in vitro amplification, it can be considered that the second stage in vitro amplification has been performed for about 1 day. For example, 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. For example, 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.

[0254] For example, the cell activator of the present invention may comprise one or more selected from the following group: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and functionally active fragments thereof. For example, the cell activator of the present invention may comprise an agonist of one or more targets selected from the following group: CD3, CD28, HVEM, CD40L, OX40, and 4-1BB. For example, the cell activator of the present invention may comprise an antibody selected from the following group: CD3, CD28, HVEM, CD40L, OX40, and 4-1BB, and an antigen-binding fragment thereof. For example, the cell activator of the present invention may comprise a CD3 agonist. For example, the cell activator of the present invention may comprise an anti-CD3 antibody and / or an antigen-binding fragment thereof, such as Miltenyi Biotech's OKT3 or BD's SP34. For example, the cell activator of the present invention may comprise a CD28 agonist. For example, 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.

[0255] For example, the cell activating agents of the present invention may comprise an anti-CD3 antibody and / or antigen-binding fragment thereof, for example, the light chain VL and heavy chain VH of Miltenyi Biotech's OKT3, or the light chain VL and heavy chain VH of BD's SP34. For example, the cell activating agents of the present invention may comprise a CD28 agonist. For example, the cell activating agents of the present invention may comprise an anti-CD28 antibody and / or antigen-binding fragment thereof, for example, the light chain VL and heavy chain VH of Merck's 15E8. For example, the cell activating agents of the present invention may comprise an anti-CD3 antibody and / or antigen-binding fragment thereof, for example, the light chain LCDR1-3 and heavy chain HCDR1-3 of Miltenyi Biotech's OKT3, or the light chain LCDR1-3 and heavy chain HCDR1-3 of BD's SP34. The anti-CD3 antibody and / or antigen-binding fragment thereof of the present invention may have CD3 binding ability. For example, the cell activating agents of the present invention may comprise a CD28 agonist. For example, 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. In the present invention, 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.

[0256] For example, 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. For example, 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. For example, 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.

[0257] For example, 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. For example, 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; for example, 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. For example, 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. For example, 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. For example, 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. For example, 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, and 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, and 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. For example, the diameter of the solid phase medium of the present invention can be about 500 nanometers to about 10 microns. For example, the diameter of the solid phase medium of the present invention can be measured by transmission electron microscopy. For example, the diameter of the solid phase medium of the present invention can be about 1 nanometer to about 500 nanometers. For example, the diameter of the solid phase medium of the present invention can be about 100 nanometers to about 500 nanometers. For example, the diameter of the solid phase medium of the present invention can be about 200 nanometers to about 500 nanometers. For example, the diameter of the solid phase medium of the present invention can be measured by transmission electron microscopy.

[0258] For example, the solid phase medium of the present invention may comprise a polymer. For example, the solid phase medium of the present invention may comprise dextran.

[0259] For example, the solid phase medium of the present invention contains at least about 25 μg of the cell activating agent of the present invention per mg.

[0260] For example, 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. For example, 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.

[0261] For example, when the diameter of the solid phase medium of the present invention is about 500 nanometers to about 10 micrometers, 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. For example, when the diameter of the solid phase medium of the present invention is about 500 nanometers to about 10 micrometers, the solid phase medium containing the cell activating agent of the present invention, such as a CD3 agonist and / or a CD28 agonist, 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.

[0262] For example, when the diameter of the solid phase medium of the present invention is about 100 nm to about 500 nm, 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. For example, when the diameter of the solid phase medium of the present invention is about 100 nm to about 500 nm, 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 For example, a solid phase medium comprising a CD28 agonist and a CD3 agonist of the present invention can be added to a cell culture medium of the cells of the present invention at a ratio of about 1:900 to about 1:2000, about 1:1000 to about 1:2000, about 1:1200 to about 1:2000, about 1:1400 to about 1:2000, about 1:1600 to about 1:2000, or about 1:1800 to about 1:2000 of the solid phase medium of the present invention to the cells of the present invention.

[0263] For example, 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.

[0264] For example, in a single stage of 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. For example, in the first stage of in vitro expansion 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. For example, in the second stage of in vitro expansion 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. For example, in the third stage of in vitro expansion 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.

[0265] For example, in a single stage of in vitro expansion 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. For example, in a single stage of in vitro expansion of the present invention, 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. For example, in a single stage of in vitro expansion of the present invention, 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. For example, in a single stage of in vitro expansion 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.

[0266] For example, in the first stage of 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 one or more cell growth factors of the present invention at substantially the same time. For example, in the second stage of in vitro expansion of the present invention, 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. For example, in the third stage of in vitro expansion of the present invention, 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.

[0267] For example, 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-γ, and functionally active fragments thereof. For example, the cell growth factor of the present invention can comprise IL-2 and / or functionally active fragments thereof. For example, 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. For example, 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.

[0268] For example, 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. For example, 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. For example, 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. , at least about 2700 IU / mL, at least about 2800 IU / mL, at least about 2900 IU / mL, at least about 3000 IU / mL, at least about 3100 IU / mL, at least about 3200 IU / mL, at least about 3300 IU / mL, at least about 3400 IU / mL, at least about 3500 IU / mL, at least about 4000 IU / mL, at least about 4500 IU / mL, at least about 5000 IU / mL, at least about 5500 IU / mL, at least about 6000 IU / mL, at least about 6500 IU / mL, at least about 7000 IU / mL, at least about 7500 IU / mL, at least about 8000 IU / mL, at least about 8500 IU / mL, or at least about 9000 IU / mL.

[0269] For example, 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. For example, the amount of IL-2 added can be reduced under the conditions of adding IL-7 and IL-15. For example, the concentration of IL-7 can be about 1 to 1000 ng / mL, preferably about 1-100 ng / mL. For example, the concentration of IL-15 can be about 1 to 1000 ng / mL, preferably about 1-100 ng / mL. For example, 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%. For example, 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. For example, 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).

[0270] For example, 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.

[0271] For example, in a single stage of in vitro expansion 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 and co-cultured with the feeder cells of the present invention. For example, 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.

[0272] For example, in the first stage in vitro expansion of the present invention, the TIL 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. For example, in the second stage in vitro expansion of the present invention, the TIL 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. For example, in the third stage in vitro expansion of the present invention, the TIL 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.

[0273] For example, in a single stage of in vitro expansion 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. For example, in the first stage in vitro expansion of the present invention, the TIL 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. For example, in the second stage in vitro expansion of the present invention, the TIL 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. For example, in the third stage in vitro expansion of the present invention, the TIL 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.

[0274] For example, in a single stage of in vitro expansion 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. For example, the certain period of time of the present invention can be at least about 1 hour. For example, 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. For example, the certain period of time of the present invention can be from about 2 hours to about 72 hours. For example, 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. For example, 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. For example, 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.

[0275] For example, the feeder cells of the present invention may comprise antigen-presenting cells. For example, 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. For example, the feeder cells of the present invention may be peripheral mononuclear cells. For example, the feeder cells of the present invention may be irradiated feeder cells. For example, 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. For example, the feeder cells of the present invention may be irradiated, for example, by gamma ray irradiation, or by X-ray irradiation.

[0276] For example, 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. For example, 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.

[0277] For example, 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. For example, 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 400:1.

[0278] In another 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 one or more cell growth factors; wherein, step (A) obtains a second TIL population; (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 BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family; wherein, step (B) obtains a third TIL population.

[0279] In another aspect, 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 through step (A); and (B) the second TIL population is 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 The expression and / or activity of members of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family are reduced and / or weakened, and the second TIL population is contacted with a T cell activator and / or a T cell growth factor, wherein a third TIL population is obtained through step (B); (C) the third TIL population is co-cultured with feeder cells, wherein a fourth TIL population is obtained through step (C).

[0280] In one embodiment, the first stage in vitro expansion of the present invention can be arbitrarily replaced with step (A) in the method of the above aspect. In one embodiment, the second stage in vitro expansion of the present invention can be arbitrarily replaced with step (B) in the method of the above aspect. In one embodiment, 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. In one embodiment, 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. In one embodiment, if necessary, 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. In one embodiment, if necessary, 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.

[0281] In another 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 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, BTB (Broad-Complex, Tramtrack and The expression and / or activity of members of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family are reduced, and the TILs are co-cultured with feeder cells; wherein a third TIL population is obtained through the step (B).

[0282] In another 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 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, BTB (Broad-Complex, Tramtrack and The expression and / or activity of a member of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced and / or weakened, and the TIL is co-cultured with feeder cells, and the 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 can include ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2, respectively; wherein, a third TIL population is obtained by the step (B).

[0283] On the other hand, the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL). 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 equipment. The serum-free culture medium of the cells can be supplemented with the CD28 antibody of the present invention, CD3 antibody comprising CD3 antibody and CD28 antibody magnetic beads (e.g., Dynabeads) and / or nanomatrix comprising CD3 antibody and CD28 antibody (e.g., transACT), IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL), and 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 The expression of members of the Bric-a-brac) protein family, the ZC3H12 protein family, or the RC3H family is reduced and / or the activity is weakened (wherein 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 TRAF-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 members may include ZBTB7B, the ZC3H12 protein family members may include ZC3H12D, and the RC3H family members may include RC3H2). For example, the TIL may be transduced with a ribonucleoprotein complex (RNP) comprising the gRNA of the present invention and the Cas protein, or an LNP comprising the gRNA and the Cas protein, or an LNP comprising a nucleic acid encoding the gRNA and the Cas protein, so that the TIL encodes a protein 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 After activating the TILs of the present invention for a period of time, irradiated PBMCs are added (TIL to PBMC ratio of approximately 1:40 to approximately 1:400) and cultured for approximately 3-14 days. Cells can be harvested from the culture medium using a cell processing system, washed, frozen, and tested. The final product can have a CD3 ratio greater than 80%, a cell viability greater than 50%, and cells greater than 80% can be memory effector cells and effector cells. Upon stimulation, they can secrete IFN-γ and / or exhibit an increased proportion of activated cells.

[0284] In one aspect, the present invention provides a cell, which can be cultured according to the culture method of the present invention. In one embodiment, 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. In one embodiment, 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.

[0285] In some embodiments, the cells expanded using the methods of the present invention can be administered to a patient as a pharmaceutical composition. In some embodiments, 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. In some embodiments, 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.

[0286] In some embodiments, any suitable dose of cells may be administered. In some embodiments, for example, when the tumor is a melanoma, about 1×109 to about 13.7×10 10 , preferably about 2.3×10 9 to about 13.7×10 10 In some embodiments, about 1×10 9 to about 12×10 10 In some embodiments, about 1.2×10 10 to about 4.3×10 10 In some embodiments, approximately 3×10 10 to about 12×10 10 In some embodiments, approximately 4×10 10 to about 10×10 10 In some embodiments, about 5×10 10 to about 8×10 10 In some embodiments, about 6×10 10 to about 8×10 10 In some embodiments, about 7×10 10 to about 8×10 10 In some embodiments, the therapeutically effective dose can 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 In some embodiments, the therapeutically effective dose can be about 1.2×10 10 to about 4.3×10 10 In some embodiments, the therapeutically effective dose can be about 3×10 10 to about 12×10 10 In some embodiments, the therapeutically effective dose can be about 4×10 10 to about 10×10 10 In some embodiments, the therapeutically effective dose can be about 5×10 10 to about 8×10 10 In some embodiments, the therapeutically effective dose can be about 6×10 10 to about 8×10 10 In some embodiments, the therapeutically effective dose can be about 7×10 10 to about 8×10 10 cells.

[0287] In some embodiments, 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 9 , about 6×10 9 , about 7×10 9 , about 8×10 9 , about 9×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11, about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , about 5×10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , or about 9×10 13 In some embodiments, the number of cells provided in the compositions of the present invention may 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 5×1013 , or about 5×10 13 to 9×10 13 .

[0288] In some embodiments, 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.

[0289] In some embodiments, 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%. .50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14%, about 13.75%, about 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75%, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%, about 9%, about 8.75%, about 8.50%, about 8.25%, about 8%, about 7.75%, about 7.5 0%, about 7.25%, about 7%, about 6.75%, about 6.50%, about 6.25%, about 6%, about 5.75%, about 5.50%, about 5.25%, about 5%, about 4.75%, about 4.50%, about 4.25%, about 4%, about 3.75%, about 3.50%, about 3.25%, about 3%, about 2.75%, about 2.50%, about 2.25%, about 2%, about 1.75%, about 1.50%, about 1.25%, 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 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 or 0.0002%, or about 0.0001% w / w, w / v or v / v.

[0290] In some embodiments, 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 21%, from about 0.2% to about 20%, from about 0.3% to about 19%, from about 0.4% to about 18%, from about 0.5% to about 17%, from about 0.6% to about 16%, from about 0.7% to about 15%, from about 0.8% to about 14%, from about 0.9% to about 12%, or from about 1% to about 10% w / w, w / v or v / v.

[0291] In some embodiments, 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.

[0292] In some embodiments, 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 0.0006 g, about 0.0005 g, about 0.0004 g, about 0.0003 g, about 0.0002 g, or about 0.0001 g.

[0293] In some embodiments, 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, about 0.0190 g, about 0.0210 g, about 0.0225 g, about 0.0237 g, about 0.0240 g, about 0.0257 g, about 0.0260 g, about 0.0275 g, about 0.0280 g, about 0.0 about 0.003g, about 0.0035g, about 0.004g, about 0.0045g, about 0.005g, about 0.0055g, about 0.006g, about 0.0065g, about 0.007g, about 0.0075g, about 0.008g, about 0.0085g, about 0.009g, about 0.0095g, about 0.01g, about 0.015g, about 0.02g, about 0.025g, about 0.03g, about 0.035g, about 0.04g, about 0.045g, about 0.05g, about 0.055g, about 0.06g, about 0.065g, about 0.07g, about 0.075g, about 0.08g, about 0.085g, about 0.09g, about 0.095g, about 0.1g, about 0.15g, about 0.2g, about 0.25g, about 0.3g, about 0.35g, about 0.4g, about 0.45g, about 0.5g, About 0.55g, about 0.6g, about 0.65g, about 0.7g, about 0.75g, about 0.8g, about 0.85g, about 0.9g, about 0.95g, about 1g, about 1.5g, about 2g, about 2.5g, about 3g, about 3.5g, about 4g, about 4.5g, about 5g, about 5.5g, about 6g, about 6.5g, about 7g, about 7.5g, about 8g, about 8.5g, about 9g, about 9.5g, or about 10g.

[0294] In some embodiments, 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.

[0295] In one aspect, the present invention provides a pharmaceutical composition, which in some embodiments may comprise the cells of the present invention and a pharmaceutically acceptable carrier.

[0296] In one aspect, 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. In another aspect, the present invention provides a kit that may include the cells of the present invention and / or the pharmaceutical composition of the present invention.

[0297] In one aspect, 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. In some embodiments, 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%.

[0298] In one aspect, 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. For example, the disease and / or symptom of the present invention may comprise a tumor. For example, the disease and / or symptom of the present invention may comprise an immune-related disease, such as an autoimmune disease. In some embodiments, the tumor of the present invention is selected from a solid tumor. In some embodiments, 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.

[0299] In one aspect, 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. For example, the disease and / or symptom of the present invention may comprise a tumor. For example, the disease and / or symptom of the present invention may comprise an immune-related disease, such as an autoimmune disease. In some embodiments, the tumor of the present invention is selected from a solid tumor. In some embodiments, 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.

[0300] In one aspect, 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. For example, the disease and / or symptom of the present invention may include a tumor. For example, the disease and / or symptom of the present invention may include an immune-related disease, such as an autoimmune disease. In some embodiments, the tumor of the present invention is selected from a solid tumor. In some embodiments, 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.

[0301] Without intending to be bound by any theory, the following examples are merely intended to illustrate the methods and uses of the present invention and are not intended to limit the scope of the present invention.

[0302] Example

[0303] Example 1

[0304] (1) TIL cell culture

[0305] 1.1 Tumor tissue receipt and processing

[0306] 1.1.1 Organization reception

[0307] Receive tumor tissue and blood samples from donors, verify and record sample information, and print corresponding sample labels.

[0308] 1.1.2 Tissue processing and culture

[0309] Use 75% alcohol to disinfect the sample tube and blood collection tube 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 thawed complete culture medium. 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 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 into a 10 cm culture dish, wash the tissue and change the culture dish. Use ophthalmic scissors and forceps to perform initial shearing, removing adipose and necrotic tissue. Each 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. Using a pipette, 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.

[0310] 1.2 Step (A) In vitro expansion and harvesting

[0311] 1.2.1 Step (A) In vitro amplification

[0312] Depending on the cell growth status, the medium should be replenished or half-replaced every 3-7 days to ensure cell nutrition. Complete culture medium can be arbitrarily selected from X-vivo 15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 (double heron and / or tetracycline) at a concentration of 300-9000 IU / mL (e.g. 1000-9000 IU / mL, such as 6000 IU / mL) can be added. 3-14 days in 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 to 5×10 8 During this time, the harvesting step (A) is entered.

[0313] 1.2.2 Results of Step (A)

[0314] 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 the TILs (second TIL population) expanded in vitro in step (A), and take samples for counting and retain about 5×10 5 to 2×10 8 cells into the subsequent in vitro expansion step; about 5×10 5 The remaining cells can be added to the cryopreservation medium and cryopreserved as cryopreserved preREP TIL in vitro cells.

[0315] 1.3 Step (B) TIL activation

[0316] Continue to culture the TILs (second TIL population) expanded in vitro in step (A), or recover the frozen preREP TIL cells in vitro and perform TIL activation in step (B).

[0317] Complete culture medium can be selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi Biotech, etc. Essential amino acids and antibiotics can be added to adjust the cell density to 5×10 5 to 2×10 6 Cells are suspended in a 24-well culture plate at a concentration of 1 mL / well and IL-2 is added at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, for example, 6000 IU / mL). T cell activators, such as CD3 agonists and / or CD28 agonists, can be added to the culture medium of each TIL population, 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 μm diameter, Thermo Fisher) at a ratio of approximately 1:2-2:1 beads to TILs, and / or transACT (Miltenyi, approximately 100 to 500 nm diameter, TILs) at a ratio of approximately 1:100-1:2000. Culture is continued for approximately 0-4 days to obtain a third TIL population.

[0318] 1.4 Step (C) TIL cell gene editing

[0319] Synthesize the sgRNA targeting each target selected from the present invention, thaw and add nuclease-free water to a concentration of about 100 μM. After incubating about 2 μL of gRNA (50 μM) at 95°C for 2 minutes to anneal, add it to P3 buffer, and add 0.3-1 μL of Cas9 (such as Kaixia, Kerui or Acro, 10 mg / mL) and incubate at 25°C for 10 minutes to form a ribonucleoprotein complex (RNP). In P3 buffer (Lonza), the above RNP is mixed with about 1×10 cells of the third TIL population using a Lonza electroporator. 6 The cells are electroporated. For example, the electroporation procedure can be human T cell stim (EO115). The electroporated cells are cultured for about 0-4 days after gene editing to obtain a fourth TIL population.

[0320] 1.5 Step (D) TIL cell culture after gene editing

[0321] Feeder cells (irradiated healthy donor PBMC T cells) are added to the fourth TIL cell population for culture. The time for contacting TIL with feeder cells needs to be several times after the TIL is contacted with IL-2 and T cell activator in step (B). n Later (for example, T n The time period can be from 0 hours to 12 days, for example, 24 hours or 48 hours. First, resuscitate the mixed feeder cells from 1-5 donors; mix the activated TIL cells and feeder cells at a ratio of approximately 1:200, transfer them to a G-Rex100 culture flask or breathable bag, and supplement with complete culture medium. Samples are taken and counted every 1-3 days, and the medium is replenished or half-filled depending on the cell status until the total number of cells is greater than 1×10 9 Alternatively, the in vitro expansion culture of step (D) is carried out for about 5 days to about 14 days, and the in vitro expansion culture of step (D) is terminated.

[0322] 1.6 Harvesting of Tumor-Infiltrating Lymphocytes

[0323] Take the cells amplified in step (D), centrifuge and discard the culture supernatant, and wash three times with PBS or saline or compound electrolyte solution to obtain TILs amplified in step (D) (fifth TIL population). Samples are counted during the third wash. According to the counting results, after the final centrifugation, discard the supernatant and take 3×10 6 The cells were sent for quality control testing; all the remaining cells were added to the freezing solution and the cell density was adjusted to 1-3×10 8 cells / mL for cryopreservation.

[0324] (2) TCR-T cell culture

[0325] T cell activation: T cells frozen in liquid nitrogen were revived and cultured, resuspended by centrifugation to 5E5 / ml in T cell culture medium RPMI 1640 (Gibco) + 10% FBS (Bovogen), and T cell TransAct (Miltenyi) was added at a dilution of 1:100. Recombinant human IL-2 was also added at a concentration of 30 IU / ml and cultured for approximately 72 hours.

[0326] TCR transduction: One day before transduction, use recombinant human fibrin fragment (Retronectin, Takara) with a final concentration of 15μg / mL to coat a 24-well suspension culture plate, 250μL per well of the 24-well plate. Protect from light and store at 4°C overnight. Take out the coated 24-well plate, discard the coating solution, add 500μL of 2% BSA blocking solution and block at room temperature for 30 minutes. Discard the blocking solution, wash the plate twice with 500μL / well of 2.5% HEPES washing solution, and discard the washing solution. The experimental group was transduced with a retrovirus carrying a specific TCR nucleic acid fragment of the NY-ESO-1 antigen peptide. Add 0.1-1mL of retrovirus solution to each well, centrifuge at 32°C, 2000g, and centrifuge for 2 hours. Discard the supernatant of the 24-well plate, add revived and activated T cells to each well of the 24-well plate, with a volume of 500-1000μL and a cell concentration of approximately 5×10 5 / mL. 30-32℃, 1000g, centrifuge for 10 minutes. After centrifugation, place the culture plate in a 37℃, 5% CO2 incubator to culture and obtain transduced cells. Culture for about 0-4 days after transduction to obtain TCR-T cell populations. After transduction, add T cell culture medium according to the cell count density and viability every 1-3 days, add recombinant human IL-2 at a concentration of 30-100 IU / ml, and adjust the initial culture cell density to 0.5-2×10 6 / ml, and continue culturing.

[0327] 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 μM. After incubating about 2 μL of gRNA (50 μM) at 95°C for 2 minutes to anneal, add it to P3 buffer, and add 0.3-1 μL of Cas9 (such as Kaixia, Kerui, Acro, 10 mg / mL), and incubate at 25°C for 10 minutes to form a ribonucleoprotein complex (RNP). In P3 buffer (Lonza), the above RNP is mixed with about 1×10 6 TCR-T cells are electroporated. For example, 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 the target gene-edited TCR-T cells of the present invention.

[0328] (III) Knockout efficiency detection

[0329] Reagents and materials used to detect the knockout efficiency of cells include: DNA extraction solution (QuickExtract DNA extraction solution, Lucigen, QE09050), RNase / DNase free water (Tiangen), EDTA (Shenggong, 0.5 M), and Recombinant DNase I (RNase-free, TAKARA).

[0330] Extract genomic DNA: About 2-7 days after T cell knockout (refer to the gene editing method in step 1.4 of Example 1 (I) or the TCR-T gene editing method in Example 1 (II)), take about 1×10 5 to about 2×10 5 Cells were washed once with PBS, and then the gene-edited cells were resuspended in 44 μL PBS. 6 μL of the prepared nuclease mixture (containing 1 μL DNase I and 5 μL 10× DNase I Buffer) was added and incubated at 37°C for 5 minutes. 2.5 μL of 0.5M EDTA was added to the sample and incubated at 80°C for 10 minutes. After centrifugation and discarding the supernatant, 50 μL of DNA extraction solution was added to the cell pellet. After a brief centrifugation, the following program was run: 75°C-10 minutes; 95°C-5 minutes; 4°C-maintain. The sample can be analyzed using a spectrophotometer (NanoDrop TM ) to detect the concentration of DNA samples.

[0331] Sequencing: 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:

[0332] And amplify according to the following PCR program:

[0333] The PCR products were analyzed by Sanger sequencing.

[0334] Analyzing Crispr Cas9 knockout efficiency

[0335] Crispr Cas9 knockout efficiency was analyzed using the Tracking of Indels by DEcomposition (Tide) method based on Sager sequencing data. For specific methods, see (Brinkman et al, Nucl. Acids Res. (2014) or shinyapps.datacurators.nl / tide / ). Knockout efficiency analysis was performed by inputting the corresponding sgRNA sequence of the present invention, the pre-knockout control sequence, and the test sequence after Crispr Cas9 knockout, with a P-value threshold of 0.001.

[0336] (IV) Amplification detection

[0337] Experimental preparation

[0338] 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.

[0339] 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 μL / well. In the non-stimulation medium group, no cell activating substances were added to the culture medium; in the CD3 antibody stimulation group, 30 ng / mL of CD3 antibody (OKT3) was added for stimulation; in the 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); in the medium group, only an equal volume of cell culture medium was added. Fluorescence was analyzed using the CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega) upon plating and three days later using the CTG kit. T cell expansion efficiency was measured by dividing the fluorescence on day 3 by the fluorescence at plating.

[0340] (V) Cell killing ability detection

[0341] Starting from the sixth day after gene editing, tumor target cells were plated in 96-well flat-bottom plates. The next day, TIL cells from each group were co-cultured with target cells at different effector-to-target ratios (T cells: target cells, E:T). Alternatively, T cells transduced with the TCR obtained in Example 1 (II) were used.

[0342] 100 μL 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.

[0343] According to the instructions for the apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL / well of the apoptosis detection reagent was added, and 25 μL / well of culture medium diluted with Caspase 3 / 7 Green Dye was added. The activity of Caspase 3 / 7 was recorded using an Incucyte recorder (Sartorius) to analyze the killing ability of TIL cells against target cells. Recording was performed every 3 hours for a total of approximately 5 days.

[0344] (6) Cell flow cytometry

[0345] The expression of 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.

[0346] 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.

[0347] Cell surface molecule detection: 1×10 5 to 5×10 5 Add a cell sample to a flow tube or a V-bottom 96-well plate. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, add 1mL / tube to the flow tube and 200μL / well to the 96-well plate, and discard the supernatant. Add the prepared antibody working solution for cell surface staining. The antibody (BD or Biolegend) concentration is 1:100 to 1:200, containing activity detection dye at 1:10000. Stain 100μL / tube of the flow tube and 50μL / well of the 96-well plate, and incubate at 2-8℃ in the dark for 30 minutes. After surface staining, wash the cells once with PBS (200μL / time for 96-well plate and 1mL / time for flow tube), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant after centrifugation. Resuspend the cells in 100-500μL PBS and perform flow cytometry detection.

[0348] For intracellular molecule detection: Prepare a mixed antibody working solution for cell surface staining of CD3 / CD4 / CD8 at a 1:100 antibody concentration and a 1:10,000 dye concentration for cell viability detection. Add 50 μL / well of a 96-well plate and 100 μL / tube to a flow cytometry tube for staining. Incubate at 2-8°C in the dark for 30 minutes. Wash cells once with PBS (200 μL / 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. Add 100 μL of Fixation / Permeabilization buffer (BD, Fixation / Permeabilization) to each well and incubate at 2-8°C in the dark for 20-40 minutes. After fixation and permeabilization, wash twice with 1× Perm / Wash Buffer (200 μL / 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 μL / well for 96-well plates, 100 μL / tube for flow cytometry staining). Incubate at 2-8°C in the dark for 30 minutes. After intracellular molecule staining, wash 1-2 times with 1× Perm / Wash Buffer (200 μL / well for 96-well plates, 1 mL / well for flow cytometry). Centrifuge at 600 g for 3 minutes and discard the supernatant. Resuspend cells in 100-500 μL PBS for flow cytometry analysis.

[0349] (VII) Flow cytometry detection of cytokine expression

[0350] The 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.

[0351] Prepare the culture medium for intracellular factor expression assays: Take TIL cell culture medium and add the following volume ratios: Golgistop 0.7:1000, Golgiplug 1:1000, and CD107a antibody 1:500, i.e., 2 μL / mL. Do not add interleukins.

[0352] Detection steps

[0353] After centrifugation, T cells from each test group were resuspended in the culture medium required for the above-mentioned intracellular factor expression assay, and the cell density was adjusted to 1×10 6Cells were plated at 200 μL / well in a 96-well plate at 1:1000 (v / v) for 1:1000 cells / mL. For CD3 antibody stimulation, 30 ng / mL of CD3 antibody (OKT3) was added to the CD3 antibody stimulation group. For TransACT stimulation, transACT (approximately 100 to 500 nm in diameter, Miltenyi) was added to the transACT working solution. For the Medium group, an equal volume of cell culture medium was added. Incubate overnight at 37°C.

[0354] Sources of main reagents and materials for cytokine flow cytometry assay:

[0355] 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.

[0356] After incubation, wash once with 200 μL / well PBS, centrifuge at 600 g for 3 minutes, and discard the supernatant. 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 assay dye concentration of 1:10,000. 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube were added for staining and incubated at 2-8°C in the dark for 30 minutes. Wash cells once with PBS (200 μL / 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. Add 100 μL of Fixation / Permeabilization buffer (BD, Fixation / Permeabilization) to each well and incubate at 2-8°C in the dark for 20-40 minutes. After fixation and permeabilization, wash twice with 1× Perm / Wash Buffer (200 μL / 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 cytokine detection antibodies (e.g., GZMB, TNF-α, IFN-γ) in 1× Perm / Wash Buffer and resuspend TIL cells (50 μL / well for 96-well plates, 100 μL / tube for flow cytometry staining). Incubate at 2-8°C in the dark for 30 minutes. After cytokine staining, wash 1-2 times with 1× Perm / Wash Buffer (200 μL / well for 96-well plates, 1 mL / well for flow cytometry). Centrifuge at 600 g for 3 minutes and discard the supernatant. Resuspend cells in 100-500 μL PBS for flow cytometry analysis.

[0357] (8) Cell apoptosis detection

[0358] 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.

[0359] The TILs in the gene knockout group or the control group (NT, no treatment) were used to detect the T cell apoptosis level using a cell apoptosis detection kit (BD 559763 Annexin V PE Apoptosis kit).

[0360] (IX) PDO model cultivation

[0361] Tumor tissue was obtained from surgery, transported and stored at 2-8°C after ex vivo, and mechanically dissociated within 24 hours. The tissue was minced into pieces of approximately 0.5 mm. 3 The organoids were then digested at 37°C using a dedicated tissue digestion solution (bioGenous) and terminated with 10% FBS. The tissue suspension was filtered through a 100μm 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 fully solidified, complete culture medium was carefully added for incubation. When the organoids reached sufficient size and density, they were passaged or frozen to obtain a PDO (patient-derived organoid) model.

[0362] Use the tip of a pipette to scrape the Matrigel and organoid mixture and transfer it to a centrifuge tube containing basal medium. Pipet to separate the organoids and Matrigel. After centrifugation, add tissue digestion buffer for passage, mix well, and place in a 37°C incubator for digestion. Add basal medium to dilute the digestion buffer and wash by centrifugation. Take a small amount of the cell suspension and add 8 times the volume of digestion buffer to digest to single cells. Stop the reaction with FBS and count. Based on the count results, aspirate the required volume of organoids and centrifuge and resuspend them in T cell culture medium. Count the cells to be tested (e.g., TIL cells or TCR-T cells) and resuspend the cells to be tested to the desired density at different effector-target ratios. Add 100 μl of PDO target cells and test cells to a flat-bottom 96-well plate, and set up three replicates for each group for PDO co-culture testing. Set up several different effector-target ratios. At the same time, set up a group containing only PDO target cells and a control group (NT) for co-culturing unedited test cells with PDO target cells.

[0363] Detection of lethality in PDO model

[0364] 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.

[0365] (10) Multiple rounds of cell killing ability detection

[0366] 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 μL / well) was taken for CBA detection; after 3 days of killing, a new A375-GFP was evenly plated in a multi-well plate. After culturing at 37°C for 4 hours, 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.

[0367] (11) CBA method for detecting cytokine release

[0368] 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.

[0369] (12) In vivo efficacy testing of gene-edited cells

[0370] Tumor tissue samples were obtained during surgery and transferred to the laboratory under sterile conditions. Necrotic tumor tissue was removed in a sterile clean bench and the tumor tissue was cut into 1-2 mm pieces. 3 The cut tumor tissue was dripped with a little Matrigel and inoculated subcutaneously into NOG mice (Vitamin Liva, strain code 408) in an SPF animal room. Each mouse was inoculated with 4-5 pieces of tissue. This batch of tumor tissue is F0 generation. When the F0 generation tumor tissue grows to 800mm under the mouse skin, 3 , surgically remove the F0 tumor tissue and divide the tumor tissue into 1-2mm 3 Part of the tissue was frozen for future use, and part was subcutaneously inoculated into the second batch of NOG mice. The tumor tissue was further passaged and expanded in the mice. This batch of tumor tissue was the F1 generation. This process was repeated until the tumor tissue reached the F3 generation in the mice. The size of the tumor tissue was measured regularly until the tumor volume grew to about 100 mm. 3 Afterwards, the mice were randomly divided into groups: the NT group of mice received intravenous injection of non-gene-edited cells, and the experimental group of mice received intravenous injection of the gene-edited cells of the present invention.

[0371] 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. Wherein, 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. When the region targeting each target is selected from 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. Preferably, the sgRNA numbering information in the embodiment is as follows:

[0372] Example 2

[0373] According to the experimental method in the embodiment of the present invention, the knockout effect of sgRNA targeting ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2 was detected.

[0374] Among them, A means the knockout efficiency is greater than or equal to 70%, and N / A means not detected.

[0375] Among them, donors 711, 105, and 505 are lung cancer donors, donors 905, 306, and 312 are cervical cancer donors, and donors 603 and 804 are melanoma donors, and are used to isolate and provide TILs.

[0376] (B) Proliferation capacity

[0377] Figure 2A shows the expansion fold of TILs edited with the gene of the present invention in the non-stimulation culture medium group.

[0378] FIG2B shows the expansion fold of TILs edited with the gene of the present invention in the TransACT stimulation group.

[0379] Significant differences are relative to the unedited NT group, with * indicating P < 0.05, ** indicating P < 0.01, *** indicating P < 0.001, and **** indicating P < 0.0001. The results demonstrate that target gene-edited T cells of the present invention exhibit significantly enhanced expansion capacity compared to the unedited control group (NT).

[0380] (C) Lethality

[0381] Figures 3A-3H show the target cell killing ability of the gene-edited TIL cells of the present invention.

[0382] Figure 3I shows the killing ability of the gene-edited TIL cells of the present invention on the autologous tumor cell PDO model.

[0383] 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.

[0384] Significant differences are relative to the unedited NT group, with * indicating P < 0.05, ** indicating P < 0.01, *** indicating P < 0.001, and **** indicating P < 0.0001. The results demonstrate that target gene-edited T cells of the present invention exhibit significantly enhanced target cell cytotoxicity and / or continuous cytotoxicity compared to the control group (NT).

[0385] (D) Cell apoptosis detection

[0386] FIG4 shows the apoptosis ratio of gene-edited TIL cells of the present invention.

[0387] The results showed that compared with the control group (NT), the target gene-edited T cells of the present invention can have a significantly reduced apoptosis ratio.

[0388] (E) Flow cytometry

[0389] Figure 5A shows that the gene-edited TIL cells of the present invention have a higher proportion of central memory T cells.

[0390] Figure 5B shows that the gene-edited TIL cells of the present invention have a higher proportion of stem cells.

[0391] Figures 5C-5D show that the gene-edited TIL cells of the present invention have a lower proportion of exhausted T cells.

[0392] For example, central memory T cells can be CD45RO positive CD62L positive cells. For example, stem cells can be CD39 negative CD69 negative cells. For example, exhausted T cells can be PD-1 positive, LAG-3 positive, TIM-3 positive, CD38 positive and / or CD101 positive cells. The results show that, relative to the non-gene-edited control group (NT), the gene-edited TIL cells of the present invention can have more favorable cell phenotypic characteristics.

[0393] (F) Cytokine expression and release

[0394] 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.

[0395] Figures 6A-6G show the cytokine expression of the gene-edited TIL cells of the unstimulated group.

[0396] Figures 6H-6O show the cytokine expression of the gene-edited TIL cells of the present invention in the TransACT stimulation group.

[0397] 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.

[0398] The results showed that compared with the control group (NT), the target gene-edited T cells of the present invention can have significantly improved cytokine expression and / or release capacity.

[0399] Example 3

[0400] According to the test method in the embodiment of the present invention, 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.

[0401] Among them, 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%, and N / A indicates not detected.

[0402] Among them, donors 505 and 105 are lung cancer donors, donors 804 and 812 are melanoma donors, and donor 306 is a cervical cancer donor, and they are used to isolate and provide TILs.

[0403] (B) Proliferation capacity

[0404] Figure 7A shows the expansion fold of TILs edited with the combination gene of the present invention in the non-stimulation culture medium group.

[0405] FIG7B shows the expansion fold of TILs edited with the combination gene of the present invention in the TransACT stimulation group.

[0406] Significant differences are relative to the unedited NT group, with * indicating P < 0.05, ** indicating P < 0.01, *** indicating P < 0.001, and **** indicating P < 0.0001. The results demonstrate that target gene-edited T cells of the present invention exhibit significantly enhanced expansion capacity compared to the unedited control group (NT).

[0407] (C) Lethality

[0408] Figures 8A-8D show the target cell killing ability of TIL cells edited by the combination of genes of the present invention.

[0409] 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.

[0410] Significant differences are relative to the unedited NT group, with * indicating P < 0.05, ** indicating P < 0.01, *** indicating P < 0.001, and **** indicating P < 0.0001. The results demonstrate that T cells edited with the target combination gene of the present invention exhibit significantly enhanced target cell cytotoxicity and / or continuous cytotoxicity compared to the control group (NT).

[0411] (D) Flow cytometry

[0412] Figures 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.

[0413] For example, central memory T cells can be CD45RO positive CD62L positive cells. The results show that, relative to the non-gene-edited control group (NT), the combined gene-edited TIL cells of the present invention can have more favorable cell phenotypic characteristics.

[0414] (E) Cytokine expression and release

[0415] 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.

[0416] Figures 10A-10C show the cytokine expression of TIL cells edited with the combination gene of the present invention in the unstimulated group.

[0417] Figures 10D-10F show the cytokine expression of TIL cells edited with the combination gene of the present invention in the TransACT stimulation group.

[0418] 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.

[0419] The results showed that compared with the control group (NT), T cells edited by the target gene combination of the present invention can have significantly improved cytokine expression and / or release capacity.

[0420] The results showed that TIL cells edited by the target combination gene of the present invention can have more significant anti-apoptosis ability.

[0421] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

[0422] Table 1A Genomic coordinates of preferred targeting subregions of human ADCY7

[0423] Table 1B Genomic coordinates of human ARIH2 preferred targeting subregions

[0424] Table 1C Genomic coordinates of human CPT2 preferred targeting subregions

[0425] Table 1D Genomic coordinates of human LNPEP preferred targeting subregions

[0426] Table 1E Genomic coordinates of human NOSIP preferred targeting subregions

[0427] Table 1F Genomic coordinates of preferred targeting subregions of human NPRL3

[0428] Table 1G Genomic coordinates of human TANK preferred targeting subregions

[0429] Table 1H Genomic coordinates of human TRAF3 preferred targeting subregions

[0430] Table II Genomic coordinates of preferred targeting subregions of human TSC1

[0431] Table 1J Genomic coordinates of human ZBTB7B preferred targeting subregions

[0432] Table 1K Genomic coordinates of human ZC3H12D preferred targeting subregions

[0433] Table 1L Genomic coordinates of preferred targeting subregions of human RC3H2

[0434] Table 2A Genomic coordinates of human ADCY7

[0435] Table 2B Human ARIH2 genomic coordinates

[0436] Table 2C Genomic coordinates of human CPT2

[0437] Table 2D Human LNPEP genomic coordinates

[0438] Table 2E NOSIP genome coordinates of the inventors

[0439] Table 2F Genomic coordinates of human NPRL3

[0440] Table 2G Genomic coordinates of the inventor's TANK

[0441] Table 2H Human TRAF3 genomic coordinates

[0442] Table 2I Genomic coordinates of human TSC1

[0443] Table 2J Genomic coordinates of human ZBTB7B

[0444] Table 2K Human ZC3H12D genomic coordinates

[0445] Table 2L Human RC3H2 genome coordinates

Claims

1. A method for culturing cells, the method comprising: reducing the expression and / or weakening the activity of a family 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family of the cells and / or a functional active fragment thereof.

2. The method according to claim 1, wherein the cells comprise immune cells.

3. The method according to claim 2, wherein the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

4. The method according to any one of claims 2-3, wherein the immune cells comprise monocytes, macrophages and / or dendritic cells.

5. The method according to any one of claims 2-4, wherein the immune cells are derived from immune cells differentiated from stem cells.

6. The method according to claim 5, wherein the stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells and / or peripheral blood stem cells.

7. The method according to any one of claims 2-6, wherein the immune cells comprise B cells, T cells, natural killer cells and / or natural killer-like T cells (NKT).

8. The method according to any one of claims 2-7, wherein the immune cells comprise αβ T cells and / or γδ T cells.

9. The method according to any one of claims 2-8, wherein the immune cells comprise tumor infiltrating lymphocytes (TIL).

10. The method according to claim 9, wherein the TIL is TIL derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis foci, fragments of adjacent cancerous tissue, pleural effusion and / or peritoneal effusion and / or TIL derived from TIL after cryopreservation and resuscitation.

11. The method according to claim 10, wherein the volume of the fragments is about 1 cubic millimeter to about 27 cubic millimeters.

12. The method according to any one of claims 2-11, wherein the immune cells comprise an engineered immune receptor displayed on the cell surface.

13. The method according to claim 12, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell.

14. The method according to any one of claims 2-13, wherein the immune cells comprise a chimeric antigen receptor and / or a T cell receptor.

15. The method according to any one of claims 1-14, wherein reducing the expression and / or attenuating the activity of a family member and / or a functionally active fragment 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family in the cell comprises an effect selected from the following group: inhibiting the function of adenylate cyclization, inhibiting the function of ubiquitination, inhibiting the function of acetylation transfer, inhibiting the function of polypeptide degradation, inhibiting the function of regulating nitric oxide production, inhibiting the function of negatively regulating the TOR signaling pathway, inhibiting the function of regulating NF-kappa-B, inhibiting the signal transduction function for TNFR, inhibiting the function of negatively regulating the mTORC1 signaling pathway, inhibiting the function of transcriptional regulation, inhibiting the function of endonuclease activity, or inhibiting the function of ubiquitin ligase.

16. The method according to any one of claims 1-15, wherein the cells obtained by reducing the expression and / or attenuating the activity of a family member and / or a functionally active fragment 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family show improved cell characteristics compared to cells in which the expression and / or activity of a family member and / or a functionally active fragment 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family is not altered.

17. The method according to claim 16, wherein the improved cell characteristics comprise one or more selected from the following group: improved cell proliferation ability, increased proportion of live cells, improved proportion of cell subsets, increased cytokine secretion ability, increased in vitro tumor cell killing ability, and increased in vivo tumor killing ability.

18. The method according to claim 17, wherein the improved proportion of cell subsets comprises one or more selected from the group consisting of: an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of apoptotic cells, and an increased proportion of stem cell-like cells.

19. The method according to any one of claims 1-18, wherein the family members of 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, and RC3H family respectively comprise a cyclase domain, a RING-type zinc finger domain, an acetyltransferase domain, an aminopeptidase domain, a nitric oxide synthase binding domain, a GTPase domain, a TRAF binding domain, a receptor cytoplasmic binding domain, a helix domain, a BTB domain, a C3H1-type zinc finger domain, and a ROQ domain.

20. The method according to any one of claims 1-19, wherein the family members of 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, and RC3H family respectively comprise ADCY7, ARIH2, CPT2, LNPEP, NOSIP, NPRL3, TANK, TRAF3, TSC1, ZBTB7B, ZC3H12D, and RC3H2.

21. The method according to any one of claims 1-20, wherein reducing the expression and / or weakening the activity of the family members of 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family in the cells comprises introducing a gene regulation system into the cells.

22. The method according to claim 21, wherein the gene regulation system disrupts family members of 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family at the DNA level; and optionally, the cell is selected from those with reduced expression and / or weakened activity of BRD4, FAS, TNFAIP3, ZC3H12A, SOCS1, CBLB, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6, TNIP1, BCL2L11, and / or PTPN2.

23. The method according to any one of claims 21-22, wherein the gene regulation system comprises a guide nucleic acid molecule and an enzyme protein.

24. The method according to claim 23, wherein reducing the expression and / or weakening the activity of family members of 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family comprises: introducing into the cell a complex comprising the guide nucleic acid molecule and the enzyme protein or a complex comprising the guide nucleic acid molecule and a nucleic acid encoding the enzyme protein.

25. The method according to any one of claims 23-24, wherein the enzyme protein comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof, preferably selected from Cas 9 and Cas 12.

26. The method according to any one of claims 23-25, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA).

27. The method according to any one of claims 23-26, wherein the guide nucleic acid molecule binds to a sequence of a family 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-associated 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.

28. The method according to any one of claims 23-27, wherein the guide nucleic acid molecule binds to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of: AGG, TGG, CGG, and GGG, or binds to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of a protospacer adjacent motif (PAM) selected from the group consisting of: NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, and NTN, where N is A, T, C, or G, Y is T or C, V is A, C, or G, R is A or G, or binds to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of a protospacer adjacent motif (PAM) selected from the group consisting of: TTTN, ATTN, GTTN, CTTN, TTC, TTG, TTA, TTT, TAN, TGN, and TCN, where N is A, T, C, or G.

29. The method according to any one of claims 23-28, wherein the guide nucleic acid molecule binds to a region defined by genomic coordinates shown in Tables 2A-2L or a fragment thereof.

30. The method according to any one of claims 23-29, wherein the guide nucleic acid molecule binds to a region or a fragment thereof selected from the group consisting of: SEQ ID NO: 4146-8290, SEQ ID NO: 10494-12696, SEQ ID NO: 13414-14130, SEQ ID NO: 15950-17768, SEQ ID NO: 19561-21352, SEQ ID NO: 22857-24360, SEQ ID NO: 25134-25906, SEQ ID NO: 29216-32524, SEQ ID NO: 34537-36548, SEQ ID NO: 39784-43018, SEQ ID NO: 44428-45836, and SEQ ID NO: 4725.

31. The method according to any one of claims 23-30, wherein the guiding nucleic acid molecule comprises a sequence as shown in SEQ ID NO: 1-4145, SEQ ID NO: 8291-10493, SEQ ID NO: 12697-13413, SEQ ID NO: 14131-15949, SEQ ID NO: 17769-19560, SEQ ID NO: 21353-22856, SEQ ID NO: 24361-25133, SEQ ID NO: 25907-29215, SEQ ID NO: 32525-34536, SEQ ID NO: 36549-39783, SEQ ID NO: 43019-44427, and SEQ ID NO: 45837-47258.

32. The method according to any one of claims 1-31, wherein the proportion of cells expressing the target gene and / or the expression level of the target gene in a single cell is reduced in cells obtained by reducing the expression and / or weakening the activity of family members of 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, compared to cells in which the expression and / or activity of family members of these families is not altered.

33. The method according to any one of claims 1-32, wherein in a cell obtained by reducing the expression and / or weakening the activity of a family member of 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-associated factor family, TSC protein family, BTB (Broad-Complex, Tramtrack and Bric-a-brac) protein family, ZC3H12 protein family, or RC3H family, the proportion of cells expressing a gene of interest is about 95% or less.

34. A cell obtained by the method according to any one of claims 1-33.

35. A pharmaceutical composition comprising the cell according to claim 34 and optionally a pharmaceutically acceptable carrier.

36. A method of affecting cell growth, comprising administering the cell according to claim 34 and / or the pharmaceutical composition according to claim 35.

37. Use of the cell according to claim 34 and / or the pharmaceutical composition according to claim 35 in the preparation of a drug for preventing and / or treating a disease and / or a symptom.

38. A drug for preventing and / or treating a disease and / or a symptom, comprising the cell according to claim 34 and / or the pharmaceutical composition according to claim 35 as an active ingredient.

39. A method of preventing and / or treating a disease and / or a symptom, comprising administering to a subject in need the cell according to claim 34 and / or the pharmaceutical composition according to claim 35.

40. The cell according to claim 34 and / or the pharmaceutical composition according to claim 35, for use in preventing and / or treating a disease and / or a symptom.

41. The use according to claim 37, the drug according to claim 38, the method according to claim 39, and / or the cell and / or pharmaceutical composition for the use according to claim 40, wherein the disease and / or symptom comprises a tumor.

42. The use according to claim 37, the drug according to claim 38, the method according to claim 39, and / or the cell and / or pharmaceutical composition for the use according to claim 40, wherein the disease and / or symptom comprises a solid tumor.

43. The use according to claim 37, the drug according to claim 38, The method according to claim 39, and / or the cell and / or pharmaceutical composition for the use according to claim 40, wherein the disease and / or symptom comprises one or more selected from the group consisting of 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.

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