Modified cell and application thereof

Abstract

The invention belongs to the field of biological medicine, provides a modified cell and application thereof, and particularly relates to a method for culturing a cell, which comprises the step of reducing expression and / or weakening activity of a peptidase C64 family member such as TNFAIP3 of the cell. The invention also provides a method for preventing and / or treating tumors by using the cultured cells.

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.

[0003] Summary of the Invention

[0004] 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.

[0005] In one aspect, the present invention provides a method for culturing cells, comprising: reducing the expression and / or attenuating the activity of a family member selected from the peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and the CBL family, and / or their functionally active fragments in the cells.

[0006] In another aspect, the present invention provides a cell obtained by the method of the present invention.

[0007] In another aspect, the present invention provides a pharmaceutical composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0008] 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.

[0009] 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.

[0010] 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 invention to which the present invention relates. Accordingly, the descriptions in the drawings and specification of the present invention are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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:

[0012] FIG1A shows the human TNFAIP3 gene editing targeting segment relative to the start codon provided by the present invention, which can be, for example, 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 20 bp away from the exon.

[0013] Figure 1B shows the human ZC3H12A gene editing targeting segment relative to the start codon provided by the present invention, for example, can be a continuous region with about 3 or more transcription factor binding numbers; and can be an exon region of the gene or an intron region about 20 bp away from the exon.

[0014] Figure 1C shows the human SOCS1 gene editing targeting segment relative to the start codon provided by the present invention, for example, can be a continuous region with about 3 or more transcription factor binding numbers; and can be an exon region of the gene or an intron region about 20 bp away from the exon.

[0015] Figure 1D shows the human CBLB gene editing targeting segment relative to the start codon provided by the present invention, for example, it can be a continuous region with about 3 or more transcription factor binding numbers; and it can be the exon region of the gene or the intron region about 20 bp away from the exon.

[0016] Figure 2A shows the fold amplification of TNFAIP3 gene-edited TCR-T cells in the non-stimulation culture medium group.

[0017] Figures 2B-2C show the fold amplification of TNFAIP3 gene-edited TCR-T cells in the TransACT stimulation group.

[0018] Figures 2D-2G show the target cell cytotoxicity of TCR-T cells derived from different donors after TNFAIP3 gene editing. Figures 2D and 2F show the cytotoxicity curves at each time point, and Figures 2E and 2G show the cytotoxicity of each experimental group at the end of the experiment, all of which were higher than those in the unedited NT group.

[0019] Figures 2H-2K show the various cytokine release capabilities of TNFAIP3 gene-edited TCR-T cells.

[0020] Figure 3A shows the expansion fold of ZC3H12A gene-edited TCR-T cells in the non-stimulation culture medium group.

[0021] Figure 3B shows the fold expansion of ZC3H12A gene-edited TCR-T cells in the TransACT stimulation group.

[0022] Figures 3C-3F show the target cell cytotoxicity of ZC3H12A gene-edited TCR-T cells. Figures 3C and 3E show the cytotoxicity curves at various time points, and Figures 3D and 3F show the cytotoxicity of each experimental group at the endpoint. All of these results were significantly higher than those of the unedited NT group.

[0023] Figures 3G-3J show the various cytokine release capabilities of ZC3H12A gene-edited TCR-T cells.

[0024] Figure 4A shows the expansion fold of SOCS1 gene-edited TCR-T cells in the non-stimulation culture medium group.

[0025] Figure 4B shows the fold expansion of SOCS1 gene-edited TCR-T cells in the TransACT stimulation group.

[0026] Figure 4C shows the expansion folds of SOCS1 gene-edited TILs from different donors in the non-stimulation culture medium group.

[0027] FIG4D shows the expansion fold of SOCS1 gene-edited TILs from donor 306 in the TransACT stimulation group.

[0028] Figures 4E-4H show the target cell killing ability of SOCS1 gene-edited TCR-T cells.

[0029] Figure 4I shows the target cell killing ability of SOCS1 gene-edited TIL cells.

[0030] Figure 4J shows the cytokine expression of SOCS1 gene-edited TCR-T cells in the unstimulated group.

[0031] Figure 4K shows the cytokine expression of SOCS1 gene-edited TCR-T cells in the CD3 antibody stimulation group.

[0032] Figure 4L shows the cytokine release capacity of SOCS1 gene-edited TCR-T cells co-cultured with A375 target cells.

[0033] Figure 4M shows the cytokine expression of SOCS1 gene-edited TIL cells in the unstimulated group.

[0034] Figure 4N shows the cytokine expression of SOCS1 gene-edited TIL cells in the TransACT stimulation group.

[0035] Figure 4O shows that TIL cells after SOCS1 gene editing have a higher proportion of stem cells.

[0036] Figure 4P shows that TIL cells after SOCS1 gene editing have a lower proportion of exhausted T cells.

[0037] Figures 5A-5B show the fold expansion of CBLB gene-edited TCR-T cells in the non-stimulation culture medium group.

[0038] Figures 5C-5D show the fold expansion of CBLB gene-edited TCR-T cells in the TransACT stimulation group.

[0039] Figures 5E-5H show the target cell cytotoxicity of CBLB gene-edited TCR-T cells. Figures 5E and 5G show the cytotoxicity curves at various time points, and Figures 5F and 5H show the cytotoxicity of each experimental group at the endpoint, all of which were higher than those of the unedited NT group.

[0040] Figures 5I-5L show the various cytokine release capabilities of CBLB gene-edited TCR-T cells.

[0041] Figure 6A shows the expansion fold of TILs gene-edited with the combination of CBLB and ZC3H12A in the non-stimulation medium group.

[0042] Figure 6B shows the expansion fold of TILs edited with the combination of SOCS1 and CBLB in the non-stimulation medium group.

[0043] Figure 6C shows the expansion fold of TILs with combined gene editing of SOCS1 and TNFAIP3 in the non-stimulation medium group.

[0044] Figure 6D shows the expansion fold of TILs with combined gene editing of SOCS1 and TNFAIP3 in the CD3 antibody stimulation group.

[0045] Figure 6E shows the expansion fold of TILs with combined gene editing of SOCS1 and ZC3H12A in the non-stimulation culture medium group.

[0046] Figure 6F shows the expansion fold of TILs edited with the combination of SOCS1 and ZC3H12A in the CD3 antibody stimulation group.

[0047] Figure 6G shows the expansion fold of TILs gene-edited with the combination of TNFAIP3 and CBLB in the non-stimulation medium group.

[0048] Figure 6H shows the expansion fold of TILs gene-edited with the combination of TNFAIP3 and ZC3H12A in the non-stimulation medium group.

[0049] Figure 6I shows the expansion fold of TILs gene-edited with the combination of TNFAIP3 and ZC3H12A in the CD3 antibody stimulation group.

[0050] Figure 6J shows the expansion fold of TILs with combined gene editing of TNFAIP3 and SOCS1 in the non-stimulation medium group.

[0051] Figure 6K shows the expansion fold of TILs with combined gene editing of TNFAIP3 and SOCS1 in the TransACT antibody stimulation group.

[0052] Figure 7A shows the target cell killing ability of TIL cells edited by the CBLB and ZC3H12A combination gene.

[0053] Figure 7B shows the target cell killing ability of TIL cells edited by the CBLB and ZC3H12A combination gene.

[0054] Figure 7C shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and CBLB genes.

[0055] Figure 7D shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and CBLB genes.

[0056] Figure 7E shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and CBLB genes.

[0057] Figure 7F shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and TNFAIP3 genes.

[0058] Figure 7G shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and TNFAIP3 genes.

[0059] Figure 7H shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and TNFAIP3 genes.

[0060] Figure 7I shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and TNFAIP3 genes.

[0061] Figure 7J shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and ZC3H12A genes.

[0062] Figure 7K shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and ZC3H12A genes.

[0063] Figure 7L shows the target cell killing ability of TIL cells edited by the combination of SOCS1 and ZC3H12A genes.

[0064] Figure 7M shows the target cell killing ability of TIL cells gene-edited with the combination of TNFAIP3 and CBLB.

[0065] Figure 7N shows the target cell killing ability of TIL cells gene-edited with the combination of TNFAIP3 and CBLB.

[0066] FIG7O shows the target cell killing ability of TIL cells gene-edited with the combination of TNFAIP3 and ZC3H12A.

[0067] Figure 7P shows the target cell killing ability of TIL cells edited by the TNFAIP3 and ZC3H12A combination gene.

[0068] Figure 7Q shows the target cell killing ability of TIL cells gene-edited with the combination of TNFAIP3 and ZC3H12A.

[0069] Figure 7R shows the target cell killing ability of TIL cells gene-edited with the combination of TNFAIP3 and ZC3H12A.

[0070] Figure 7S shows the target cell killing ability of TIL cells edited by the TNFAIP3 and SOCS1 combination genes.

[0071] Figure 7T shows the killing ability of TIL cells gene-edited with TNFAIP3 and SOCS1 combination against autologous tumor organoids.

[0072] Figure 8A shows that TIL cells after combined gene editing of CBLB and ZC3H12A have a lower proportion of exhausted T cells.

[0073] Figure 8B shows that TIL cells after combined gene editing of SOCS1 and CBLB have a higher proportion of central memory T cells.

[0074] Figure 8C shows that TIL cells after combined gene editing of SOCS1 and CBLB have a lower proportion of exhausted T cells.

[0075] Figure 8D shows that TIL cells after combined gene editing of SOCS1 and TNFAIP3 have a higher proportion of central memory T cells.

[0076] Figure 8E shows that TIL cells after combined gene editing of SOCS1 and TNFAIP3 have a lower proportion of exhausted T cells.

[0077] Figure 8F shows that TIL cells after combined gene editing of SOCS1 and ZC3H12A have a higher proportion of central memory T cells.

[0078] Figures 8G-8H show that TIL cells after combined gene editing of SOCS1 and ZC3H12A have a lower proportion of exhausted T cells.

[0079] Figure 8I shows that TIL cells after combined gene editing of TNFAIP3 and CBLB have a lower proportion of exhausted T cells.

[0080] Figure 8J shows that TIL cells after combined gene editing of TNFAIP3 and ZC3H12A have a higher proportion of naive T cells.

[0081] Figure 8K shows that TIL cells after combined gene editing of TNFAIP3 and ZC3H12A have a higher proportion of central memory T cells.

[0082] Figures 8L-8M show that TIL cells after combined gene editing of TNFAIP3 and ZC3H12A have a lower proportion of exhausted T cells.

[0083] Figure 8N shows that TIL cells after combined gene editing of TNFAIP3 and SOCS1 have a higher proportion of stem cells.

[0084] Figure 8O shows that TIL cells after combined gene editing of TNFAIP3 and SOCS1 have a lower proportion of exhausted T cells.

[0085] Figure 9A shows that TIL cells after gene editing of the CBLB and ZC3H12A combination in the CD3 antibody stimulation group had a higher cytokine expression ratio.

[0086] Figure 9B shows that TIL cells after gene editing of SOCS1 and CBLB combination in the unstimulated Medium group had a higher cytokine expression ratio.

[0087] Figure 9C shows that TIL cells after gene editing of SOCS1 and CBLB combination in the CD3 antibody stimulation group had a higher cytokine expression ratio.

[0088] Figure 9D shows that TIL cells after gene editing of SOCS1 and TNFAIP3 combination in the unstimulated Medium group had a higher cytokine expression ratio.

[0089] Figure 9E shows that TIL cells after SOCS1 and TNFAIP3 combination gene editing in the CD3 antibody stimulation group had a higher cytokine expression ratio.

[0090] Figure 9F shows that TIL cells after gene editing of SOCS1 and ZC3H12A combination in the unstimulated Medium group have a higher cytokine expression ratio.

[0091] Figure 9G shows that TIL cells after gene editing of SOCS1 and ZC3H12A combination in the CD3 antibody stimulation group had a higher cytokine expression ratio.

[0092] Figure 9H shows that TIL cells after gene editing of TNFAIP3 and CBLB combination in the unstimulated Medium group had a higher cytokine expression ratio.

[0093] Figure 9I shows that TIL cells after gene editing of TNFAIP3 and CBLB combination in the CD3 antibody stimulation group have a higher cytokine expression ratio.

[0094] Figures 9J-9K show that TIL cells in the unstimulated Medium group after gene editing of the TNFAIP3 and ZC3H12A combination have a higher cytokine expression ratio.

[0095] Figure 9L shows that TIL cells after gene editing of TNFAIP3 and ZC3H12A combination in the CD3 antibody stimulation group have a higher cytokine expression ratio.

[0096] Figure 9M shows that TIL cells after TNFAIP3 and SOCS1 combination gene editing co-cultured with autologous tumor organoids have higher cytokine release capacity.

[0097] FIG. 10 shows the results of apoptosis detection of TIL cells derived from donor 504. DETAILED DESCRIPTION

[0098] 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.

[0099] Definition of terms

[0100] In the present invention, 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 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. For example, the CBL family members of the present invention may include functionally active fragments thereof as well as any other domains.

[0101] In the present invention, 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 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 STAT-induced STAT inhibitor (SSI) family member of the present invention may include functionally active fragments thereof and any other structural domains.

[0102] In the present invention, 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 the present invention may also include functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing the functionally active fragments thereof produced after processing and / or modification in cells. For example, the peptidase C64 family member of the present invention may include functionally active fragments thereof as well as any other domains.

[0103] In the present invention, 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 the present invention may also include functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances containing the functionally active fragments thereof produced after processing and / or modification in cells. For example, the ZC3H12 family members of the present invention may include functionally active fragments thereof as well as any other domains.

[0104] 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.

[0105] In the present invention, 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 the present invention, TNFAIP3 can include unprocessed TNFAIP3, any form of processed TNFAIP3, TNFAIP3 variants, or substances comprising functionally active fragments of TNFAIP3.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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).

[0110] 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. Antigen binding domains that can be used in the present disclosure may include, for example, scFv, antibodies, antigen-binding regions of antibodies, variable regions of heavy / light chains, and / or single-chain antibodies.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] In the present invention, "CD4 + Cells" generally refer to CD4-positive cells, such as T cells. The term "CD4 + Cells”, “CD4 positive cells” can be used synonymously. These cells can be identified by methods known in the art, such as by staining the cells with fluorescently labeled antibodies against CD4 and using fluorescence activated cell sorting. 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.

[0129] 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.

[0130] 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).

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] In the present invention, the term "in vivo" generally refers to events that occur within the body of a subject.

[0142] In the present invention, the term "in vitro" generally refers to events that occur outside the body of a subject.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] In the present invention, the term "specific binding" generally refers to a binding substance that recognizes a specific target substance but does not substantially recognize or bind to other molecules in the sample. 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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 Th17CD4 +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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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 - CD62L + To identify stem cell-like T cells. For example, CD39 - 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.

[0177] 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.

[0178] 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 components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents and / or excipients.

[0179] 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 formulations 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.

[0180] 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.

[0181] 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 may be 3575), ITGA4 (the gene encoding it may be 3676), VLA1 (the gene encoding it may be 3672), CD49a (the gene encoding it may be 3672), IA4 (the gene encoding it may be 3732), CD49D (the gene encoding it may be 3676), ITGA6 (the gene encoding it may be 3655), VLA-6 (the gene encoding it may be 3655), CD49f (the gene encoding it may be 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), CD 103 (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 3681 ... 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. ,

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0195] Detailed Description of the Invention

[0196] TNFAIP3 knockout

[0197] The present invention provides a method for reducing the expression and / or weakening the activity of peptidase C64 family members and / or functionally active fragments thereof in the cell.

[0198] In one aspect, the present invention provides a method for culturing cells to reduce the expression and / or attenuate the activity of a peptidase C64 family member and / or a functionally active fragment thereof. For example, the peptidase C64 family member may comprise a ubiquitin binding domain. For example, the peptidase C64 family member may comprise TNFAIP3.

[0199] For example, the target gene of the present invention can be a gene encoding a peptidase C64 family member and / or a functionally active fragment thereof. For example, compared with cells whose expression and / or activity of the target gene are unchanged, the cells obtained by reducing the expression and / or weakening the activity of at least one target gene of the cell can show improved cell characteristics. In one embodiment, the cells whose expression and / or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and / or weakened the activity of at least one target gene of the cell. In one embodiment, the cells whose expression and / or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and / or weakened the activity of other genes (e.g., knocking out the other genes, which have substantially no effect on cell function) other than the target gene of the cell.

[0200] For example, the cells include immune cells. For example, the cells include immune effector cells. For example, the cells include immune effector T cells, immune effector NK cells, immune effector NKT cells. For example, the cells include phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0201] For example, the cells comprise monocytes, macrophages and / or dendritic cells.

[0202] 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.

[0203] 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."

[0204] 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.

[0205] 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.

[0206] 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.

[0207] In one aspect, the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: reducing the expression and / or activity of a peptidase C64 family member and / or a functionally active fragment thereof in the TIL.

[0208] For example, TILs derived from tumor tissue, pleural effusion and / or ascites and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0209] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of the peptidase C64 family members and / or their functionally active fragments in the TILs can be reduced.

[0210] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0211] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0212] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0213] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0214] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0215] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0216] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0217] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0218] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of peptidase C64 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0219] For example, cells obtained by reducing the expression and / or attenuating the activity of a peptidase C64 family member exhibit improved cell properties compared to cells in which the expression and / or activity of the peptidase C64 family member is not altered.

[0220] For example, improved cell number of the present invention means that the cell number of the cells of the present invention in which the expression and / or activity of the peptidase C64 family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold, compared to cells in which the expression and / or activity of the peptidase C64 family member is not altered.

[0221] For example, an increased proportion of live cells can be expressed as an increase in cell viability. For example, an increased proportion of live cells of the present invention can mean that the proportion of live cells of the cells of the present invention in which the expression and / or activity of the peptidase C64 family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about At least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0222] For example, the improved cytokine secretion capacity of the present invention may refer to an improved cytokine secretion capacity of a cell selected from the group consisting of IL-2, IL-6, CD107a, GZMB, TNF-α, and IFN-γ. For example, the improved cytokine secretion capacity of the present invention may refer to an increase in the proportion of cells secreting cytokines in the cells of the present invention in which the expression and / or activity of the peptidase C64 family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage compared to cells in which the expression and / or activity of the peptidase C64 family member is not altered, which can 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. For example, the improved cytokine secretion capacity of the present invention may mean that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and / or activity of the peptidase C64 family member is reduced and / or the activity is attenuated during at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, or more compared to cells in which the expression and / or activity of the peptidase C64 family member is not changed. 0%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0223] For example, the improved tumor cell killing ability of the present invention can mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the peptidase C64 family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to cells in which the expression and / or activity of the peptidase C64 family member is not changed. For example, the improved tumor cell killing ability of the present invention can mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the peptidase C64 family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, compared to cells in which the expression and / or activity of the peptidase C64 family member is not changed. , at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%. For example, the tumor cell killing rate of the cells of the present invention can be measured by the IncuCyte system or CFSE and DAPI staining. For example, tumor cell killing of the cells of the present invention can refer to the ability of the cells to kill solid tumor cells.

[0224] For example, the improved cell subpopulation ratio of the present invention may comprise one or more selected from the following groups: increased CD8 + cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of regulatory cells, an increased proportion of activated cells, an increased proportion of tumor-specific cells, and an increased proportion of stem-like cells.

[0225] For example, in cells CD8 +The proportion of cells, central memory cells and / or naive cells, activated cells, tumor-specific cells and / or stem-like cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0226] For example, the reduced ratio of exhausted cells in the present invention can be + 、LAG-3 + 、TIM-3 + , and / or CD39 + For example, the ratio of regulatory cells decreased in the present invention can be an increase in the ratio of CD4 + CD25 + Foxp3 + For example, the ratio of apoptotic cells reduced in the present invention can be the ratio of CD95 + caspass3 + cells and / or CD95 + DR5 + A decrease in the proportion of cells.

[0227] For example, the proportion of exhausted cells, regulatory cells and / or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 19%, at least about 18 ... The amount of the active ingredient in the present invention may be about 4% less, at least about 3% less, at least about 2% less, at least about 1% less, at least about 0.5% less, at least about 0.4% less, at least about 0.3% less, at least about 0.2% less, or at least about 0.1% less, or may be at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold less.

[0228] 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.

[0229] For example, the gene regulatory system can destroy the target gene at the DNA level. For example, the gene regulatory system can destroy the region or fragment of the target gene in the genome of the cell. For example, after using the gene regulatory system, the DNA region or fragment where the target gene is located in the cell is sheared, and the expression ability of the target gene is reduced or the activity of the target gene is inhibited. For example, the editing effect of the gene regulatory system on the target gene can be long-term and continuous. The genomic region of the present invention is determined based on the human reference genome version hg38.

[0230] 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.

[0231] For example, the present invention can reduce the expression and / or weaken the activity of at least one target gene of a cell by introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell. For example, the enzyme protein can include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the guide nucleic acid molecule can include a guide RNA (gRNA). For example, the guide nucleic acid molecule can include 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.

[0232] 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.

[0233] For example, the editing target region of the present invention may be a region preceding a promoter. 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 bindings. For example, the editing target region of the present invention may be a continuous region with approximately 3 or more transcription factor bindings.

[0234] 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.

[0235] For example, when the gene editing system includes CRISPR / Cas12, the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G. 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 downstream of the 3' end 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, 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] For example, the guide nucleic acid molecule can comprise a target sequence consisting of about 10 to about 30 nucleotides before the PAM region represented by AGG, TGG, GGG and / or CGG in the DNA encoding a peptidase C64 family member and / or a functionally active fragment thereof. For example, the guide nucleic acid molecule can comprise a target sequence capable of binding to a gene encoding a peptidase C64 family member and / or a functionally active fragment thereof, comprising about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides preceding the PAM region represented by AGG, TGG, GGG and / or CGG.

[0242] For example, the target sequence can be selected from a region defined by the genomic coordinates shown in Table 1A, or a fragment thereof.

[0243] For example, the target sequence of the present invention can be the OUT structural functional domain of TNFAIP3. For example, the target sequence of the present invention can be the zinc finger structural functional domain of TNFAIP3. For example, the target sequence of the present invention can be chr6:137871529-137871637, chr6:137874734-137874807, chr6:137874895-137874943, chr6:137875008-137875040, chr6:137875046-137875149, chr6:137875614-137875650, chr6:137875666-137875724, chr6:1 37875789-137875816、chr6:137875844-137876075、chr6:137879031-137879264、chr6:137879337-137879460、chr 6:137879958-137880090, chr6:137880155-137880268, chr6:137880275-137880377, chr6:137880940-137881384.

[0244] For example, the guide nucleic acid molecule can include a targeting domain that is complementary to a target sequence selected from the group consisting of SEQ ID NOs: 107-212, 1562-2532.

[0245] For example, the guide nucleic acid molecule can include a targeting domain, which can comprise a sequence as shown in SEQ ID NOs: 1-106, 591-1561, 7267-7324, 7419, 7420.

[0246] For example, the guide nucleic acid molecule can include a targeting domain, which can comprise a sequence as shown in SEQ ID NOs: 7267-7324, 7419, 7420.

[0247] For example, compared to cells in which the expression and / or activity of at least one target gene of the cells 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.

[0248] For example, in the methods of the present invention, the proportion of cells expressing the product of the target gene is reduced by at least about 5% compared to cells in which the expression and / or activity of the target gene is not altered. For example, the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and / or functionally active fragment thereof is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%. For example, the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and / or its functionally active fragment can be reduced from the observed proportion of cells to 0%. For example, the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and / or its functionally active fragment can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%. For example, the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and / or its functionally active fragment can be detected by flow cytometry.

[0249] For example, in the method of the present invention, the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and / or its functionally active fragment obtained by reducing the expression and / or attenuating the activity of at least one target gene in the cells can be at most about 95%. For example, the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and / or its functionally active fragment can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%. For example, the proportion of cells expressing the product of the gene encoding the peptidase C64 family member and / or its functionally active fragment can be detected by flow cytometry.

[0250] For example, in the methods of the present invention, the expression and / or activity of at least one target gene in the cell is reduced and / or the activity is attenuated, and the expression of the target gene in a single cell can be reduced by at least about 5% compared to a cell 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%, 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 an observable amount to 0%. For example, the expression level of the gene of interest in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.

[0251] For example, in the method of the present invention, the expression level of the target gene in a single cell obtained by reducing the expression and / or attenuating the activity of at least one target gene in the cell can be up to about 95% of that in the cell in which the expression and / or activity of the target gene is not changed. For example, the expression level of the gene encoding the peptidase C64 family member and / or functionally active fragment thereof (e.g., the gene encoding TNFAIP3) in a single cell can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5% of that in a cell in which the expression and / or activity of the gene encoding the peptidase C64 family member and / or functionally active fragment thereof is not altered.

[0252] For example, the method of the present invention comprises: subjecting the cells to at least one stage of in vitro expansion, wherein, during at least one stage of in vitro expansion, the expression and / or activity of a peptidase C64 family member in the cells is reduced.

[0253] 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 of peptidase C64 family members of the TILs expanded in vitro in the first stage is reduced and / or the activity is attenuated.

[0254] 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.

[0255] For example, during a single stage of in vitro expansion of the present invention, the cells can be contacted with the one or more cell activators and the expression and / or activity of a peptidase C64 family member and / or a functionally active fragment thereof can be reduced in the cells. For example, the cell activator can comprise an agonist of one or more targets selected from the group consisting of CD3, CD28, HVEM, CD40L, OX40, and 4-1BB. For example, during a single stage of in vitro expansion, the expression and / or activity of a peptidase C64 family member in the cells of the present invention can be reduced and / or the activity can be attenuated, and the cells can be contacted with the one or more cell activators of the present invention. For example, during the first stage of in vitro expansion of the present invention, the TILs of the present invention can be contacted with the expression and / or activity of a peptidase C64 family member and / or the activity can be attenuated, and the cells can be contacted with the one or more cell activators of the present invention. For example, during the second stage of in vitro expansion of the present invention, the expression and / or activity of a peptidase C64 family member in the TILs of the present invention can be reduced and / or the activity can be attenuated, and the cells can be contacted with the one or more cell activators of the present invention. For example, during the third stage of in vitro expansion of the present invention, the expression and / or activity of a peptidase C64 family member in the TILs of the present invention can be reduced and / or the activity can be attenuated, and the cells can be contacted with the one or more cell activators of the present invention.

[0256] For example, in a single stage of in vitro expansion of the present invention, the cells of the present invention can be exposed to reduced expression and / or attenuated activity of a peptidase C64 family member 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 exposed to reduced expression and / or attenuated activity of a peptidase C64 family member, for example, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours prior to exposure, and then exposed to 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 exposed to one or more cell activators of the present invention, for example, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours prior to exposure, and then exposed to reduced expression and / or attenuated activity of a peptidase C64 family member.

[0257] For example, in the first stage of in vitro expansion of the present invention, the TILs of the present invention can be made to reduce the expression and / or weaken the activity of the peptidase C64 family member and contact one or more cell activators of the present invention 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 made to reduce the expression and / or weaken the activity of the peptidase C64 family member and contact one or more cell activators of the present invention 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 made to reduce the expression and / or weaken the activity of the peptidase C64 family member and contact one or more cell activators of the present invention at substantially the same time.

[0258] On the other hand, 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, pleural effusion and / or ascites and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and / or weakening the activity of a peptidase C64 family member in the second TIL population; wherein, a third TIL population is obtained through step (B).

[0259] In the terms of 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 the terms of 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 the terms of 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 population obtained by step (A) in the method of the above aspect. In the terms of 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 population obtained by step (B) in the method of the above aspect. In the terms of 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 the terms of 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 population obtained by any additional step (C) in the method of the above aspect.

[0260] On the other hand, 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, pleural effusion, and / or ascites and not expanded in vitro with a plurality of cell growth factors; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with a plurality of cell growth factors, with a plurality of cell activators, reducing the expression and / or attenuating the activity of a peptidase C64 family member, and co-culturing the TILs with feeder cells; wherein, a third TIL population is obtained through step (B).

[0261] On the other hand, 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, pleural effusion, and / or ascites and not expanded in vitro with a cell growth factor; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with a cell growth factor, with a cell activator, reducing the expression and / or attenuating the activity of a peptidase C64 family member, and co-culturing the TILs with feeder cells, wherein the peptidase C64 family member may include TNFAIP3; wherein, a third TIL population is obtained through step (B).

[0262] On the other hand, the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL). The method for obtaining TIL cells from a subject's tissue sample can be to obtain an in situ tumor sample or a metastatic tumor sample from a patient during surgery, which can weigh at least about 1g, or multiple tissues can be combined. Tumor tissue, pleural effusion and / or peritoneal effusion are transported in a sample transport fluid, such as a commercially available tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, at about 2-8 degrees Celsius 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, such as 6000 IU / mL). The cells in the culture medium are collected and transferred into a breathable culture bag, a Grex, or a Xuri device. The serum-free culture medium of the cells can be supplemented with the CD28 antibody, CD3 antibody, and CD28 antibody of the present invention, magnetic beads (e.g., Dynabeads) containing CD3 antibody and CD28 antibody, and / or nanomatrix (e.g., transACT) containing CD3 antibody and CD28 antibody, and IU / mL at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL). L-2 and reducing the expression and / or activity of peptidase C64 family members (peptidase C64 family members may include TNFAIP3, for example, by transducing with a ribonucleoprotein complex (RNP) containing the gRNA of the present invention and the Cas protein to make the proportion of cells encoding the gene of the peptidase C64 family member in the TIL be about 95% or less), after activating the TIL of the present invention for a certain period of time, adding irradiated PBMC (TIL to PBMC at a ratio of about 1:40-about 1:400), and expanding and culturing for about 3-14 days. The cells in the culture medium can be collected using a cell processing system, washed, frozen, and tested. The final product CD3 ratio can be greater than 80%, the cell viability can be greater than 50%, and greater than 80% of the cells can be memory effector cells and effector cells. After stimulation, IFN-γ can be secreted, and / or it can have the characteristic of an increased proportion of activated cells.

[0263] ZC3H12A knockout

[0264] 1. A method for culturing cells, comprising: reducing the expression and / or attenuating the activity of a ZC3H12 family member and / or a functionally active fragment thereof in the cells.

[0265] 2. The method of embodiment 1, wherein the cells comprise immune cells.

[0266] 3. The method according to embodiment 2, wherein the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0267] 4. The method of any one of embodiments 2-3, wherein the immune cells comprise monocytes, macrophages and / or dendritic cells.

[0268] 5. The method according to any one of embodiments 2-4, wherein the immune cells are derived from immune cells differentiated from stem cells.

[0269] 6. The method of embodiment 5, wherein the stem cells comprise induced pluripotent stem cells (iPSCs).

[0270] 7. The method of any one of embodiments 2-6, wherein the immune cells comprise B cells, T cells, natural killer cells and / or natural killer-like T cells (NKT).

[0271] 8. The method of any one of embodiments 2-7, wherein the immune cells comprise αβT cells and / or γδT cells.

[0272] 9. The method of any one of embodiments 2-8, wherein the immune cells comprise tumor infiltrating lymphocytes (TILs).

[0273] 10. The method according to embodiment 9, wherein the TILs are derived from fragments of tumor tissue, TILs from pleural effusion and / or ascites and / or TILs revived after cryopreservation.

[0274] 11. The method of embodiment 10, wherein the fragments have a volume of about 1 cubic millimeter to about 27 cubic millimeters.

[0275] 12. The method of any one of embodiments 2-11, wherein the immune cell comprises an engineered immune receptor displayed on the cell surface.

[0276] 13. A method according to embodiment 12, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell.

[0277] 14. The method of any one of embodiments 2-13, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0278] 15. The method of any one of embodiments 1-14, wherein reducing the expression and / or attenuating the activity of a ZC3H12 family member in the cell comprises inhibiting the function of a nuclease.

[0279] 16. The method of any one of embodiments 1-15, wherein the cells obtained by reducing the expression and / or attenuating the activity of the ZC3H12 family member exhibit improved cellular properties compared to cells in which the expression and / or activity of the ZC3H12 family member is not altered.

[0280] 17. A method according to embodiment 16, wherein the improved cell characteristics include one or more selected from the following groups: improved cell proliferation ability, increased proportion of living cells, improved proportion of cell subpopulations, increased cytokine secretion ability and improved tumor cell killing ability.

[0281] 18. A method according to embodiment 17, wherein 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.

[0282] 19. The method of any one of embodiments 1-18, wherein the ZC3H12 family member comprises a C3H1-type zinc finger domain.

[0283] 20. The method of any one of embodiments 1-19, wherein the ZC3H12 family member comprises ZC3H12A.

[0284] 21. The method of any one of embodiments 1-20, wherein reducing the expression and / or attenuating the activity of a ZC3H12 family member in the cell comprises introducing a gene regulatory system into the cell.

[0285] 22. The method of embodiment 21, wherein the gene regulatory system is capable of disrupting the ZC3H12 family member at the DNA level.

[0286] 23. The method of any one of embodiments 21-22, wherein the gene regulatory system comprises a guide nucleic acid molecule and an enzyme protein.

[0287] 24. The method of embodiment 23, wherein reducing the expression and / or attenuating the activity of the ZC3H12 family member comprises: introducing into the cell a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and a Cas protein.

[0288] 25. The method of any one of embodiments 23-24, wherein the enzyme protein comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

[0289] 26. The method of any one of embodiments 23-25, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA).

[0290] 27. The method of any one of embodiments 23-26, wherein the guide nucleic acid molecule is capable of binding to the sequence of the ZC3H12 family member.

[0291] 28. The method of any one of embodiments 23-27, wherein the guide nucleic acid molecule is capable of binding to a region defined by the genomic coordinates selected from those shown in Table 1B, or a fragment thereof.

[0292] 29. The method of any one of embodiments 23-28, wherein the guide nucleic acid molecule is capable of binding to a region or fragment thereof selected from the group consisting of SEQ ID NOs: 281-348, 3116-3698.

[0293] 30. A method according to any one of embodiments 23-29, wherein the guide nucleic acid molecule is capable of binding 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, CGG and GGG.

[0294] 31. The method of any one of embodiments 23-30, wherein the guide nucleic acid molecule comprises a targeting domain comprising a sequence as shown in any one of SEQ ID NOs: 213-280, 2533-3115, 7325-7345, 7416, 7417.

[0295] 32. The method of any one of embodiments 1-31, wherein the proportion of cells expressing the product of the gene of interest is reduced and / or the expression level of the gene of interest in individual cells is decreased, compared to cells in which the expression and / or activity of the ZC3H12 family member is not altered.

[0296] 33. The method of any one of embodiments 1-32, wherein the proportion of cells expressing the gene of interest in the cells obtained by reducing the expression and / or attenuating the activity of the ZC3H12 family member is about 95% or less.

[0297] 34. A cell obtained by the method of any one of embodiments 1-33.

[0298] 35. A composition comprising the cell of embodiment 34.

[0299] 36. A pharmaceutical composition comprising the cell of embodiment 34 and / or the composition of embodiment 35, and optionally a pharmaceutically acceptable carrier.

[0300] 37. A method of influencing cell growth comprising administering the cell of embodiment 34, the composition of embodiment 35, and / or the pharmaceutical composition of embodiment 36.

[0301] 38. Use of the cell of embodiment 34, the composition of embodiment 35, and / or the pharmaceutical composition of embodiment 36 in the preparation of a medicament for preventing and / or treating a disease and / or symptom.

[0302] 39. The use according to embodiment 38, wherein the disease and / or condition comprises a tumor.

[0303] 40. The use according to any one of embodiments 38-39, wherein the disease and / or condition comprises a solid tumor.

[0304] 41. The use according to any one of embodiments 38-40, wherein the disease and / or symptoms comprise 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.

[0305] The present invention provides a method for reducing the expression and / or attenuating the activity of ZC3H12 family members and / or functionally active fragments thereof in the cells.

[0306] In one aspect, the present invention provides a method for culturing cells to reduce the expression and / or attenuate the activity of a ZC3H12 family member and / or a functionally active fragment thereof in the cells. For example, the ZC3H12 family member may comprise a C3H1-type zinc finger domain. For example, the ZC3H12 family member may comprise ZC3H12A.

[0307] For example, the target gene of the present invention can be a gene encoding a ZC3H12 family member and / or a functionally active fragment thereof. For example, compared with cells in which the expression and / or activity of the target gene are unchanged, the cell obtained by reducing the expression and / or weakening the activity of at least one target gene of the cell can show improved cell characteristics. In one embodiment, the cell in which the expression and / or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and / or weakened the activity of at least one target gene of the cell. In one embodiment, the cell in which the expression and / or activity of the target gene are unchanged can refer to cells derived from the same donor and have not reduced the expression and / or weakened the activity of other genes (such as knocking out these other genes, having no substantial effect on cell function) other than the target gene of the cell.

[0308] For example, the cells include immune cells. For example, the cells include immune effector cells. For example, the cells include immune effector T cells, immune effector NK cells, immune effector NKT cells. For example, the cells include phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0309] For example, the cells comprise monocytes, macrophages and / or dendritic cells.

[0310] 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.

[0311] 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."

[0312] 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.

[0313] 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.

[0314] 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.

[0315] In one aspect, the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL), which may comprise: reducing the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TIL.

[0316] For example, TILs derived from tumor tissue, pleural effusion and / or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, during the at least one stage of in vitro expansion, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0317] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0318] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0319] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced. In the second stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0320] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0321] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0322] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0323] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced. In the second stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0324] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced. In the third stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0325] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion. In the second stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced. In the third stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0326] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced. In the second stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced. In the third stage of in vitro expansion of the present invention, the expression and / or activity of ZC3H12 family members and / or functionally active fragments thereof in the TILs can be reduced.

[0327] For example, cells obtained by reducing the expression and / or attenuating the activity of a ZC3H12 family member exhibit improved cellular properties compared to cells in which the expression and / or activity of the ZC3H12 family member is not altered.

[0328] For example, improved cell number of the present invention means that the cell number of the cells of the present invention having reduced expression and / or attenuated activity of the ZC3H12 family member in at least one in vitro expansion stage can be increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold compared to cells in which the expression and / or activity of the ZC3H12 family member is not altered.

[0329] For example, an increased proportion of live cells can be expressed as an increase in cell viability. For example, an increased proportion of live cells of the present invention can mean that the proportion of live cells of the cells of the present invention in which the expression and / or activity of the ZC3H12 family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, or more, compared to cells in which the expression and / or activity of the ZC3H12 family member is not altered. At least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0330] For example, the improved cytokine secretion capacity of the present invention may refer to an improved cytokine secretion capacity of the cell selected from the group consisting of IL-2, IL-6, CD107a, GZMB, TNF-α, and IFN-γ. For example, the improved cytokine secretion capacity of the present invention may refer to an increase in the proportion of cells secreting cytokines in the cells of the present invention in which the expression and / or activity of the ZC3H12 family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage compared to cells in which the expression and / or activity of the ZC3H12 family member is not altered, which is 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. For example, the improved cytokine secretion capacity of the present invention may mean that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and / or activity of the ZC3H12 family member is reduced and / or the activity is attenuated during at least one in vitro expansion stage is increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0331] For example, the improved tumor cell killing ability of the present invention can mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the ZC3H12 family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold compared to cells in which the expression and / or activity of the ZC3H12 family member is not altered. For example, the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the ZC3H12 family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, or more compared to cells in which the expression and / or activity of the ZC3H12 family member is not changed. At least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 1%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%. For example, the tumor cell killing rate of the cells of the present invention can be measured by the IncuCyte system or CFSE and DAPI staining. For example, tumor cell killing of the cells of the present invention can refer to the ability of the cells to kill solid tumor cells.

[0332] For example, the improved cell subpopulation ratio of the present invention may comprise one or more selected from the following groups: increased CD8 + cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of regulatory cells, an increased proportion of activated cells, an increased proportion of tumor-specific cells, and an increased proportion of stem-like cells.

[0333] For example, in cells CD8 +The proportion of cells, central memory cells and / or naive cells, activated cells, tumor-specific cells and / or stem-like cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0334] For example, the reduced ratio of exhausted cells in the present invention can be + 、LAG-3 + 、TIM-3 + , and / or CD39 + For example, the ratio of regulatory cells decreased in the present invention can be an increase in the ratio of CD4 + CD25 + Foxp3 + For example, the ratio of apoptotic cells reduced in the present invention can be the ratio of CD95 + caspass3 + cells and / or CD95 + DR5 + A decrease in the proportion of cells.

[0335] For example, the proportion of exhausted cells, regulatory cells and / or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, or more. %, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced 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.

[0336] 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.

[0337] For example, the gene regulatory system can destroy the target gene at the DNA level. For example, the gene regulatory system can destroy the region or fragment of the target gene in the genome of the cell. For example, after using the gene regulatory system, the DNA region or fragment where the target gene is located in the cell is sheared, and the expression ability of the target gene is reduced or the activity of the target gene is inhibited. For example, the editing effect of the gene regulatory system on the target gene can be long-term and continuous. The genomic region of the present invention is determined based on the human reference genome version hg38.

[0338] 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.

[0339] For example, the present invention can reduce the expression and / or weaken the activity of at least one target gene of a cell by introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell. For example, the enzyme protein can include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the guide nucleic acid molecule can include a guide RNA (gRNA). For example, the guide nucleic acid molecule can include 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.

[0340] 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.

[0341] For example, the editing target region of the present invention may be a region preceding a promoter. 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 bindings. For example, the editing target region of the present invention may be a continuous region with approximately 3 or more transcription factor bindings.

[0342] 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.

[0343] For example, when the gene editing system includes CRISPR / Cas12, the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G. 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 downstream of the 3' end 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, 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.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] For example, the guide nucleic acid molecule can comprise a target sequence capable of binding to the 10 to about 30 nucleotides preceding the PAM region represented by AGG, TGG, GGG and / or CGG in the DNA encoding the ZC3H12 family member and / or its functionally active fragment. For example, the guide nucleic acid molecule can comprise a target sequence capable of binding to a gene encoding a ZC3H12 family member and / or a functionally active fragment thereof, comprising about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides preceding the PAM region represented by AGG, TGG, GGG, and / or CGG in DNA of the gene encoding the ZC3H12 family member and / or a functionally active fragment thereof.

[0350] For example, the target sequence can be selected from a region defined by the genomic coordinates shown in Table 1B, or a fragment thereof.

[0351] For example, the target sequence of the present invention can be the C3H1 type zinc finger structure functional domain of ZC3H12A.For example, the target sequence of the present invention can be R_22_chr1:37482635-37482714.

[0352] For example, the guide nucleic acid molecule can include a targeting domain that is complementary to a target sequence selected from the group consisting of SEQ ID NOs: 281-348, 3116-3698.

[0353] For example, the guide nucleic acid molecule can include a targeting domain, which can comprise a sequence as shown in SEQ ID NOs: 213-280, 2533-3115, 7325-7345, 7416, 7417.

[0354] For example, the guide nucleic acid molecule can include a targeting domain, which can comprise a sequence as shown in SEQ ID NOs: 7325-7345, 7416, 7417.

[0355] For example, compared to cells in which the expression and / or activity of at least one target gene of the cells 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.

[0356] For example, in the methods of the present invention, the proportion of cells expressing the product of the gene of interest is reduced by at least about 5% compared to cells in which the expression and / or activity of the gene of interest is unchanged. For example, the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%. For example, the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof can be reduced from the observed proportion of cells to 0%. For example, the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%. For example, the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof can be detected by flow cytometry.

[0357] For example, in the methods of the present invention, wherein the expression and / or activity of at least one target gene in the cells is reduced, the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof can be at most about 95%. For example, the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%. For example, the proportion of cells expressing the product of the gene encoding the ZC3H12 family member and / or the functionally active fragment thereof can be detected by flow cytometry.

[0358] For example, in the methods of the present invention, the expression and / or activity of at least one target gene in the cell is reduced and / or the activity is attenuated, and the expression of the target gene in a single cell can be reduced by at least about 5% compared to a cell 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%, 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 an observable amount to 0%. For example, the expression level of the gene of interest in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.

[0359] For example, in the method of the present invention, the expression level of the target gene in a single cell obtained by reducing the expression and / or attenuating the activity of at least one target gene in the cell can be up to about 95% of that in the cell in which the expression and / or activity of the target gene is not changed. For example, the expression level of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof (e.g., the gene encoding ZC3H12A) in a single cell may be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5% of that in a cell in which the expression and / or activity of the gene encoding the ZC3H12 family member and / or functionally active fragment thereof is not altered.

[0360] For example, the method of the present invention comprises: subjecting the cell to at least one stage of in vitro expansion, wherein, during at least one stage of in vitro expansion, the expression and / or activity of a ZC3H12 family member in the cell is reduced.

[0361] 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 ZC3H12 family members of the TILs expanded in vitro in the first stage are reduced.

[0362] 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.

[0363] For example, in a single stage of in vitro expansion of the present invention, the cells may be contacted with the one or more cell activators and the expression and / or activity of a ZC3H12 family member and / or a functionally active fragment thereof in the cells may be reduced. For example, the cell activator may comprise an agonist of one or more targets selected from the group consisting of 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 ZC3H12 family member in the cells of the present invention may be reduced and / or the activity may be attenuated, and the cells may be contacted with the one or more cell activators of the present invention. For example, in the first stage of in vitro expansion of the present invention, the TILs of the present invention may be contacted with the expression and / or activity of a ZC3H12 family member of the present invention and / or the activity may be attenuated, and the cells may be contacted with the one or more cell activators of the present invention. For example, in the second stage of in vitro expansion of the present invention, the expression and / or activity of a ZC3H12 family member in the TILs of the present invention may be reduced and / or the activity may be attenuated, and the cells may be contacted with the one or more cell activators of the present invention. For example, in the third stage of in vitro expansion of the present invention, the expression and / or activity of the ZC3H12 family members of the TIL of the present invention can be reduced and / or attenuated and the TIL can be contacted with one or more cell activators of the present invention.

[0364] For example, in a single stage of in vitro expansion of the present invention, the cells of the present invention can be exposed to reduced expression and / or attenuated activity of a ZC3H12 family member and one or more cell activators of the present invention substantially simultaneously. For example, in a single stage of in vitro expansion of the present invention, the cells of the present invention can be exposed to reduced expression and / or attenuated activity of a ZC3H12 family member, for example, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours prior to exposure, and then exposed to 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 exposed to one or more cell activators of the present invention, for example, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours prior to exposure, and then exposed to reduced expression and / or attenuated activity of a ZC3H12 family member.

[0365] For example, in the first stage of in vitro expansion of the present invention, the TILs of the present invention can be caused to substantially simultaneously reduce the expression and / or attenuate the activity of a ZC3H12 family member and contact 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 can be caused to substantially simultaneously reduce the expression and / or attenuate the activity of a ZC3H12 family member and contact 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 can be caused to substantially simultaneously reduce the expression and / or attenuate the activity of a ZC3H12 family member and contact one or more cell activators of the present invention.

[0366] 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, pleural effusion, and / or ascites and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and / or weakening the activity of ZC3H12 family members in the second TIL population; wherein, a third TIL population is obtained through step (B).

[0367] In the terms of 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 the terms of 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 the terms of 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 population obtained by step (A) in the method of the above aspect. In the terms of 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 population obtained by step (B) in the method of the above aspect. In the terms of 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 the terms of 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 population obtained by any additional step (C) in the method of the above aspect.

[0368] 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, pleural effusion, and / or ascites and not expanded in vitro with a plurality of cell growth factors; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with a plurality of cell growth factors, with a plurality of cell activators, reducing the expression and / or attenuating the activity of ZC3H12 family members, and co-culturing the TILs with feeder cells; wherein, a third TIL population is obtained through step (B).

[0369] 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, pleural effusion, and / or ascites and not expanded in vitro with a cell growth factor; wherein, step (A) obtains a second TIL population; (B) contacting the second TIL population with a cell growth factor, with a cell activator, reducing the expression and / or attenuating the activity of a ZC3H12 family member, and co-culturing the TILs with feeder cells, wherein the ZC3H12 family member may include ZC3H12A; wherein, step (B) obtains a third TIL population.

[0370] On the other hand, the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL). The method for obtaining TIL cells from a subject's tissue sample can be to obtain an in situ tumor sample or a metastatic tumor sample from a patient during surgery, which can weigh at least about 1g, or multiple tissues can be combined. Tumor tissue, pleural effusion and / or peritoneal effusion are transported in a sample transport fluid, such as a commercially available tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, at about 2-8 degrees Celsius 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, such as 6000 IU / mL). The cells in the culture medium are collected and transferred into a breathable culture bag, a Grex, or a Xuri device. The serum-free culture medium for the cells can be supplemented with the CD28 antibody, CD3 antibody, and CD28 antibody of the present invention, magnetic beads (e.g., Dynabeads) containing CD3 antibody and CD28 antibody, and / or nanomatrix (e.g., transACT) containing CD3 antibody and CD28 antibody, and IL-10 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL). 2 and reducing the expression and / or activity of ZC3H12 family members (ZC3H12 family members may include ZC3H12A, for example, by transducing with a ribonucleoprotein complex (RNP) containing the gRNA of the present invention and the Cas protein to make the proportion of cells encoding genes of ZC3H12 family members in the TIL be about 95% or less), after activating the TIL of the present invention for a certain period of time, adding irradiated PBMC (TIL to PBMC at a ratio of about 1:40-about 1:400), and expanding and culturing for about 3-14 days. The cells in the culture medium can be collected using a cell processing system, washed, frozen, and tested. The final product CD3 ratio can be greater than 80%, the cell viability can be greater than 50%, and greater than 80% of the cells can be memory effector cells and effector cells. After stimulation, IFN-γ can be secreted, and / or it can have the characteristic of an increased proportion of activated cells.

[0371] SOCS1 knockout

[0372] 1. A method for culturing cells, comprising: reducing the expression and / or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof in the cells.

[0373] 2. The method of embodiment 1, wherein the cells comprise immune cells.

[0374] 3. The method according to embodiment 2, wherein the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0375] 4. The method of any one of embodiments 2-3, wherein the immune cells comprise monocytes, macrophages and / or dendritic cells.

[0376] 5. The method according to any one of embodiments 2-4, wherein the immune cells are derived from immune cells differentiated from stem cells.

[0377] 6. The method of embodiment 5, wherein the stem cells comprise induced pluripotent stem cells (iPSCs).

[0378] 7. The method of any one of embodiments 2-6, wherein the immune cells comprise B cells, T cells, natural killer cells and / or natural killer-like T cells (NKT).

[0379] 8. A method according to any one of embodiments 2-7, wherein the immune cells comprise αβT cells and / or γδT cells.

[0380] 9. A method according to any one of embodiments 2-8, wherein the immune cells comprise tumor infiltrating lymphocytes (TILs).

[0381] 10. The method according to embodiment 9, wherein the TILs are derived from fragments of tumor tissue, TILs from pleural effusion and / or ascites and / or TILs revived after cryopreservation.

[0382] 11. The method of embodiment 10, wherein the fragments have a volume of about 1 cubic millimeter to about 27 cubic millimeters.

[0383] 12. The method of any one of embodiments 2-11, wherein the immune cell comprises an engineered immune receptor displayed on the cell surface.

[0384] 13. A method according to embodiment 12, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell.

[0385] 14. The method of any one of embodiments 2-13, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0386] 15. The method according to any one of embodiments 1-14, wherein reducing the expression and / or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member of the cell comprises inhibiting the function of negative regulation of cytokine signaling.

[0387] 16. The method of any one of embodiments 1-15, wherein cells obtained by reducing the expression and / or attenuating the STAT-induced STAT inhibitor (SSI) family member exhibit improved cellular properties compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not altered.

[0388] 17. A method according to embodiment 16, wherein the improved cell characteristics include one or more selected from the following groups: improved cell proliferation ability, increased proportion of living cells, improved proportion of cell subpopulations, increased cytokine secretion ability and improved tumor cell killing ability.

[0389] 18. A method according to embodiment 17, wherein 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.

[0390] 19. The method of any one of embodiments 1-18, wherein the STAT-induced STAT inhibitor (SSI) family member comprises an SH2 domain.

[0391] 20. The method of any one of embodiments 1-19, wherein the STAT-induced STAT inhibitor (SSI) family member comprises SOCS1.

[0392] 21. The method of any one of embodiments 1-20, wherein reducing the expression and / or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member in the cell comprises introducing a gene regulatory system into the cell.

[0393] 22. The method of embodiment 21, wherein the gene regulatory system is capable of disrupting the STAT-induced STAT inhibitor (SSI) family member at the DNA level.

[0394] 23. The method of any one of embodiments 21-22, wherein the gene regulatory system comprises a guide nucleic acid molecule and an enzyme protein.

[0395] 24. A method according to embodiment 23, wherein reducing the expression and / or weakening the activity of the STAT-induced STAT inhibitor (SSI) family member comprises: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and an encoding a Cas protein into the cell.

[0396] 25. The method of any one of embodiments 23-24, wherein the enzyme protein comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

[0397] 26. The method of any one of embodiments 23-25, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA).

[0398] 27. The method of any one of embodiments 23-26, wherein the guide nucleic acid molecule is capable of binding to a sequence of the STAT-induced STAT inhibitor (SSI) family member.

[0399] 28. The method of any one of embodiments 23-27, wherein the guide nucleic acid molecule is capable of binding to a region defined by the genomic coordinates selected from those shown in Table 1C, or a fragment thereof.

[0400] 29. The method of any one of embodiments 23-28, wherein the guide nucleic acid molecule is capable of binding to a region or fragment thereof selected from the group consisting of SEQ ID NOs: 399-448, 4393-5086.

[0401] 30. A method according to any one of embodiments 23-29, wherein the guide nucleic acid molecule is capable of binding 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, CGG and GGG.

[0402] 31. The method of any one of embodiments 23-30, wherein the guide nucleic acid molecule comprises a targeting domain comprising a sequence as shown in any one of SEQ ID NOs: 349-398, 3699-4392, 7346-7375, 7418.

[0403] 32. A method according to any one of embodiments 1-31, wherein the proportion of cells expressing the product of the target gene is reduced and / or the expression level of the target gene in a single cell is decreased in the cells obtained by reducing the expression and / or attenuating the STAT-induced STAT inhibitor (SSI) family member compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not changed.

[0404] 33. A method according to any one of embodiments 1-32, wherein the proportion of cells expressing the target gene in the cells obtained by reducing the expression and / or attenuating the activity of the STAT-induced STAT inhibitor (SSI) family member is about 95% or less.

[0405] 34. A cell obtained by the method of any one of embodiments 1-33.

[0406] 35. A composition comprising the cell of embodiment 34.

[0407] 36. A pharmaceutical composition comprising the cell of embodiment 34 and / or the composition of embodiment 35, and optionally a pharmaceutically acceptable carrier.

[0408] 37. A method of influencing cell growth comprising administering the cell of embodiment 34, the composition of embodiment 35, and / or the pharmaceutical composition of embodiment 36.

[0409] 38. Use of the cell of embodiment 34, the composition of embodiment 35, and / or the pharmaceutical composition of embodiment 36 in the preparation of a medicament for preventing and / or treating a disease and / or symptom.

[0410] 39. The use according to embodiment 38, wherein the disease and / or condition comprises a tumor.

[0411] 40. The use according to any one of embodiments 38-39, wherein the disease and / or condition comprises a solid tumor.

[0412] 41. The use according to any one of embodiments 38-40, wherein the disease and / or symptoms comprise 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.

[0413] The present invention provides a method for reducing the expression and / or attenuating the activity of a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof in the cell.

[0414] In one aspect, the present invention provides a method for culturing cells to reduce the expression and / or attenuate the activity of a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof. For example, the STAT-induced STAT inhibitor (SSI) family member may comprise an SH2 domain. For example, the STAT-induced STAT inhibitor (SSI) family member may comprise SOCS1.

[0415] For example, the target gene of the present invention can be a gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof. For example, compared to cells in which the expression and / or activity of the target gene is unchanged, the cells obtained by reducing the expression and / or weakening the activity of at least one target gene of the cell can show improved cell characteristics. In one embodiment, the cells in which the expression and / or activity of the target gene is unchanged can refer to cells derived from the same donor and in which the expression and / or activity of at least one target gene of the cell is not reduced and / or weakened. In one embodiment, the cells in which the expression and / or activity of the target gene is unchanged can refer to cells derived from the same donor and in which the expression and / or activity of other genes other than the target gene of the cell (e.g., knocking out the other gene has substantially no effect on cell function) is not reduced and / or weakened.

[0416] For example, the cells include immune cells. For example, the cells include immune effector cells. For example, the cells include immune effector T cells, immune effector NK cells, immune effector NKT cells. For example, the cells include phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0417] For example, the cells comprise monocytes, macrophages and / or dendritic cells.

[0418] 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.

[0419] 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."

[0420] For example, the cell comprises a B cell, a T cell, a natural killer cell and / or a natural killer-like T cell (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, a non-targeted gRNA, an interfering siRNA, etc.). For example, the cell comprises an αβT cell and / or a γδT cell. For example, the cell comprises a tumor infiltrating lymphocyte (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.

[0421] 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.

[0422] 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.

[0423] In one aspect, the present invention provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: reducing the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or functionally active fragments thereof in the TIL.

[0424] For example, TILs derived from tumor tissue, pleural effusion and / or ascites and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or functionally active fragments thereof contained in the TILs can be reduced.

[0425] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0426] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0427] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0428] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0429] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0430] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0431] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0432] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0433] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TILs can be reduced.

[0434] For example, the TIL of the present invention that is derived from tumor tissue, pleural effusion and / or ascites and has not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion, and in the first stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TIL can be reduced, and in the second stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TIL can be reduced, and in the third stage of in vitro expansion of the present invention, the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or their functionally active fragments in the TIL can be reduced.

[0435] For example, cells obtained by reducing the expression and / or attenuating the STAT-induced STAT inhibitor (SSI) family member exhibit improved cell properties compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not altered.

[0436] For example, improved cell number of the present invention means that the cell number of the cells of the present invention in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is reduced in at least one in vitro expansion stage can be increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold, compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not altered.

[0437] For example, an increased proportion of live cells can be expressed as an increase in cell viability. For example, an increased proportion of live cells in the present invention can refer to a decrease in the expression and / or activity of a STAT-induced STAT inhibitor (SSI) family member in at least one in vitro expansion stage compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not changed. The proportion of viable cells of the cells of the invention with or without reduced activity can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0438] For example, the improved cytokine secretion capacity of the present invention can refer to the improvement of the cytokine secretion capacity of the cell 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 improvement of the cytokine secretion capacity of the cell 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 the STAT inhibitor (SSI) family member of STAT induction compared to the cell in which the expression and / or activity of the STAT inhibitor (SSI) family member of STAT induction is not changed, and the cell ratio of the cell secreting cytokine in the cell of the present invention in which the expression and / or activity of the STAT inhibitor (SSI) family member of the STAT induction is reduced and / or the activity is weakened in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times. For example, the improved cytokine secretion capacity of the present invention may mean that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, or more compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not changed. 0%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0439] For example, the improved tumor cell killing ability of the present invention can mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is reduced in at least one in vitro expansion stage can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not changed. For example, the improved tumor cell killing ability of the present invention can mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is reduced in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, compared to cells in which the expression and / or activity of the STAT-induced STAT inhibitor (SSI) family member is not changed. , at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%. For example, the tumor cell killing rate of the cells of the present invention can be measured by the IncuCyte system or CFSE and DAPI staining. For example, tumor cell killing of the cells of the present invention can refer to the ability of the cells to kill solid tumor cells.

[0440] For example, the improved cell subpopulation ratio of the present invention may comprise one or more selected from the following groups: increased CD8 + cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of regulatory cells, an increased proportion of activated cells, an increased proportion of tumor-specific cells, and an increased proportion of stem-like cells.

[0441] For example, in cells CD8 +The proportion of cells, central memory cells and / or naive cells, activated cells, tumor-specific cells and / or stem-like cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0442] For example, the reduced ratio of exhausted cells in the present invention can be + 、LAG-3 + 、TIM-3 + , and / or CD39 + For example, the ratio of regulatory cells decreased in the present invention can be an increase in the ratio of CD4 + CD25 + Foxp3 + For example, the ratio of apoptotic cells reduced in the present invention can be the ratio of CD95 + caspass3 + cells and / or CD95 + DR5 + A decrease in the proportion of cells.

[0443] For example, the proportion of exhausted cells, regulatory cells and / or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 19%, at least about 18 ... The amount of the active ingredient in the present invention may be about 4% less, at least about 3% less, at least about 2% less, at least about 1% less, at least about 0.5% less, at least about 0.4% less, at least about 0.3% less, at least about 0.2% less, or at least about 0.1% less, or may be at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold less.

[0444] 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.

[0445] For example, the gene regulatory system can destroy the target gene at the DNA level. For example, the gene regulatory system can destroy the region or fragment of the target gene in the genome of the cell. For example, after using the gene regulatory system, the DNA region or fragment where the target gene is located in the cell is sheared, and the expression ability of the target gene is reduced or the activity of the target gene is inhibited. For example, the editing effect of the gene regulatory system on the target gene can be long-term and continuous. The genomic region of the present invention is determined based on the human reference genome version hg38.

[0446] 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.

[0447] For example, the present invention can reduce the expression and / or weaken the activity of at least one target gene of a cell by introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell. For example, the enzyme protein can include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the guide nucleic acid molecule can include a guide RNA (gRNA). For example, the guide nucleic acid molecule can include 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.

[0448] 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.

[0449] For example, the editing target region of the present invention may be a region preceding a promoter. 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 bindings. For example, the editing target region of the present invention may be a continuous region with approximately 3 or more transcription factor bindings.

[0450] 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.

[0451] For example, when the gene editing system includes CRISPR / Cas12, the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G. 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 downstream of the 3' end 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, 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.

[0452] 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.

[0453] 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.

[0454] 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.

[0455] 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.

[0456] 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.

[0457] For example, the guide nucleic acid molecule can comprise a target sequence consisting of about 10 to about 30 nucleotides before the PAM region represented by AGG, TGG, GGG and / or CGG in the DNA encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof. For example, the guide nucleic acid molecule can comprise a target sequence that is capable of binding to a gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof, and consisting of about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides preceding the PAM region represented by AGG, TGG, GGG and / or CGG.

[0458] For example, the target sequence can be selected from a region defined by the genomic coordinates shown in Table 1C, or a fragment thereof.

[0459] For example, the guide nucleic acid molecule can include a targeting domain that is complementary to a target sequence selected from the group consisting of SEQ ID NOs: 399-448, 4393-5086.

[0460] For example, the guide nucleic acid molecule can include a targeting domain, which can comprise a sequence as shown in SEQ ID NOs: 349-398, 3699-4392, 7346-7375, 7418.

[0461] For example, the guide nucleic acid molecule can include a targeting domain, which can comprise a sequence as shown in SEQ ID NOs: 7346-7375, 7418.

[0462] For example, compared to cells in which the expression and / or activity of at least one target gene of the cells 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.

[0463] For example, in the methods of the present invention, the proportion of cells expressing the product of the gene of interest is reduced by at least about 5% compared to cells in which the expression and / or activity of the gene of interest is unchanged. For example, the proportion of cells expressing the product of a gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%. For example, the proportion of cells expressing the product of the gene encoding a STAT-inducible STAT inhibitor (SSI) family member and / or a functionally active fragment thereof can be decreased from the proportion of cells that can be observed to 0%. For example, the proportion of cells expressing the product of the gene encoding a STAT-inducible STAT inhibitor (SSI) family member and / or a functionally active fragment thereof can be decreased to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%. For example, the proportion of cells expressing the product of the gene encoding the STAT-induced STAT inhibitor (SSI) family member and / or its functionally active fragment can be detected by flow cytometry.

[0464] For example, in the method of the present invention, the expression of at least one target gene in the cells is reduced and / or the activity is attenuated, and the proportion of cells expressing the product of the gene encoding the STAT-induced STAT inhibitor (SSI) family member and / or its functionally active fragment can be up to about 95%. For example, the proportion of cells expressing the product of the gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%. For example, the proportion of cells expressing the product of the gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof can be detected by flow cytometry.

[0465] For example, in the methods of the present invention, the expression and / or activity of at least one target gene in the cell is reduced and / or the activity is attenuated, and the expression of the target gene in a single cell can be reduced by at least about 5% compared to a cell 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%, 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 an observable amount to 0%. For example, the expression level of the gene of interest in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.

[0466] For example, in the method of the present invention, the expression level of the target gene in a single cell obtained by reducing the expression and / or attenuating the activity of at least one target gene in the cell can be up to about 95% of that in the cell in which the expression and / or activity of the target gene is not changed. For example, the expression level of the gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof (e.g., a gene encoding SOCS1) in a single cell can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5% of the expression and / or activity of the gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof in a cell in which the expression and / or activity is not altered.

[0467] For example, the method of the present invention comprises: subjecting the cells to at least one stage of in vitro expansion, wherein, during at least one stage of in vitro expansion, the expression and / or activity of a STAT-induced STAT inhibitor (SSI) family member of the cells is reduced.

[0468] For example, the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and / or peritoneal effusion and not expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion, the expression of STAT-induced STAT inhibitor (SSI) family members of the TILs expanded in vitro in the first stage is reduced and / or the activity is attenuated.

[0469] 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.

[0470] For example, the cells can be contacted with the one or more cell activators and the expression and / or activity of STAT-induced STAT inhibitor (SSI) family members and / or functionally active fragments thereof in the cells can be reduced during a single stage of in vitro expansion of the present invention. 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 STAT-induced STAT inhibitor (SSI) family members of the cells of the present invention are reduced and / or weakened and 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 STAT-induced STAT inhibitor (SSI) family members of the TILs of the present invention can be reduced and / or weakened and contacted 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 expression and / or activity of STAT-induced STAT inhibitor (SSI) family members of the TILs of the present invention can be reduced and / or weakened and contacted 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 expression and / or activity of STAT-induced STAT inhibitor (SSI) family members of the TILs of the present invention can be reduced and / or attenuated and contacted with one or more cell activators of the present invention.

[0471] For example, in a single stage of in vitro expansion of the present invention, the cells of the present invention can be exposed to reduced expression and / or attenuated activity of a STAT-induced STAT inhibitor (SSI) family member 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 exposed to reduced expression and / or attenuated activity of a STAT-induced STAT inhibitor (SSI) family member, for example, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours in advance, etc., and then exposed to 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 exposed to one or more cell activators of the present invention, for example, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours in advance, etc., and then exposed to reduced expression and / or attenuated activity of a STAT-induced STAT inhibitor (SSI) family member.

[0472] For example, in the first stage of in vitro expansion of the present invention, the TILs of the present invention can be made to reduce the expression and / or weaken the activity of STAT-induced STAT inhibitor (SSI) family members and contact one or more cell activators of the present invention at 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 made to reduce the expression and / or weaken the activity of STAT-induced STAT inhibitor (SSI) family members and contact one or more cell activators of the present invention at 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 made to reduce the expression and / or weaken the activity of STAT-induced STAT inhibitor (SSI) family members and contact one or more cell activators of the present invention at the same time.

[0473] On the other hand, 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, pleural effusion and / or ascites and not expanded in vitro with one or more cell growth factors; wherein, a second TIL population is obtained through step (A); (B) reducing the expression and / or weakening the activity of STAT-induced STAT inhibitor (SSI) family members in the second TIL population; wherein, a third TIL population is obtained through step (B).

[0474] In the terms of 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 the terms of 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 the terms of 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 population obtained by step (A) in the method of the above aspect. In the terms of 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 population obtained by step (B) in the method of the above aspect. In the terms of 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 the terms of 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 population obtained by any additional step (C) in the method of the above aspect.

[0475] On the other hand, 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, pleural effusion and / or ascites and not expanded in vitro with multiple cell growth factors; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with multiple cell growth factors, multiple cell activators, reducing the expression and / or attenuating the activity of STAT-induced STAT inhibitor (SSI) family members, and co-culturing the TILs with feeder cells; wherein, a third TIL population is obtained through step (B).

[0476] On the other hand, 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, pleural effusion and / or ascites and not expanded in vitro with a cell growth factor; wherein, a second TIL population is obtained through step (A); (B) contacting the second TIL population with a cell growth factor, with a cell activator, reducing the expression and / or weakening the activity of a STAT-induced STAT inhibitor (SSI) family member, and co-culturing the TILs with feeder cells, wherein the STAT-induced STAT inhibitor (SSI) family member may include SOCS1; wherein, a third TIL population is obtained through step (B).

[0477] On the other hand, the present invention provides a method for culturing tumor infiltrating lymphocytes (TIL). The method for obtaining TIL cells from a subject's tissue sample can be to obtain an in situ tumor sample or a metastatic tumor sample from a patient during surgery, which can weigh at least about 1g, or multiple tissues can be combined. Tumor tissue, pleural effusion and / or peritoneal effusion are transported in a sample transport fluid, such as a commercially available tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, at about 2-8 degrees Celsius 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, such as 6000 IU / mL). The cells in the culture medium are collected and transferred into a breathable culture bag, a Grex, or a Xuri device. The serum-free culture medium of the cells can be supplemented with the CD28 antibody, CD3 antibody, and CD28 antibody of the present invention, magnetic beads (e.g., Dynabeads) containing CD3 antibody and CD28 antibody, and / or nanomatrix (e.g., transACT) containing CD3 antibody and CD28 antibody, IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL), and a STAT inhibitor that induces STAT. The expression and / or activity of (SSI) family members are reduced (STAT-induced STAT inhibitor (SSI) family members can include SOCS1, for example, by transducing with a ribonucleoprotein complex (RNP) containing the gRNA of the present invention and the Cas protein to form a STAT-induced STAT inhibitor (SSI) family member so that the proportion of cells encoding the gene of the STAT-induced STAT inhibitor (SSI) family member in the TIL is about 95% or less), after activating the TIL of the present invention for a certain period of time, irradiated PBMCs are added (TIL and PBMC are at a ratio of about 1:40 to about 1:400), and the cells are expanded and cultured for about 3-14 days. A cell processing system can be used to collect cells in the culture medium, wash, freeze, and detect. The final product CD3 ratio can be greater than 80%, the cell viability can be greater than 50%, and greater than 80% of the cells can be memory effector cells and effector cells. After stimulation, IFN-γ can be secreted, and / or it can have the characteristic of an increased proportion of activated cells.

[0478] CBLB knockout

[0479] 1. A method for culturing cells, comprising: reducing the expression and / or attenuating the activity of a CBL family member and / or a functionally active fragment thereof in the cells.

[0480] 2. The method of embodiment 1, wherein the cells comprise immune cells.

[0481] 3. The method according to embodiment 2, wherein the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0482] 4. The method of any one of embodiments 2-3, wherein the immune cells comprise monocytes, macrophages and / or dendritic cells.

[0483] 5. The method according to any one of embodiments 2-4, wherein the immune cells are derived from immune cells differentiated from stem cells.

[0484] 6. The method of embodiment 5, wherein the stem cells comprise induced pluripotent stem cells (iPSCs).

[0485] 7. The method of any one of embodiments 2-6, wherein the immune cells comprise B cells, T cells, natural killer cells and / or natural killer-like T cells (NKT).

[0486] 8. A method according to any one of embodiments 2-7, wherein the immune cells comprise αβT cells and / or γδT cells.

[0487] 9. A method according to any one of embodiments 2-8, wherein the immune cells comprise tumor infiltrating lymphocytes (TILs).

[0488] 10. The method according to embodiment 9, wherein the TILs are derived from fragments of tumor tissue, TILs from pleural effusion and / or ascites and / or TILs revived after cryopreservation.

[0489] 11. The method of embodiment 10, wherein the fragments have a volume of about 1 cubic millimeter to about 27 cubic millimeters.

[0490] 12. The method of any one of embodiments 2-11, wherein the immune cell comprises an engineered immune receptor displayed on the cell surface.

[0491] 13. A method according to embodiment 12, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell.

[0492] 14. The method of any one of embodiments 2-13, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0493] 15. The method according to any one of embodiments 1-14, wherein reducing the expression and / or attenuating the activity of a CBL family member in the cell comprises inhibiting the function of an E3 ubiquitin protein ligase.

[0494] 16. The method of any one of embodiments 1-15, wherein the cells obtained by reducing the expression and / or attenuating the activity of the CBL family member exhibit improved cellular properties compared to cells in which the expression and / or activity of the CBL family member is not altered.

[0495] 17. A method according to embodiment 16, wherein the improved cell characteristics include one or more selected from the following groups: improved cell proliferation ability, increased proportion of living cells, improved proportion of cell subpopulations, increased cytokine secretion ability and improved tumor cell killing ability.

[0496] 18. A method according to embodiment 17, wherein 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.

[0497] 19. The method of any one of embodiments 1-18, wherein the CBL family member comprises an SH3 domain.

[0498] 20. The method of any one of embodiments 1-19, wherein the CBL family member comprises CBLB.

[0499] 21. The method of any one of embodiments 1-20, wherein reducing the expression and / or attenuating the activity of a CBL family member in the cell comprises introducing a gene regulatory system into the cell.

[0500] 22. The method of embodiment 21, wherein the gene regulatory system is capable of disrupting the CBL family member at the DNA level.

[0501] 23. The method of any one of embodiments 21-22, wherein the gene regulatory system comprises a guide nucleic acid molecule and an enzyme protein.

[0502] 24. The method according to embodiment 23, wherein reducing the expression and / or attenuating the activity of the CBL family member comprises: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and a Cas protein into the cell.

[0503] 25. The method of any one of embodiments 23-24, wherein the enzyme protein comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

[0504] 26. The method of any one of embodiments 23-25, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA).

[0505] 27. The method of any one of embodiments 23-26, wherein the guide nucleic acid molecule is capable of binding to a sequence of the CBL family member.

[0506] 28. The method of any one of embodiments 23-27, wherein the guide nucleic acid molecule is capable of binding to a region defined by the genomic coordinates selected from those shown in Table 1D, or a fragment thereof.

[0507] 29. The method of any one of embodiments 23-28, wherein the guide nucleic acid molecule is capable of binding to a region or fragment thereof selected from the group consisting of SEQ ID NOs: 520-590, 6177-7266.

[0508] 30. A method according to any one of embodiments 23-29, wherein the guide nucleic acid molecule is capable of binding 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, CGG and GGG.

[0509] 31. The method of any one of embodiments 23-30, wherein the guide nucleic acid molecule comprises a targeting domain comprising a sequence as shown in any one of SEQ ID NOs: 449-519, 5087-6176, 7376-7413, 7414, 7415.

[0510] 32. The method according to any one of embodiments 1 to 31, wherein the proportion of cells expressing the product of the target gene is reduced and / or the expression level of the target gene in individual cells is decreased, compared to cells in which the expression and / or activity of the CBL family member is not altered.

[0511] 33. The method according to any one of embodiments 1 to 32, wherein the proportion of cells expressing the target gene among the cells obtained by reducing the expression and / or attenuating the activity of the CBL family member is about 95% or less.

[0512] 34. A cell obtained by the method of any one of embodiments 1-33.

[0513] 35. A composition comprising the cell of embodiment 34.

[0514] 36. A pharmaceutical composition comprising the cell of embodiment 34 and / or the composition of embodiment 35, and optionally a pharmaceutically acceptable carrier.

[0515] 37. A method of influencing cell growth comprising administering the cell of embodiment 34, the composition of embodiment 35, and / or the pharmaceutical composition of embodiment 36.

[0516] 38. Use of the cell of embodiment 34, the composition of embodiment 35, and / or the pharmaceutical composition of embodiment 36 in the preparation of a medicament for preventing and / or treating a disease and / or symptom.

[0517] 39. The use according to embodiment 38, wherein the disease and / or condition comprises a tumor.

[0518] 40. The use according to any one of embodiments 38-39, wherein the disease and / or condition comprises a solid tumor.

[0519] 41. The use according to any one of embodiments 38-40, wherein the disease and / or symptoms comprise 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.

[0520] The present invention provides a method for reducing the expression and / or attenuating the activity of CBL family members and / or functionally active fragments thereof in the cells.

[0521] In one aspect, the present invention provides a method for culturing cells to reduce the expression and / or attenuate the activity of a CBL family member and / or a functionally active fragment thereof in the cells. For example, the CBL family member may comprise an SH3 domain. For example, the CBL family member may comprise CBLB.

[0522] For example, the target gene of the present invention can be a gene encoding a CBL family member and / or a functionally active fragment thereof. For example, a cell obtained by reducing the expression and / or attenuating the activity of at least one target gene in the cell can exhibit improved cellular properties compared to a cell in which the expression and / or activity of the target gene is unchanged. In one embodiment, a cell in which the expression and / or activity of the target gene is unchanged can refer to a cell derived from the same donor in which the expression and / or activity of at least one target gene in the cell has not been reduced and / or attenuated. In one embodiment, a cell in which the expression and / or activity of the target gene is unchanged can refer to a cell derived from the same donor in which the expression and / or activity of another gene other than the target gene in the cell has not been reduced and / or attenuated (e.g., knocking out the other gene has no substantial effect on cellular function).

[0523] For example, the cells include immune cells. For example, the cells include immune effector cells. For example, the cells include immune effector T cells, immune effector NK cells, immune effector NKT cells. For example, the cells include phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0524] For example, the cells comprise monocytes, macrophages and / or dendritic cells.

[0525] 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.

[0526] 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."

[0527] 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.

[0528] 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.

[0529] 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.

[0530] In one aspect, the present invention provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: reducing the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TIL.

[0531] For example, TILs derived from tumor tissue, pleural effusion, and / or ascites and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, during the at least one stage of in vitro expansion, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0532] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion. In the second stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0533] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion. During the first stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0534] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced. In the second stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0535] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion, and a third stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0536] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion and a third stage of in vitro expansion. In the second stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0537] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion, and a third stage of in vitro expansion. In the third stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0538] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion, and a third stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced. In the second stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0539] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion, and a third stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced. In the third stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0540] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion, and a third stage of in vitro expansion. In the second stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced. In the third stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0541] For example, the TILs of the present invention that are derived from tumor tissue, pleural effusion, and / or ascites and have not been expanded in vitro can be subjected to a first stage of in vitro expansion, a second stage of in vitro expansion, and a third stage of in vitro expansion. In the first stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced. In the second stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced. In the third stage of in vitro expansion of the present invention, the expression and / or activity of CBL family members and / or functionally active fragments thereof in the TILs can be reduced.

[0542] For example, cells obtained by reducing the expression and / or attenuating the activity of a CBL family member exhibit improved cellular properties compared to cells in which the expression and / or activity of the CBL family member is not altered.

[0543] For example, improved cell number of the present invention means that the cell number of the cells of the present invention having reduced expression and / or attenuated activity of a CBL family member in at least one stage of in vitro expansion can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to cells in which the expression and / or activity of the CBL family member is not altered.

[0544] For example, an increased proportion of live cells can be expressed as an increase in cell viability. For example, an increased proportion of live cells of the present invention can mean that the proportion of live cells of the cells of the present invention in which the expression and / or activity of the CBL family member is reduced during at least one in vitro expansion stage is increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 30%, or at least about 40%. At least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0545] For example, the improved cytokine secretion capacity of the present invention may refer to an improved cytokine secretion capacity of the cells selected from the group consisting of IL-2, IL-6, CD107a, GZMB, TNF-α, and IFN-γ. For example, the improved cytokine secretion capacity of the present invention may refer to an increase in the proportion of cells secreting cytokines in the cells of the present invention in which the expression and / or activity of the CBL family member is reduced and / or the activity is attenuated during at least one in vitro expansion stage compared to cells in which the expression and / or activity of the CBL family member is not altered, 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. For example, the improved cytokine secretion capacity of the present invention may mean that the proportion of cells secreting cytokines in the cells of the present invention in which the expression and / or activity of the CBL family member is reduced and / or the activity is attenuated during at least one in vitro expansion stage is increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, compared to cells in which the expression and / or activity of the CBL family member is not changed. , at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0546] For example, the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the CBL family member is reduced and / or the activity is attenuated during at least one in vitro expansion stage is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold, compared to cells in which the expression and / or activity of the CBL family member is not altered. For example, the improved tumor cell killing ability of the present invention may mean that the tumor cell killing rate of the cells of the present invention in which the expression and / or activity of the CBL family member is reduced and / or the activity is attenuated in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about In some embodiments, the cells of the present invention can kill at least about 1% of the cells of the present invention, or at least about 1% of the cells of the present invention. In some embodiments, the cells of the present invention can kill at least about 1% of the cells of the present invention, or at least about 1% of the cells of the present invention. In some embodiments, the cells of the present invention can kill at least about 1% of the cells of the present invention, or at least about 1% of the cells of the present invention. In some embodiments, the cells of the present invention can kill at least about 1% of the cells of the present invention, or at least about 1% of the cells of the present invention. In some embodiments, the cells of the present invention can kill at least about 1% of the cells of the present invention, or at least about 1% of the cells of the present invention.

[0547] For example, the improved cell subpopulation ratio of the present invention may comprise one or more selected from the following groups: increased CD8 + cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of regulatory cells, an increased proportion of activated cells, an increased proportion of tumor-specific cells, and an increased proportion of stem-like cells.

[0548] For example, in cells CD8 +The proportion of cells, central memory cells and / or naive cells, activated cells, tumor-specific cells and / or stem-like cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.

[0549] For example, the reduced ratio of exhausted cells in the present invention can be + 、LAG-3 + 、TIM-3 + , and / or CD39 + For example, the ratio of regulatory cells decreased in the present invention can be an increase in the ratio of CD4 + CD25 + Foxp3 + For example, the ratio of apoptotic cells reduced in the present invention can be the ratio of CD95 + caspass3 + cells and / or CD95 + DR5 + A decrease in the proportion of cells.

[0550] For example, the proportion of exhausted cells, regulatory cells and / or apoptotic cells in a cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, or more. %, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced 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.

[0551] 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.

[0552] For example, the gene regulatory system can destroy the target gene at the DNA level. For example, the gene regulatory system can destroy the region or fragment of the target gene in the genome of the cell. For example, after using the gene regulatory system, the DNA region or fragment where the target gene is located in the cell is sheared, and the expression ability of the target gene is reduced or the activity of the target gene is inhibited. For example, the editing effect of the gene regulatory system on the target gene can be long-term and continuous. The genomic region of the present invention is determined based on the human reference genome version hg38.

[0553] 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.

[0554] For example, the present invention can reduce the expression and / or weaken the activity of at least one target gene of a cell by introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell. For example, the enzyme protein can include a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the guide nucleic acid molecule can include a guide RNA (gRNA). For example, the guide nucleic acid molecule can include 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.

[0555] 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.

[0556] For example, the editing target region of the present invention may be a region preceding a promoter. 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 bindings. For example, the editing target region of the present invention may be a continuous region with approximately 3 or more transcription factor bindings.

[0557] 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.

[0558] For example, when the gene editing system includes CRISPR / Cas12, the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G. 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 downstream of the 3' end 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, 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.

[0559] 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.

[0560] 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.

[0561] 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.

[0562] 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.

[0563] 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.

[0564] For example, the guide nucleic acid molecule can comprise a target sequence capable of binding to a PAM region of about 10 to about 30 nucleotides preceding the AGG, TGG, GGG and / or CGG region in the DNA encoding a CBL family member and / or a functionally active fragment thereof. For example, the guide nucleic acid molecule can comprise a target sequence capable of binding to a gene encoding a CBL family member and / or a functionally active fragment thereof, comprising about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides preceding the PAM region represented by AGG, TGG, GGG, and / or CGG.

[0565] For example, the target sequence can be a region or fragment thereof defined by the genomic coordinates shown in Table ID. For example, the target sequence of the present invention can be the PTB structural and functional domain of CBLB. For example, the target sequence of the present invention can be the zinc finger stru...

Claims

1. A method for culturing cells, comprising: reducing the expression and / or attenuating the activity of a peptidase C64 family member and / or a functionally active fragment thereof in the cells.

2. The method of 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 to 4, wherein the immune cells are derived from immune cells differentiated from stem cells. The method of claim 5 , wherein the stem cells comprise induced pluripotent stem cells (iPSCs).

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

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

10. The method according to claim 9, wherein the TILs are derived from tumor tissue fragments, pleural effusion and / or ascites and / or TILs revived after cryopreservation.

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

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

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

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

15. The method according to any one of claims 1 to 14, wherein reducing the expression and / or attenuating the activity of a peptidase C64 family member in the cell comprises inhibiting the function of a deubiquitinating enzyme and / or a zinc finger nuclease.

16. The method according to any one of claims 1 to 15, wherein the cells obtained by reducing the expression and / or attenuating the activity of the peptidase C64 family member show improved cell properties compared to cells in which the expression and / or activity of the peptidase C64 family member is not altered.

17. The method according to claim 16, wherein the improved cell properties comprise one or more selected from the group consisting of improved cell proliferation ability, increased proportion of living cells, improved proportion of cell subpopulations, enhanced cytokine secretion ability, and enhanced tumor cell killing ability.

18. The method according to claim 17, wherein 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.

19. The method of any one of claims 1-18, wherein the peptidase C64 family member comprises a ubiquitin binding domain.

20. The method of any one of claims 1-19, wherein the peptidase C64 family member comprises TNFAIP3.

21. The method according to any one of claims 1 to 20, wherein reducing the expression and / or attenuating the activity of a member of the peptidase C64 family in the cell comprises introducing a gene regulatory system into the cell.

22. The method of claim 21, wherein the gene regulatory system is capable of disrupting the peptidase C64 family member at the DNA level.

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

24. The method of claim 23, wherein reducing the expression and / or attenuating the activity of the peptidase C64 family member comprises: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein, an LNP comprising a gRNA and a Cas protein, or an LNP comprising a nucleic acid encoding a gRNA and a Cas protein into the cell.

25. The method of any one of claims 23-24, wherein the enzyme protein comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

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

27. The method of any one of claims 23-26, wherein the guide nucleic acid molecule is capable of binding to the sequence of the peptidase C64 family member.

28. The method of any one of claims 23-27, wherein the guide nucleic acid molecule is capable of binding to a region defined by the genomic coordinates selected from those shown in Table 1A, or a fragment thereof.

29. The method of any one of claims 23-28, wherein the guide nucleic acid molecule is capable of binding to a region or a fragment thereof selected from the group consisting of SEQ ID NOs: 107-212, 1562-2532.

30. The method of any one of claims 23-29, wherein the guide nucleic acid molecule is capable of binding 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, CGG, and GGG.

31. The method of any one of claims 23-30, wherein the guide nucleic acid molecule comprises a targeting domain comprising a sequence as shown in any one of SEQ ID NOs: 1-106, 591-1561, 7267-7324, 7419, 7420.

32. The method according to any one of claims 1 to 31, wherein the proportion of cells expressing the product of the target gene is reduced and / or the expression level of the target gene in individual cells is decreased, compared to cells in which the expression and / or activity of the peptidase C64 family member is not altered.

33. The method according to any one of claims 1 to 32, wherein the proportion of cells expressing the target gene in the cells obtained by reducing the expression and / or attenuating the activity of the peptidase C64 family member is about 95% or less.

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

35. A composition comprising the cell of claim 34.

36. A pharmaceutical composition comprising the cell of claim 34 and / or the composition of claim 35, and optionally a pharmaceutically acceptable carrier.

37. A method of influencing cell growth comprising administering the cell of claim 34, the composition of claim 35 and / or the pharmaceutical composition of claim 36.

38. Use of the cell according to claim 34, the composition according to claim 35 and / or the pharmaceutical composition according to claim 36 in the preparation of a medicament, wherein the medicament is for preventing and / or treating a disease and / or symptom.

39. The use according to claim 38, wherein the disease and / or condition comprises a tumor.

40. The use according to any one of claims 38-39, wherein the disease and / or condition comprises a solid tumor.

41. The use according to any one of claims 38 to 40, wherein the disease and / or symptoms comprise 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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