Modified cell and use thereof

Abstract

A modified cell and the use thereof. The present invention particularly relates to a cell culture method, comprising reducing the expression and / or activity of a target gene of a cell. The present invention further relates to a method for preventing and / or treating tumors by means of using cultured cells.

Description

Modified cells and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT International Application No. PCT / CN2024 / 119541 filed September 19, 2024, the contents of which are hereby incorporated by reference in their entirety for all purposes. TECHNICAL FIELD

[0003] The present application relates to the field of biomedicine, in particular to a modified cell and uses thereof. BACKGROUND

[0004] Currently, immunotherapy is an effective method for treating patients with poor prognosis. However, the immune cells used in immunotherapy have the problem of weak cell function or weak proliferation and survival ability after in vivo reinfusion. Therefore, how to provide a modified immune cell and a robust and reliable method for culturing and editing immune cells is an urgent problem to be solved. SUMMARY

[0005] The present application 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 naive cells, reduced proportion of apoptotic cells, and increased proportion of stem cell-like cells. The method of the present application can also improve the knockout efficiency of the target and / or improve the cell characteristics of the cells.

[0006] In one aspect, the present application provides a method for culturing cells, comprising: reducing and / or weakening the expression and / or activity of a family member selected from the group consisting of Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof in the cells by a CRISPR / Cas12 editing system.

[0007] In another aspect, the present application provides a cell obtained by the method of the present application.

[0008] In another aspect, the present application provides a pharmaceutical composition comprising the cell of the present application, and optionally a pharmaceutically acceptable carrier.

[0009] In another aspect, the present application provides a method for affecting cell growth, comprising administering the cell of the present application and / or the pharmaceutical composition of the present application.

[0010] In another aspect, the present application provides use of the cells of the present application and / or the pharmaceutical composition of the present application in the manufacture of a medicament for preventing and / or treating a disease and / or a symptom.

[0011] In one aspect, the present application provides a method of culturing a cell, the method comprising: reducing expression and / or attenuating activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof, in the cell by a Cas12 nuclease.

[0012] In some embodiments, the cell comprises an immune cell.

[0013] In some embodiments, the immune cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil, and / or a basophil.

[0014] In some embodiments, the immune cell comprises a monocyte, a macrophage, and / or a dendritic cell.

[0015] In some embodiments, the immune cell is derived from a stem cell differentiated immune cell.

[0016] In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC).

[0017] In some embodiments, the immune cell comprises a B cell, a T cell, a natural killer cell, and / or a natural killer T cell (NKT).

[0018] In some embodiments, the immune cell comprises an alpha beta T cell and / or a gamma delta T cell.

[0019] In some embodiments, the immune cell comprises a tumor infiltrating lymphocyte (TIL).

[0020] In some embodiments, the TIL is a TIL derived from a fragment of tumor tissue, a pleural effusion, and / or an ascitic fluid and / or a TIL derived from a post-thaw recovery.

[0021] In some embodiments, the fragment has a volume of about 1 cubic millimeter to about 27 cubic millimeters.

[0022] In some embodiments, the immune cell comprises an engineered immune receptor displayed on the cell surface.

[0023] In some embodiments, the engineered immune receptor specifically binds to an antigen expressed on a target cell.

[0024] In some embodiments, the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0025] In some embodiments, reducing expression and / or attenuating activity of a member of the Peptidase C64 family in the cell comprises inhibiting the function of a deubiquitinase, reducing expression and / or attenuating activity of a member of the ZC3H12 family in the cell comprises inhibiting the function of a nuclease, and / or reducing expression and / or attenuating activity of a member of the STAT-induced STAT inhibitor (SSI) family in the cell comprises inhibiting the function of a negative regulator of cytokine signaling.

[0026] In some embodiments, the cell obtained by reducing expression and / or attenuating activity of one or more of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family exhibits improved cell properties compared to a cell in which expression and / or activity of one or more of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family is not altered.

[0027] In some embodiments, the improved cell properties comprise one or more selected from the group consisting of: improved cell proliferation capacity, increased proportion of viable cells, improved proportion of cell subpopulations, increased cytokine secretion capacity, and increased tumor cell killing capacity.

[0028] In some embodiments, the improved proportion of cell subpopulations comprises one or more selected from the group consisting of: increased proportion of activated cells, decreased proportion of regulatory cells, decreased proportion of exhausted cells, increased proportion of central memory cells and / or naive cells, decreased proportion of apoptotic cells, and increased proportion of stem cell-like cells.

[0029] In some embodiments, the member of the Peptidase C64 family, the member of the ZC3H12 family, the member of the STAT-induced STAT inhibitor (SSI) family respectively comprises a ubiquitin binding domain, a C3H1 type zinc finger domain, a SH2 domain.

[0030] In some embodiments, the member of the Peptidase C64 family, the member of the ZC3H12 family, the member of the STAT-induced STAT inhibitor (SSI) family respectively comprises TNFAIP3, ZC3H12A, SOCS1.

[0031] In some embodiments, reducing expression and / or attenuating activity of one or more of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family in the cell comprises introducing a gene regulation system into the cell.

[0032] In some embodiments, the gene-regulating system is capable of disrupting one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family at the DNA level.

[0033] In some embodiments, the gene-regulating system comprises a guide nucleic acid molecule and a Cas12 nuclease.

[0034] In some embodiments, reducing expression and / or attenuating activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family comprises introducing into the cell a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the Cas12 nuclease, an LNP comprising a gRNA and a Cas12 nuclease, or an LNP comprising a nucleic acid encoding a gRNA and a nucleic acid encoding a Cas12 nuclease.

[0035] In some embodiments, the Cas12 nuclease comprises an AaCas12b nuclease or a variant or functional derivative thereof.

[0036] In some embodiments, the guide nucleic acid molecule comprises a guide RNA (gRNA) capable of binding to a Cas12 nuclease.

[0037] In some embodiments, the guide nucleic acid molecule is capable of binding to a sequence of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family.

[0038] In some embodiments, the guide nucleic acid molecule is capable of binding to a region selected from the group consisting of regions defined by genomic coordinates set forth in Table 7A-7C, or a fragment thereof.

[0039] In some embodiments, the guide nucleic acid molecule is capable of binding to a region selected from the group consisting of SEQ ID NOs: 1709-3416, 4058-4698, 4904-5108, or a fragment thereof.

[0040] In some embodiments, the guide nucleic acid molecule comprises a guide sequence comprising a sequence as set forth in any one of SEQ ID NOs: 1-1708, 3417-4057, 4699-4903, 5110-5136.

[0041] In some embodiments, the guide nucleic acid molecule further comprises a scaffold sequence, 3’ end of which is linked to the 5’ end of the guide sequence, the scaffold sequence being for binding to the Cas12 nuclease, optionally the scaffold sequence comprises a base modification.

[0042] In some embodiments, the guide nucleic acid molecule comprises a base modification.

[0043] In some embodiments, the guide nucleic acid molecule comprises one or more nucleotides with 2'-0-methyl modification, and / or 3' phosphorothioate internucleoside linkage added to the 3' end of the guide sequence.

[0044] In another aspect, the present application provides a cell obtained by any of the methods of the present application.

[0045] In another aspect, the present application provides a composition comprising any of the cells of the present application.

[0046] In another aspect, the present application provides a pharmaceutical composition comprising the cells and / or compositions of the present application, and optionally a pharmaceutically acceptable carrier.

[0047] In another aspect, the present application provides a method of affecting cell growth, comprising administering the cells, compositions and / or pharmaceutical compositions of the present application.

[0048] Use of the cells, compositions and / or pharmaceutical compositions of the present application in the manufacture of a medicament for the prevention and / or treatment of a disease and / or a condition.

[0049] In some embodiments, the disease and / or condition comprises a tumor.

[0050] In some embodiments, the disease and / or condition comprises a solid tumor.

[0051] In some embodiments, the disease and / or condition comprises one or more selected from the group consisting of melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0052] In another aspect, the present application provides a composition comprising one or more guide nucleic acid molecules comprising a guide sequence selected from the group consisting of the guide sequences set forth in SEQ ID NOs: 1-1708, 3417-4057, 4699-4903, and 5110-5136.

[0053] In some embodiments, the composition further comprises a Cas12 nuclease.

[0054] In another aspect, the present application provides an LNP comprising the composition of the present application.

[0055] In another aspect, the present application provides a cell comprising the composition or LNP of the present application.

[0056] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description in combination with the figures. The illustrative embodiments described in the detailed description, given merely for the purposes of illustration, are not to be construed as limiting the spirit and scope of the application. The drawings, given by way of example, and not limitation, solely for the purpose of illustration, are a part of the detailed description. It is noted that scales of various elements in the figures can be exaggerated for illustrative purposes and therefore the drawings should not be construed as limiting the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0057] The features and advantages of the application involved will be better understood from the detailed description of the exemplary embodiments with reference to the drawings. The drawings are briefly described as follows:

[0058] FIG. 1A-1C show a continuous region with more than about 3 transcription factor binding sites before the start codon (e.g., the region shown by the black line segment above 3 on the y-axis).

[0059] FIG. 1D shows the T7E1 enzyme digestion electrophoresis results of the SOCS1 candidate site amplification products.

[0060] FIG. 1E shows the T7E1 enzyme digestion electrophoresis results of the TNFAIP3 candidate site (mismatch 2 = 0) amplification products.

[0061] FIG. 1F shows the T7E1 enzyme digestion electrophoresis results of the TNFAIP3 candidate site (mismatch 2 < 4) amplification products.

[0062] FIG. 1G shows the T7E1 enzyme digestion electrophoresis results of the ZC3H12A candidate site amplification products.

[0063] FIG. 1H shows the verification of candidate sgRNAs in PBMCs.

[0064] FIG. 1I shows the T7E1 enzyme digestion electrophoresis results of the amplification sites after the modified sgRNAs were electroporated into PBMCs.

[0065] FIG. 1J shows the knockout efficiency of some sgRNAs on target genes.

[0066] FIG. 2A shows the target cell killing ability of TIL cells with single target gene editing.

[0067] FIG. 2B shows the target cell killing ability of TIL cells with combined gene editing.

[0068] FIG. 2C shows the target cell killing ability of TIL cells with single target gene editing.

[0069] FIG. 3A shows the TIL cell expansion fold of TIL cells with single target gene editing.

[0070] Figure 3B shows the TIL cell expansion fold of TIL cells with combinatorial gene editing in two batches of experiments.

[0071] Figure 3C shows the TIL cell expansion fold of TIL cells with single target gene editing and combinatorial editing in the absence of IL-2 in the culture medium.

[0072] Figure 4A shows the apoptosis level of TIL cells with single target gene editing.

[0073] Figure 4B shows the apoptosis level of TIL cells with combinatorial gene editing.

[0074] Figure 5A shows that TIL cells with single target gene editing have higher stem cell proportion.

[0075] Figure 5B shows that TIL cells with combinatorial gene editing have higher stem cell proportion.

[0076] Figure 5C shows that TIL cells with single target gene editing have higher central memory T cell proportion.

[0077] Figures 5D, 5E show that TIL cells with combinatorial gene editing have higher central memory T cell proportion.

[0078] Figure 5F shows that TIL cells with single target gene editing have lower exhausted T cell proportion.

[0079] Figures 5G, 5H show that TIL cells with combinatorial gene editing have lower exhausted T cell proportion.

[0080] Figure 6A shows the cytokine expression proportion of TIL cells with various combinatorial gene editing in the absence of stimulation medium.

[0081] Figures 6B, 6C show the cytokine expression proportion of TIL cells with various combinatorial gene editing in the TransAct (cell stimulator with CD3 antibody and CD28 antibody) stimulation group.

[0082] Figure 6D shows the cytokine release ability of TIL cells with combinatorial gene editing co-cultured with A375 tumor cells.

[0083] Figure 6E shows the cytokine release ability of TIL cells with combinatorial gene editing co-cultured with autologous tumor cells of the TIL cell donor.

[0084] Figure 6F shows the cytokine release ability of TIL cells with combinatorial gene editing co-cultured with A375 tumor cells, relative to Cas9 gene editing, the target of the present application for Cas12 gene edited cells.

[0085] FIG. 7A shows the killing ability of Cas12b edited TILs compared to Cas9 edited TILs on target cells.

[0086] FIG. 7B shows the proliferative ability of Cas12b edited TILs compared to Cas9 edited TILs.

[0087] FIG. 7C shows the cytokine independent T cell survival ability of Cas12b edited TILs compared to Cas9 edited TILs.

[0088] FIG. 7D shows the apoptosis level of Cas12b edited TILs compared to Cas9 edited TILs.

[0089] FIG. 7E shows the T cell exhaustion marker expression of Cas12b edited TILs compared to Cas9 edited TILs.

[0090] FIG. 7F shows the central memory T cell proportion of Cas12b edited TILs compared to Cas9 edited TILs.

[0091] FIG. 7G shows the expression of T cell activation markers of Cas12b edited TILs compared to Cas9 edited TILs.

[0092] FIG. 7H shows the stem-like T cell proportion (CD39 CD69 in CD8+ TILs) of Cas12b edited TILs compared to Cas9 edited TILs.

[0093] FIG. 8A shows the killing ability of Cas12b edited TILs compared to unedited TILs on target cells.

[0094] FIG. 8B shows the ability of Cas12b edited TILs compared to unedited TILs to continuously kill target cells.

[0095] FIG. 8C-8D shows the proliferative ability of Cas12b edited TILs compared to unedited TILs.

[0096] FIG. 8E-8H shows the expression of T cell activation markers of Cas12b edited TILs compared to unedited TILs.

[0097] FIG. 81 shows the T cell cytokine IFN-γ expression level of Cas12b edited TILs compared to unedited TILs.

[0098] FIG. 8J shows the T cell cytokine TNF-a expression level of Cas12b edited TILs compared to unedited TILs.

[0099] FIG. 8K shows T cell cytokine GZMB expression levels of Casl2b edited TILs compared to unedited TILs.

[0100] FIG. 8L shows T cell cytokine IFN-g expression levels of Casl2b edited TILs compared to unedited TILs following TranAct activation.

[0101] FIG. 8M shows T cell cytokine TNF-a expression levels of Casl2b edited TILs compared to unedited TILs following TranAct activation.

[0102] FIG. 8N shows T cell cytokine GZMB expression levels of Casl2b edited TILs compared to unedited TILs following TranAct activation.

[0103] FIG. 8O shows the proportion of central memory T cells of Casl2b edited TILs compared to unedited TILs.

[0104] FIG. 8P shows T cell stemness molecule TCF-1 expression of Casl2b edited TILs compared to unedited TILs. DETAILED DESCRIPTION

[0105] The present application is explained by the following specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure herein.

[0106] TERMS

[0107] In the present invention, the term "Cas12" generally refers to an RNA-guided nuclease or a fragment thereof, which comprises Cas12, preferably comprises Cas12b, C2cl protein (e.g., a protein comprising the active, inactive, or partially active DNA cleavage domain of Cas12b and / or the gRNA binding domain of Cas12b), a derivative or variant thereof (e.g., an engineered Cas12b, dCas12b, or engineered Cas12b effector protein), and a functional fragment thereof, such as an oligonucleotide binding fragment. Cas12b homologs include, but are not limited to, Alicyclobacillus acidoterrestris, Alicyclobacillus acidophilus, Bacillus hisashi, and Bacillus sp. In some embodiments, a protein comprising Cas12b or a fragment thereof is referred to as a "Cas12b variant". A Cas12b variant shares homology with Cas12b or a fragment thereof. For example, a Cas12b variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas12b. For example, an exemplary Cas12b polypeptide in the present invention can be AaCas12b (a Cas12b nuclease from Alicyclobacillus acidophilus).

[0108] In the present invention, the term "STAT-induced STAT inhibitor (SSI) family member" generally refers to a family member protein having a SH2 domain or a functionally active fragment thereof. For example, a STAT-induced STAT inhibitor (SSI) family member can comprise SOCS1. For example, the UniProt number of a STAT-induced STAT inhibitor (SSI) family member can be 0155524. A STAT-induced STAT inhibitor (SSI) family member of the present invention can also encompass a functionally active fragment thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or substances comprising the functionally active fragment resulting from processing and / or modification of the same in a cell. For example, a STAT-induced STAT inhibitor (SSI) family member of the present invention can comprise a functionally active fragment thereof as well as any other arbitrary domain.

[0109] 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 inclusion of a peptidase C64 family member can include TNFAIP3. For example, the UniProt number of a peptidase C64 family member can be P21580. The peptidase C64 family member of the present invention can 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 resulting from processing and / or modification in cells. For example, the peptidase C64 family member of the present invention can include functionally active fragments thereof as well as any other arbitrary domains.

[0110] 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, the inclusion of a ZC3H12 family member can include ZC3H12A. For example, the UniProt number of a ZC3H12 family member can be Q5D1E8. The ZC3H12 family member of the present invention can 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 resulting from processing and / or modification in cells. For example, the ZC3H12 family member of the present invention can include functionally active fragments thereof as well as any other arbitrary domains.

[0111] In the present invention, the term "immune cell" generally refers to a cell involved in innate and adaptive immune responses. For example, it can 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 cell is a T cell, such as a CD4+ T cell, a CD8+ T cell (also known as a cytotoxic T cell or CTL), a regulatory T cell (Treg), a Thl cell, a Th2 cell, a Thl7 cell, an alpha beta T cell, and / or a gamma delta T cell. For example, the immune cell of the present invention also includes an immune cell derived from differentiation of a stem cell. For example, the immune cell of the present invention also includes an immune cell derived from differentiation of a pluripotent stem cell. For example, the stem cell of the present invention can be obtained by induction. For example, the above-mentioned stem cell of the present invention can include an induced pluripotent stem cell (iPSC).

[0112] In the present application, the term "chimeric antigen receptor" generally refers to an engineered antigen receptor. For example, a CAR can comprise an extracellular antigen binding domain fused via a hinge and transmembrane domain to a cytoplasmic domain comprising a signaling domain. In some embodiments, the CAR extracellular domain can bind to an antigen expressed by a target cell in an MHC independent manner, resulting in activation and proliferation of the cell. In some embodiments, the extracellular domain of a CAR can recognize a tag fused to an antibody or antigen binding fragment thereof. For example, a single CAR construct can be made so that it can be targeted to multiple different antigens by substituting one antibody for another. In some embodiments, the extracellular domain of a CAR can comprise an antigen binding fragment derived from an antibody. Antigen binding domains for use in the present application can include, for example, scFv, antibodies, antigen binding regions of antibodies, variable regions of heavy chains / light chains, and / or single chain antibodies.

[0113] In the present application, the term "T cell receptor" generally refers to an engineered antigen receptor. For example, a TCR can comprise a TCRa and / or TCRP chain that has been isolated and cloned from a population of T cells that recognize a particular target antigen. For example, the TCRa and / or TCRP genes (i.e., TRAC and TRBC) can be cloned from a population of T cells isolated from an individual with a particular malignancy or from a population of T cells that have been isolated from a humanized mouse that has been immunized with a specific tumor antigen or tumor cell. An engineered TCR can recognize an antigen through the same mechanism as its endogenous counterpart (e.g., by recognizing its cognate antigen presented in the context of a major histocompatibility complex (MHC) protein expressed on the surface of a target cell), which can result in activation and proliferation of the TCR engineered cell.

[0114] In the present application, the term "gene regulation system" generally refers to a system that modulates the expression or activity of a target gene. For example, a gene regulation system can comprise a gene regulation molecule. For example, a gene regulation system can modulate the expression or activity of a gene, such as placing the gene in an inactive or active state, increasing or decreasing the number of the gene, placing the gene in a state of increased or decreased transcription, and / or placing the transcription product of the gene in an inactive or active state; for example, a gene regulation system can modulate the expression or activity of a gene, such as increasing or decreasing the amount of expression product of the gene in a single cell and / or increasing or decreasing the number of cells that express the expression product of the gene.

[0115] In the present disclosure, 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, guiding 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 location within a target DNA. For example, where hybridization between a gRNA and a DNA target sequence promotes formation of a CRISPR complex, perfect complementarity can not necessarily be required, for example, so long as there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0116] In the present disclosure, the term "enzyme protein" generally refers to a protein having enzymatic activity. For example, an enzyme protein can refer to a Cas protein. For example, a Cas protein can comprise at least one RNA recognition or binding domain that can interact with a gRNA. A Cas protein can also comprise a nuclease domain (e.g., a DNAse or RNAse domain), a DNA binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and / or other domains. A nuclease domain can have catalytic activity for nucleic acid cleavage. Cleavage can include breakage of a covalent bond of a nucleic acid molecule. A Cas protein can be a wild-type protein (i.e., a protein that occurs in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. A Cas protein can also be an active variant or fragment of a wild-type or modified Cas protein. In the present disclosure, a Cas protein can encompass an unprocessed Cas protein, a Cas protein in any form of processing, a variant of a Cas protein, or a substance comprising a functionally active fragment of a Cas protein.

[0117] In the present disclosure, the term "ribonucleoprotein complex" generally refers to a complex of a protein and a nucleic acid. For example, a protein in a ribonucleoprotein complex can have nuclease activity. For example, a ribonucleoprotein complex can cleave a target sequence under the guidance of a nucleic acid. For example, a ribonucleoprotein complex can be a complex of a Cas protein and a gRNA.

[0118] In the present invention, the term "lipid nanoparticle (LNP)" generally refers to a lipid-nucleic acid particle or a nucleic acid-lipid particle. For example, LNP indicates a particle made of lipids (e.g., cationic lipids, non-cationic lipids, and conjugated lipids to prevent aggregation of the particle) and nucleic acids (e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, self-amplifying RNA, or plasmid, including a plasmid from which an interfering RNA or mRNA is transcribed) encapsulated in the lipids. For example, a protein can be encapsulated in the LNP, for example, a Cas protein known in the art can be encapsulated in the LNP. For example, the lipids in the LNP include (1) "simple lipids" including fats and oils and waxes; (2) "complex lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids. For example, the lipids in the LNP can also include lipid derivatives, for example, lipids covalently or non-covalently bound to a protein or polypeptide. For example, the components in the LNP can also include a polypeptide component, in which the polypeptide component can replace one or more lipid components in a conventional LNP while maintaining or improving the delivery ability of the LNP.

[0119] In the present invention, the term "exon" generally refers to a portion of a gene that can be expressed as a protein. For example, the exon can refer to a portion having the ability to be expressed as a protein in the process of protein biosynthesis. For example, editing the sequence of an exon of a target gene can reduce the activity or function of the target gene.

[0120] In the present invention, the term "intron" generally refers to a segment in DNA that does not encode part or all of an expressed protein. The intron is generally transcribed into an RNA molecule under endogenous conditions, but is spliced out from the endogenous RNA before being translated into a protein. For example, targeting the position of an intron for editing can reduce the activity or function of the target gene. For example, targeting the intron region at the junction with an exon, such as about 0 bp to about 100 bp, preferably about 0 bp to about 20 bp, upstream or downstream of the exon for editing can reduce the activity or function of the target gene.

[0121] In the present invention, the term "initiation codon" generally refers to a unit ('codon') of adjacent nucleotides on a gene that can define the start of protein synthesis (mRNA translation). For example, targeting a region 0 bp to 1500 bp upstream of the initiation codon, preferably 0 bp to 100 bp upstream of the initiation codon for editing can reduce the activity or function of the target gene.

[0122] In the present application, the term "protospacer adjacent motif (PAM)" generally refers to a short sequence following a target sequence. For example, when a Cas9 cleaves a target DNA at a site-specific manner, the PAM sequence can be used to determine the cleavage site. For example, once the region of PAM is determined, one skilled in the art can easily determine the appropriate target sequence location, and can easily design a gRNA sequence for cleaving the target sequence.

[0123] In the present application, the term "expression reduction" generally refers to a reduction in the amount of expression of a product or its gene and / or a reduction in the proportion of cells capable of expressing the product (e.g., at least about 5-100%). For example, it can be a reduction in the amount of the product expressed by the gene in a cell or a reduction in the proportion of cells containing the product expressed by the gene, or a reduction in the proportion of cells secreting the product expressed by the gene. For example, a reduction in the expression of a gene can be indirectly indicated by detecting the amount of knockout of the gene in the genome of a cell. For example, a reduction in the expression of a gene can be indirectly indicated by detecting the proportion of cells in a cell population in which the gene is knocked out.

[0124] In the present application, the term "activity" generally refers to the biological function of a substance. For example, the activity of a gene can refer to the transcription and / or translation state of the gene. For example, a reduction in the activity of a gene (e.g., at least about 5-100%) can refer to a reduction in the transcription function of the gene, the inability of the gene to be normally transcribed, or the inhibition of the function of the gene transcription product.

[0125] In the present application, the term "CD80" generally refers to a cell stimulatory molecule. For example, CD80 can be a ligand of CD28. For example, CD80 can be found in GenBank Accession No. P33681. The CD80 protein of the present application can also encompass functionally active fragments thereof, not limited to substances containing functionally active fragments of CD80 produced after processing and / or modification occurring in cells. For example, the CD80 of the present application can contain functionally active fragments of CD80 and other arbitrary domains.

[0126] In the present application, the term "CD86" generally refers to a cell stimulatory molecule. For example, CD86 can be a ligand of CD28. For example, CD86 can be found in GenBank Accession No. P42081. The CD86 protein of the present application can also encompass functionally active fragments thereof, not limited to substances containing functionally active fragments of CD86 produced after processing and / or modification occurring in cells. For example, the CD86 of the present application can contain functionally active fragments of CD86 and other arbitrary domains.

[0127] In the present disclosure, the term "secreted" generally refers to a substance that can be located in the extracellular space of a cell. For example, a secreted substance can be transported to the extracellular space of a cell after being synthesized in the cell. For example, whether a substance is a secreted substance can be detected by enzyme-linked immunosorbent assay or other detection methods.

[0128] In the present disclosure, the term "T cell receptor" or "TCR" generally refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of antigens. The TCR can be responsible for recognizing antigens bound to molecules of the major histocompatibility complex. The TCR can consist of a heterodimer of alpha (a) and beta (b) chains, or be composed of gamma and delta (g / d) chains. The TCR can exist in a / b and g / d forms, which are structurally similar but have unique anatomical locations and functions. For example, the TCR can be a TCR modified on any cell expressing the TCR. For example, the species of the TCR can be analyzed by TCR subtype analysis reagents.

[0129] In the present disclosure, the term "clonal diversity" generally refers to a certain substance having multiple clonal types. For example, the clonal diversity of TCR can mean that TCR can have different sequence structures and / or antigen recognition capabilities. For example, the diversity of TCR, which is often distinguished by b chain subtypes, can include Vb23, Vb7.2, Vb5.2, Vb11, Vb16, Vb3, etc. When a T cell population has more b chain subtypes, it can be considered that the T cell population has higher clonal diversity.

[0130] In the present disclosure, "CD4 + cells" generally refer to CD4-positive cells, which can be T cells, for example. The term "CD4 + cells", "CD4-positive cells" can be used synonymously. These cells can be identified by methods known in the art, for example, by staining cells with a fluorescently labeled antibody against CD4 and using fluorescence-activated cell sorting. For example, existing data can demonstrate that an increase in the proportion of CD4 + cells can increase the ability of the cell population to secrete IFN and / or TNF, and can improve the effect of the T cell population on promoting tumor suppression. For example, see Tay, R. E., Richardson, E. K., et al. (2020). Cancer Gene Therapy, 1-13. However, there is a lack of a method for increasing the proportion of CD4 + cells in the art, and the present disclosure can provide a method for affecting the proportion of CD4 + cells.

[0131] In the present disclosure, "CD8 +"Cell" generally refers to a CD8 positive cell, e.g., which can be a T cell. The term "CD8 + "Cell", "CD8 positive cell" can be used synonymously. These cells can be identified by methods known in the art, e.g., by staining the cells with a fluorescently labeled antibody to CD8 and using fluorescence activated cell sorting.

[0132] In the present application, the term "IC 50 Value" or "IC50 value" generally refers to the concentration of a subject that achieves a 50% inhibition of a biological process. IC50values can be converted to absolute inhibition constants (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22:3099).

[0133] In the present application, the term "K D Value" or "KD value" generally refers to the dissociation constant, which can be determined by surface plasmon resonance. Typically, surface plasmon resonance analysis uses the BIAcore system (Pharmacia Biosensor, Piscataway, NJ) to measure real-time binding interactions 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 by immobilizing the analyte (a substance on a biosensor matrix) and presenting the ligand.

[0134] In the present application, the term "encoding" generally refers to the ability to directly or indirectly infer, from the structural or compositional information of one molecule, the structural or compositional information of another type of molecule associated therewith according to substantially established rules. For example, the nucleotide sequence can be inferred from the sequence of amino acids, e.g., the property of a deoxyribonucleic acid to transcribe a complementary nucleic acid, including a nucleic acid that is translated into a polypeptide. For example, a deoxyribonucleic acid can encode RNA transcribed from the deoxyribonucleic acid. The deoxyribonucleic acid can similarly encode a polypeptide translated from the RNA transcribed from the deoxyribonucleic acid.

[0135] In the present application, the term "small molecule compound" generally refers to a peptide, a peptide mimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, a nucleotide, a nucleotide analog, an organic or inorganic compound (i.e., including heteroorganic and organometallic compounds) having a molecular weight of less than about 10,000 g / mole, an organic or inorganic compound having a molecular weight of less than about 5,000 g / mole, an organic or inorganic compound having a molecular weight of less than about 1,000 g / mole, an organic or inorganic compound having a molecular weight of less than about 500 g / mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0136] In the present application, the term "NK cell" also known as "natural killer cell" generally refers to a cell with large granules in the cytoplasm. NK cells develop from bone marrow lymphoid stem cells and can differentiate, develop depending on the bone marrow or thymic microenvironment. In the present application, the proportion of NK cells in TIL cells can be changed by the method of the present application.

[0137] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced biologically or by recombinant DNA methods. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact" as in "intact antibody", for the purposes of the present application, 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 composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 of the basic heterotetramer units along with an additional polypeptide called J chain, and contain 10 antigen binding sites. IgA antibodies, which are present as a dimmer in secretions, and as a tetramer in the blood, contain 2-5 basic 4-chain units which can aggregate to form polyvalent assemblies and can also contain J-chain. For IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while each of the H chains is disulfide ly linked to the other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains, or four CH domains for μ and ε isotypes. Each L chain has a variable domain at its N-terminus (VL), followed by a constant domain at its other end. The VL is aligned with the VHand the CL is aligned with a first constant domain of a heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The VHand VLpair together to form a single antigen binding site. The L chain from any vertebrate species can be readily identified by using an antibody that specifically detects the light chain type in question. Based on the amino acid sequences of their constant domains, the antibodies can be assigned to one of two clearly distinct types, called kappa and lambda. Depending on the amino acid sequences of the constant domains of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively.

[0138] In the present application, the term "antigen binding fragment" generally refers to one or more polypeptide fragments that are capable of specifically binding to an antigen. In the present application, the antigen binding fragment can include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb.

[0139] In the present application, the term "expression" generally refers to the process of transcription and / or translation of a gene encoding a polypeptide of interest that occurs within a cell. The level of transcription of a gene encoding a polypeptide of interest in a host cell can be determined by measuring the amount of the corresponding mRNA present in the cell. For example, the mRNA transcribed from a gene encoding a polypeptide of interest can be quantitatively measured by PCR or by RNA hybridization. The level of translation of a gene encoding a polypeptide of interest can be measured by a variety of methods, for example, by ELISA, by a polypeptide bioassay, or by Western blot or radioimmunoassay. In the present application, the term "expression" can also generally refer to the process of transcription and / or translation of a product. For example, the expression of a cytokine can be the process of transcription and / or translation of the cytokine by a cell. For example, the expression of a cytokine can be determined by detecting the amount of the corresponding mRNA present in the cell or by detecting the amount of the cytokine produced by the cell, or both.

[0140] In the present application, the term "one stage of in vitro expansion", "single stage of in vitro expansion", or "first stage of in vitro expansion" and the like generally refers to a period of expansion process that the TILs go through 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 is increased by at least about 1-fold, 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 is increased by at least about 1-50-fold, such as 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, 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 condition of TIL cell culture. In one embodiment, when T cell activators and / or T cell growth factors are added or supplemented in the cell culture medium, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In one embodiment, when the TIL cells are subjected to centrifugation and / or cell washing, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the number of days of TIL cell culture. In one embodiment, when the TIL cells are cultured in vitro for about 1-100 days, such as about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days, or about 100 days, it can be considered that the TIL cells have entered the next stage of in vitro expansion.

[0141] In the present application, the term "first stage ex vivo expansion" generally refers to the stage of expansion using T cell growth factors after the primary TILs are obtained from the tissue. In one embodiment, the tissue of the present application can be selected from the group consisting of tumor tissue, tumor associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of peritumoral tissue, pleural effusion and / or peritoneal effusion, the pleural effusion of the present application can be from a patient with metastatic cancer. In one embodiment, the expansion of the present application can be autologous or allogeneic in vivo expansion, or can be ex vivo expansion. The first stage ex vivo expansion of the present application can also be referred to as the pre-REP (rapid expansion pre) stage. For example, TILs derived from tumor tissue and not subjected to ex vivo expansion can be referred to as the first TIL population. For example, TILs obtained after the first stage ex vivo expansion in the two-step method divided culture of the present application can be referred to as the second TIL population.

[0142] In the present application, the term "second stage ex vivo expansion" generally refers to the stage of expansion after the tissue is removed from the subject and expanded, and then expanded again. In one embodiment, the TILs of the present application that are subjected to the second stage ex vivo expansion have an increased number of cells compared to the TILs that are subjected to the first stage ex vivo expansion, for example, the number of cells can be increased by at least about 10 fold (or at least about 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. In one embodiment, the second stage ex vivo expansion can be different from the culture conditions of the first stage ex vivo expansion, for example, the culture material added can be different. For example, the second stage ex vivo expansion in the two-step method divided culture of the present application can also be referred to as the REP (rapid expansion) stage. For example, TILs obtained after the second stage ex vivo expansion in the two-step method divided culture of the present application can be referred to as the third TIL population.

[0143] In the present application, the term "in vivo" generally refers to an event that occurs in the body of a subject.

[0144] In the present application, the term "ex vivo" generally refers to an event that occurs outside the body of a subject.

[0145] In the present application, the term "ex vivo" generally refers to an event that occurs outside the body of a subject.

[0146] In the present application, the term "secretion capacity" generally refers to the ability of a cell to express a polypeptide or a protein and to transfer the polypeptide or the protein of the present application to the extracellular environment.

[0147] In the present application, the term "irradiation" generally refers to the treatment of a substance by a beam of rays. For example, in one embodiment, irradiation can refer to the irradiation of a substance by X-rays, alpha rays, beta rays, or gamma rays.

[0148] In the present application, the term "engineered cell" generally refers to a cell that has been genetically modified by the addition of extra genetic material in the form of DNA or RNA to the total genetic material of the cell. In one embodiment, an engineered cell can be genetically modified to express a T cell activator and / or a T cell growth factor of the present application for a TIL.

[0149] In the present application, the term "co-culturing" generally refers to the culturing of two or more different populations of cells with some degree of contact between them. The "contact" between two or more different populations of cells of the present application can be by direct contact, in one embodiment, i.e., where the cells of one population are in direct physical contact with the cells of another population. Or it can be by indirect contact mediated by a shared medium, in one embodiment. The shared medium of the present application can contain metabolic products produced and released by at least one of the co-cultured populations of cells and used to culture the other population of cells.

[0150] In the present application, the term "contacting" generally refers to the contacting together of two or more different types of substances in any order, in any manner, and for any length of time. It can be by direct contact, in one embodiment, e.g., one or more feeder cells, a T cell activator, and / or a T cell growth factor can be added to the culture medium of TIL cells, e.g., a culture medium comprising one or more feeder cells, a T cell activator, and / or a T cell growth factor can be added to and / or replace the culture medium of TIL cells, e.g., a culture medium comprising one or more feeder cells, a T cell activator, and / or a T cell growth factor can be used for the culturing of TIL cells. It can be by indirect contact, in one embodiment, e.g., metabolic products produced and released by feeder cells can be used to culture TIL cells.

[0151] In the present application, the terms "simultaneous contacting", "co-contacting", "contacting simultaneous with", "simultaneously", and "co" generally refer to the administration of two or more substances to a subject and / or cells such that the substances are present in the environment of the subject and / or cell culture at the same time. Simultaneous contacting can include administration in different compositions at the same time, administration in different compositions at different times, or administration in a composition in which two or more active pharmaceutical ingredients are present. For example, "simultaneous contacting" in the present application can generally refer to substantially simultaneous contacting.

[0152] In the present application, the term "expansion" generally refers to an increase in the number of cells by several fold 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. In the present application, the term "expanded" generally refers to cells of the present application that have been subjected to one or more of the above-described expansions.

[0153] In the present application, the term "polymer" generally refers to a molecule composed of individual chemical moieties linked together, the polymer moieties of the present application can be the same or different. In one embodiment, the term "polymer" can refer to individual chemical moieties linked tail-to-tail to form a linear molecule, as well as individual chemical moieties linked together in a branched (e.g., "multi-armed" or "star") structure. In one embodiment, the polymer can include, for example, a polysaccharide, a dextran, a hydrogel, a polyethylene glycol, or a poloxamer. Poloxamers are non-ionic triblock copolymers 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 application can be formulated with, or administered with, any of the polymers described herein or known in the art.

[0154] In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region of a murine antibody is fused to the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To create a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody can be created, and the variable region gene can be cloned from the murine hybridoma cells, and the constant region gene of a human antibody can be cloned as needed, and the murine variable region gene and the human constant region gene can be linked into a chimeric gene and inserted into an expression vector, and the chimeric antibody molecule can be expressed in a eukaryotic system or a prokaryotic system.

[0155] In the present application, the term "humanized antibody", also known as CDR-grafted antibody, generally refers to an antibody in which the CDR sequences of a murine antibody are grafted into the variable region framework of a human antibody, i.e., an antibody generated from different types of human germline antibody framework sequences. The heterogeneity reaction induced by the chimeric antibody due to the presence of a large amount of murine protein components can be overcome. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database.

[0156] In the present application, the term "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody can be human, to remove immunogenicity and side effects. The development of monoclonal antibodies has undergone four stages, namely: murine monoclonal antibody, chimeric monoclonal antibody, humanized monoclonal antibody and fully human monoclonal antibody. The antibody or ligand described in the present application can be a fully human monoclonal antibody. The related technology for preparing fully human antibodies can be: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology and single B cell antibody preparation technology, etc.

[0157] In the present application, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain 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 E. A. et al., Chothia et al. and MacCallum et al. As used in the present application, the Kabat definition of CDR can be applied to CDR1, CDR2 and CDR3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), and CDR1, CDR2 and CDR3 of the heavy chain variable domain (CDR H1, CDR H2, CDR H3 or H1, H2, H3).

[0158] In the present application, the term "IL-2" or "IL2" generally refers to a T cell growth factor called interleukin 2, and includes all forms of IL-2, which can include in one embodiment human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, or active fragments thereof. The GeneID of the IL-2 gene can be 3558.

[0159] In the present application, the term "antigen presenting cell", "antigen presenting cell" or "APC" generally refers to an immune cell that displays foreign antigens complexed with major histocompatibility complexes (MHCs) on its surface, such as accessory cells (e.g., B cells, dendritic cells, etc.). T cells can recognize these complexes using their T cell receptors (TCRs). APCs can process and present antigens to T cells. In one embodiment, antigen presenting cells can include a group selected from peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.

[0160] In the present application, the term "TIL properties" generally refers to the properties of TIL cells obtained by the culture methods of the present application. Changes in TIL properties can include: increased TIL cell numbers, increased proportion of viable cells, increased survival capacity, improved T cell subpopulation proportions, improved cytokine secretion capacity, improved in vitro tumor cell killing capacity, improved in vivo tumor killing capacity, improved T cell receptor (TCR) clonal diversity, and improved TIL cell numbers in tissues, or any combination thereof. Changes in the present application can be increases or decreases.

[0161] In the present application, the term "survival" generally refers to the presence of cells in vitro and / or in a subject. For example, an increase in TIL cell survival capacity can refer to an increase in the time period that TIL cells are present in vivo. For example, an increase in survival capacity can refer to an increase in the time period that cells are present in a subject tissue, such as a tumor, spleen, bone marrow, lung tissue, and blood. For example, an increase in survival capacity can refer to an increase in TIL cell survival capacity after removal of IL-2 from the culture medium.

[0162] In the present application, the term "artificial antigen presenting cell" generally refers to an artificially constructed immune cell for presenting an exogenous antigen, for example, the manner of presenting an exogenous antigen can be that the surface of the artificial antigen presenting cell comprises a complex of an exogenous antigen and a major histocompatibility complex (MHC). In one embodiment, an isolated artificial antigen presenting cell (aAPC) can be included, which can comprise a cell expressing HLA-A / B / C (the gene encoding it can be GeneID 3105, 3106, or 3107), CD64 (the gene encoding it can be GeneID 2209), CD80 (the gene encoding it can be GeneID 941), ICOS-L (the gene encoding it can be GeneID 23308), and CD58 (the gene encoding it can be GeneID 965), and can be modified to express one or more T cell activators.

[0163] In the present application, the term "fusion protein" generally refers to a polypeptide or protein that contains the amino acid sequence of a first polypeptide or protein or fragment, analog or derivative thereof and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein or fragment, analog or derivative thereof that is different from the first polypeptide or protein or fragment, analog or derivative thereof, or is not normally part of the first polypeptide or protein or fragment, analog or derivative thereof). In certain instances, a fusion protein can comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide. In this regard, the heterologous protein, polypeptide or peptide of the present application can or can not be of the same type as the 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 certain instances, the fusion protein can retain or improve activity compared to the activity of the original polypeptide or protein prior to fusion with the heterologous protein, polypeptide or peptide.

[0164] In the present application, the term "killing capacity" generally refers to the ability to kill a target cell by contacting the cell with an effective amount of a substance of the present application. In one embodiment, the substance of the present application can be a TIL cell. Killing by the present application can include killing of a cell by itself or by facilitating CDC, apoptosis, ADCC and / or phagocytosis by other cells or substances, or by a combination of two or more of these mechanisms.

[0165] In the present application, the term "administering" or "administration" generally refers to the delivery of 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 can include intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral and / or mucosal.

[0166] In the present application, 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 application. For example, a kit comprising TIL cells of the present application.

[0167] In the present application, the term "subject" generally refers to a cell or an animal, which can be a mammal, such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs) and a laboratory animal (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, juvenile and adult human subjects. Subjects include animal disease models, such as tumor animal models, and other animal models known to those skilled in the art.

[0168] In the present application, the term "feeder" generally refers to a culture cell that can be used to support the growth of another cell of interest in culture. For example, a feeder cell can be grown in vitro and secrete at least one factor into the culture medium. In one embodiment, a feeder cell can include an antigen presenting cell.

[0169] In the present application, the term "specifically binds" generally refers to a binding substance that recognizes a specific target substance, but does not substantially recognize or bind other molecules in a sample. For example, if a binding substance can specifically bind to a specific target substance of the present application from one species, the binding substance of the present application can also specifically bind to a target substance or a homologous target substance of the present application from one or more other species. Such inter-species reactivity can not change the classification of the binding substance as specific per se. In some cases, a binding substance that specifically binds to a target substance can also bind to different variant forms of the target substance.

[0170] In the present application, the term "complete culture process" generally refers to a complete process of isolating cells from a tumor tissue isolated from a patient, starting from isolation, through one or more than one expansion, and finally obtaining cells that can be administered to a subject.

[0171] In the present application, the term "cell culture medium" generally refers to a nutrient solution in which cells, such as mammalian cells, are grown. The formulation of cell culture media is well known in the art. Typically, cell culture media include buffers, salts, carbohydrates, amino acids, vitamins, and essential trace elements. Cell culture media can or can not contain serum, peptone, and / or proteins. Cell culture media can 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.

[0172] In the present application, the term "pharmaceutical composition" or "pharmaceutical preparation" generally refers to a preparation that allows the biologically active effectiveness of the active ingredient of the present application, and can not contain additional components that are not acceptable for the subject to which the preparation will be administered. Such preparations are sterile. "Pharmaceutically acceptable" excipients (carriers, additives) are those excipients that can be reasonably administered to a subject mammal to provide an effective dose of the active ingredient used.

[0173] In the present application, the term "tumor infiltrating lymphocyte" or "TIL" generally refers to a population of cells originally obtained as white blood cells, the cells of the present application have left the bloodstream of a subject and migrated into a tumor. TILs can include, but are not limited to, CD8 + Cytotoxic T cells (lymphocytes), Thl and Thl 7 CD4 +T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs can include primary TILs and secondary TILs. "Primary TILs" can be those TIL cells obtained from a subject tissue sample, and "secondary TILs" can be any TIL population that has been expanded or is expanded in the present application. In some embodiments, the tumor infiltrating lymphocytes of the present application can be unpurified or can be interpenetrating with tumor cells. In one embodiment, the TILs of the present application can refer to a population of TILs.

[0174] In the present application, the term "central memory T cells" generally refers to T cells that have long-term memory and are capable of accepting antigen re-stimulation. Central memory T cells can have a phenotype of CD45RO + CD62L + , for example, central memory T cells can be identified by CD45RO + and CD62L + . Central memory T cells can have a stronger ability to resist tumor growth than ordinary T cells.

[0175] In the present application, the term "regulatory T cells" generally refers to a subset of T cells that control the body's autoimmune response. Regulatory T cells can have a phenotype of CD4 + CD25 + Foxp3 + , for example, regulatory T cells can be identified by CD4 + , CD25 + , and Foxp3 + . Regulatory T cells can have an ability to suppress the anti-tumor growth of T cells.

[0176] In the present application, the term "activated T cells" generally refers to T cells that have been activated and can have an ability to resist tumor growth. Activated T cells can have a phenotype of PD-1 + (PD1 + ), LAG-3 + (LAG3 + ), or CD28 + , for example, activated T cells can be identified by PD-1 + , LAG-3 + , or CD28 + . Activated T cells can have an ability to resist tumor growth.

[0177] In the present application, the term "tumor-specific T cells" generally refers to T cells that can specifically resist tumor growth. Tumor-specific T cells can have a phenotype of CD103 + CD39 + , for example, tumor-specific T cells can be identified by CD103+ and CD39 + Tumor-specific T cells can have a more specific ability to fight tumor growth than ordinary T cells.

[0178] In the present application, the term "stem cell-like T cell" generally refers to a type of T cell that can have the potential to self-renew and / or differentiate. For example, a cell that has the potential to differentiate and / or the ability to continue to proliferate in the present application can be considered a stem cell-like cell. For example, a naive T cell (CD45RO - CD62L + ) can be considered a stem cell-like cell. For example, a naive T cell can have the phenotype of CD45RO - CD62L + . For example, a stem cell-like T cell can be identified by CD45RO - CD62L + . For example, a stem cell-like T cell can be identified by CD39 - CD69 - . For example, a stem cell-like T cell can have the phenotype of TCF1 + . For example, a stem cell-like T cell can be identified by TCF1 + . A stem cell-like T cell can have a stronger and / or longer term ability to fight tumor growth than ordinary T cells.

[0179] In the present application, the term "tumor "fragment" generally refers to a tumor fragment that can be formed by mechanical disruption, enzymatic digestion, and / or other disruption methods after tumor tissue is removed from a subject.

[0180] In the present application, the term "composition" or "pharmaceutical composition" generally refers to a mixture of at least one cell and at least one and optionally more than one other pharmaceutically acceptable chemical component such as a carrier, a stabilizer, a diluent, a dispersant, a suspending agent, a thickening agent, and / or an excipient.

[0181] In the present application, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the activity of the active ingredient. For example, a pharmaceutically acceptable carrier can not interfere with the biological activity of the active ingredient; for example, a pharmaceutically acceptable carrier can not interfere with the effectiveness of the biological activity possessed by the active ingredient. Such formulations can routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations can also contain compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that can be used in the formulations described herein can include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), 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. In the present application, "pharmaceutically acceptable carrier" can be understood as a vector that does not contain nucleic acid used for genetic engineering.

[0182] In the present application, 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 can retain or partially retain the ability of the full-length protein to bind to another molecule.

[0183] In the present application, the term "T cell activating agent" generally refers to a substance that binds to a corresponding binding receptor on a T cell and mediates a T cell costimulatory response. The T cell activating agent can be a substance other than an antigen receptor that is required for a T cell to produce an effective immune response. The T cell activating agent can refer to a T cell costimulatory molecule. For example, the T cell activating agent of the present application can include a variant thereof, a homolog thereof, or any substance comprising a functionally active fragment thereof. The T cell activating agent can include, but is not limited to, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signal lymphocyte activation molecule (SLAM protein), an NK cell activating receptor, BTLA (the gene encoding which can be Gene ID 151888), a Toll ligand receptor, OX40 (the gene encoding which can be Gene ID 7293), CD2 (the gene encoding which can be Gene ID 914), CD7 (the gene encoding which can be Gene ID 924), CD27 (the gene encoding which can be Gene ID 939), CD28 (the gene encoding which can be Gene ID 940), CD30 (the gene encoding which can be Gene ID 943), CD40 (the gene encoding which can be Gene ID 958), CDS, ICAM-1 (the gene encoding which can be Gene ID 3383), LFA-1 (CD11a / CD18) (the gene encoding which can be Gene ID 3689), 4-1BB (CD137) (the gene encoding which can be Gene ID 3604), B7-H3 (the gene encoding which can be Gene ID 80381), ICOS (CD278) (the gene encoding which can be Gene ID 29851), GITR (the gene encoding which can be Gene ID 8784), BAFFR (the gene encoding which can be Gene ID 115650), LIGHT (the gene encoding which can be Gene ID 8740), HVEM (LIGHTR) (the gene encoding which can be Gene ID 8764), KIRDS2 (the gene encoding which can be Gene ID 100132285), SLAMF7 (the gene encoding which can be Gene ID 57823), NKp80 (KLRF1) (the gene encoding which can be Gene ID 51348), NKp44 (the gene encoding which can be Gene ID 9436), NKp30 (the gene encoding which can be Gene ID 259197), NKp46 (the gene encoding which can be Gene ID 9437), CD19 (the gene encoding which can be Gene ID 930), CD4 (the gene encoding which can be Gene ID 920), CD8a (the gene encoding which can be Gene ID 925), CD8b (the gene encoding which can be Gene ID 926),IL-2Rβ, IL-2Rγ, IL7Rα (GeneID of the gene encoding it can be 3575), ITGA4 (GeneID of the gene encoding it can be 3676), VLA1 (GeneID of the gene encoding it can be 3672), CD49a (GeneID of the gene encoding it can be 3672), IA4 (GeneID of the gene encoding it can be 3732), CD49D (GeneID of the gene encoding it can be 3676), ITGA6 (GeneID of the gene encoding it can be 3655), VLA-6 (GeneID of the gene encoding it can be 3655), CD49f (GeneID of the gene encoding it can be 3655), ITGAD (GeneID of the gene encoding it can be 3681), CD11d (GeneID of the gene encoding it can be 3681), ITGAE (GeneID of the gene encoding it can be 3682), CD103 (GeneID of the gene encoding it can be 3682), ITGAL (GeneID of the gene encoding it can be 3683), CD11a (GeneID of the gene encoding it can be 3683), LFA-1 (GeneID of the gene encoding it can be 3683), ITGAM (GeneID of the gene encoding it can be 3684), CD11b (GeneID of the gene encoding it can be 3684), ITGAX (GeneID of the gene encoding it can be 3687), CD11c (GeneID of the gene encoding it can be 3687), ITGB1 (GeneID of the gene encoding it can be 3688), CD29 (GeneID of the gene encoding it can be 3688), ITGB2 (GeneID of the gene encoding it can be 3689), CD18 (GeneID of the gene encoding it can be 3689), LFA-1 (GeneID of the gene encoding it can be 3689), ITGB7 (GeneID of the gene encoding it can be 3695), NKG2D (GeneID of the gene encoding it can be 22914), NKG2C (GeneID of the gene encoding it can be 3822), TNFR2 (GeneID of the gene encoding it can be 7133), TRANCE / RANKL (GeneID of the gene encoding it can be 8600), DNAM1 (CD226) (GeneID of the gene encoding it can be 10666), SLAMF4 (CD244, 2B4) (GeneID of the gene encoding it can be 51744), CD84 (GeneID of the gene encoding it can be 8832), CD96 (Tactile) (GeneID of the gene encoding it can be 10225), CEACAM1 (GeneID of the gene encoding it can be 634),CRTAM (GeneID of the gene encoding it can be 56253), Ly9 (CD229) (GeneID of the gene encoding it can be 4063), CD160 (BY55) (GeneID of the gene encoding it can be 11126), PSGL1 (GeneID of the gene encoding it can be 6404), CD100 (SEMA4D) (GeneID of the gene encoding it can be 10507), CD69 (GeneID of the gene encoding it can be 969), SLAMF6 (NTB-A, Ly108) (GeneID of the gene encoding it can be 114836), SLAM (SLAMF1, CD150, IPO-3) (GeneID of the gene encoding it can be 6504), BLAME (SLAMF8) (GeneID of the gene encoding it can be 56833), SELPLG (CD162) (GeneID of the gene encoding it can be 6404), LTBR (GeneID of the gene encoding it can be 4055), LAT (GeneID of the gene encoding it can be 27040), GADS (GeneID of the gene encoding it can be 9402), SLP-76 (GeneID of the gene encoding it can be 3937), PAG / Cbp (GeneID of the gene encoding it can be 55824), CD19a, a ligand that specifically binds CD3, a ligand that specifically binds CD28, a ligand that specifically binds HVEM, a ligand that specifically binds CD40L, a ligand that specifically binds OX40, and a ligand that specifically binds 4-1BB. A costimulatory intracellular signaling domain can refer to the intracellular portion of a T cell activator. An intracellular signaling domain can comprise the entire intracellular portion of a molecule from which it is derived or the entire native intracellular signaling domain or a functional fragment thereof.

[0184] In the present application, 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 application can comprise a variant thereof, a homolog thereof, or any agent comprising a functionally active fragment thereof. In one embodiment, the T cell growth factor can be selected from one or more of the following group: IL-2 (the gene encoding which can be GeneID 3558), IL-4 (the gene encoding which can be GeneID 3565), IL-6 (the gene encoding which can be GeneID 3569), IL-7 (the gene encoding which can be GeneID 3574), IL-10 (the gene encoding which can be GeneID 3586), IL-12 (the gene encoding which can be GeneID 3592 or 3593), IL-15 (the gene encoding which can be GeneID 3600), IL-21 (the gene encoding which can be GeneID 59067), TNF-a (the gene encoding which can be GeneID 100137091), gamma interferon (the gene encoding which can be GeneID 3458), GZMB (the gene encoding which can be GeneID 3002), CD 107a (the gene encoding which can be GeneID 6499), and the like.

[0185] In the present application, the term "substantially simultaneously" generally refers to a period of time during which the TILs can be contacted with two or more agents simultaneously, but can not be limited to the entire period of contact during which the TILs are always contacted with two or more agents. In one embodiment, substantially simultaneously can refer to a period of time during which the TILs can be contacted with each of the two or more agents simultaneously for at least 10-95%, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%.

[0186] In the present application, the term "dendritic cell" generally refers to an antigen presenting cell that is present in vivo, in vitro, ex vivo, or in a host or subject, or can be derived from hematopoietic stem cells or monocytes. Dendritic cells and their precursors can be isolated from various lymphoid organs, such as the spleen, lymph nodes, and bone marrow and peripheral blood. The dendritic cells of the present application can have a characteristic morphology, such as thin sheets (lamellipodia) extending in multiple directions from the dendritic cell body. In general, dendritic cells can express high levels of MHC and costimulatory (e.g., B7-1 and B7-2) molecules. Dendritic cells can induce antigen-specific differentiation of T cells in vitro, and are capable of priming primary T cell responses in vitro and in vivo.

[0187] In the present invention, the term "in vitro expansion" generally refers to a change in the number of cells through culturing, and the expanded cells can also have a change in the number and / or ratio of cells, a change in the secretion ability, a change in the killing ability, or a change in the expression ability, or any combination thereof. The change in the present invention can be an increase or a decrease. In the present invention, in vitro expansion can be for the purpose of expansion; a procedure performed on TIL cells for the purpose of detecting the function of TIL cells, such as detecting the cytokine releasing ability of TIL cells (e.g., adding one or more substances to the culture medium of TIL cells to detect the cytokine releasing ability of TIL cells), can not belong to the in vitro expansion of the present invention.

[0188] In the present invention, the term "peripheral mononuclear cell" or "peripheral blood mononuclear cell" generally refers to a cell having a single nucleus in the peripheral blood. For example, in the present invention, the peripheral blood mononuclear cell of the present invention can include a lymphocyte, a monocyte, and / or a dendritic cell.

[0189] In the present invention, the term "cytokine" generally refers to a protein released by one cell 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 an interleukin (IL) such as IL-1, IL-1a, 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 a protein from a natural source or from a recombinant cell culture, a biologically active equivalent of a naturally occurring sequence cytokine, and a functionally active fragment thereof.

[0190] In the present invention, the term "diameter" generally refers to the diameter of the 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 the average diameter of the largest cross section of the substance of the present invention. The method for determining the diameter of the substance can be a method generally used in the art, such as transmission electron microscopy.

[0191] In the present invention, the term "tumor" generally refers to any new pathological tissue proliferation. The tumor of the present invention can be benign or malignant. The tumor of the present invention can be solid or hematological. The term "tumor" can be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and renal cancer.

[0192] In the present application, the term "tumor tissue" generally refers to a sample of any tissue from a tumor in a subject, including any tissue of any solid tumor and / or non-solid tumor in a subject.

[0193] In the present application, the term "T cell subpopulation ratio" generally refers to the ratio of different T cell subpopulations in TIL cells or TIL population. For example, different T cell subpopulations in the present application have different immune activities and / or differentiation abilities. For example, T cell subpopulations in the present application can be distinguished according to T cell surface markers. For example, central memory T cells can have the phenotype of CD45RO + CD62L + . For example, naive T cells can have the phenotype of CD45RO - CD62L + . For example, regulatory T cells can have the phenotype of CD4 + CD25 + Foxp3 + . For example, activated T cells can have the phenotype of CD25 + , CD28 + , PD-1 + or 41BB + . For example, tumor-specific T cells can have the phenotype of CD103 + CD39 + . For example, stem cell-like T cells can have the phenotype of TCF1 + .

[0194] In the present application, the term "TIL cell number" generally refers to the number of cells in TIL cells in the present application. In the present application, TIL cell number can refer to the number of cells in a TIL population obtained at any stage of the present application. For example, TIL cell number can refer to the number of cells in a first TIL population derived from tumor tissue and not subjected to in vitro expansion. For example, TIL cell number can refer to the number of cells in a second TIL population subjected to in vitro expansion at a first stage. For example, TIL cell number can refer to the number of cells in a third TIL population subjected to in vitro expansion at a second stage. For example, TIL cell number can refer to the number of cells in TILs finally obtained by any one of the culture methods of the present application. In the present application, TIL cell number can be measured by methods commonly used in the art, for example, can include but are not limited to manual cell counting by cell counting plate and / or automatic cell counter counting.

[0195] In the present invention, the terms "about" and "approximately" generally refer to a range of values that is statistically meaningful. Such a range can be within one order of magnitude of a given value or range, can include within 50%, preferably within 20%, more preferably within 10%, most preferably within 5%. The allowable variation encompassed by the terms "about" or "approximately" can depend on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0196] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0197] DETAILED DESCRIPTION

[0198] Cas12 nuclease

[0199] The present invention provides engineered Cas12 nucleases, which have improved activities, such as improved gene editing activities and / or improved cell properties of cells after gene editing, when used for target genes provided in the present invention.

[0200] In one aspect, the Cas12 nuclease editing system of the present invention can comprise a Cas12 nuclease and / or a guide nucleic acid. For example, the Cas12 nuclease is capable of associating with each other in the presence of a guide nucleic acid (e.g., guide RNA) comprising a guide sequence (targeting domain, a domain capable of binding to a target sequence) to form a CRISPR complex. For example, the CRISPR complex can specifically bind to a target nucleic acid comprising a target sequence complementary to the guide sequence. For example, the Cas12 nuclease of the present invention can comprise a Cas12b (also known as C2c1) nuclease or a variant or functional derivative thereof. For example, the Cas12 nuclease of the present invention can comprise a AaCas12b (Cas12b nuclease from Alicyclobacillus acidophilus).

[0201] For example, the present invention comprises an engineered Cas12b nuclease or a variant or functional derivative thereof, which can function as a genome editing enzyme, such as a single-stranded or double-stranded nuclease that cleaves double-stranded nucleic acid, when forms a complex with a target DNA and a guide RNA. For example, the sequence cleaved by Cas12b can have a sticky end. For example, Cas9 nuclease is known to comprise a HNH nuclease subdomain and a RuvC1 subdomain. For example, Cas12b nuclease can not comprise a HNH nuclease subdomain. For example, Cas12b nuclease can not comprise a HNH nuclease subdomain with nuclease activity.

[0202] or for example, the application includes engineered Cas12b nuclease can be a catalytically dead Cas12b (dCas12b), which is essentially a DNA binding protein that can have little or no catalytic activity due to typical mutations in its catalytic domain. Mutations of one or more amino acid residues in the active site of an exemplary Cas12b can result in a catalytically dead Cas12b, such as D570A, E848A, R785A, E848A, R911A, and / or D977A mutants of AaCas12b.

[0203] In another aspect, the application also provides engineered Cas12b effector proteins that further comprise additional protein domains and / or components, such as linkers, nuclear localization / export sequences, functional domains, and / or reporter proteins. For example, the engineered Cas12b nuclease in the engineered Cas12b effector protein can be a dCas12b. For example, the engineered Cas12b effector protein, upon binding to a target nucleic acid, can have a function of epigenetic modification, increasing gene expression, and / or decreasing memory expression. In some embodiments, the engineered Cas12b effector protein is a protein complex. In some embodiments, the engineered Cas12b effector protein of the application can comprise or associate with one or more functional domains (e.g., via a fusion protein, such as via one or more peptide linkers, e.g., a GS peptide linker, etc.), such as via co-expression of multiple proteins. In some embodiments, the one or more functional domains is an enzymatic domain. These functional domains can have various activities, such as DNA and / or RNA methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and switch activity (e.g., light-induced). In some embodiments, the one or more functional domains is a transactivation domain (i.e., transactivation domain) or a repressor domain. In some embodiments, the transactivation domain or the repressor domain can recruit chromatin modifiers. In some embodiments, the one or more functional domains is a histone modification domain. In some embodiments, the one or more functional domains is a transposase domain, an HR (homologous recombination) machinery domain, a recombinase domain, and / or an integrase domain.

[0204] In some embodiments, the functional domain is Krüppel-associated box (KRAB), VP64, VP16, Fok1, P65, HSF1, MyoD1, biotin-APEX, APOBEC1, AID, PmCDA1, Tad1, and M-MLV reverse transcriptase. In some embodiments, the functional domain is selected from the group consisting of a translational promoter domain, a transcriptional repressor domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., a CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), and a nuclease domain. In some embodiments, the functional domain is a KRAB domain, such as the KRAB domain of ZIM3. In some embodiments, the positioning of one or more functional domains in the engineered Cas12b effector protein can allow the functional domain to be in the correct spatial orientation, thereby affecting the function and / or activity of the target sequence.

[0205] In some embodiments, the engineered Cas12b effector protein is a transcriptional activator. In some embodiments, the transactivation domain is selected from the group consisting of VP64, p65, HSF1, VP16, MyoD1, HSF1, RTA, SET7 / 9, and combinations thereof. In some embodiments, the transactivation domain comprises VP64, p65, and HSF1.

[0206] In some embodiments, the engineered Cas12b effector protein is a transcriptional repressor. In some embodiments, the engineered Cas12b effector protein comprises an enzymatically inactive variant of any of the engineered Cas12b nucleases described herein fused to a transcriptional repressor domain (e.g., KRAB). In some embodiments, the transcriptional repressor domain is selected from the group consisting of Krüppel-associated box (KRAB), EnR, NuE, NcoR, SID, SID4X, and combinations thereof.

[0207] In some embodiments, the engineered Cas12b effector protein is a base editor, such as a cytosine editor or an adenosine editor. In some embodiments, the engineered Cas12b effector protein comprises an enzymatically inactive variant of any of the engineered Cas12b nucleases described herein fused to an editing nucleobase editing domain, such as a cytosine base editing (CBE) domain or an adenosine base editing (ABE) domain.

[0208] In some embodiments, the engineered Cas12b effector protein comprises one or more nuclear localization sequences (NLS) and / or one or more nuclear export sequences (NES). In some embodiments, the engineered Cas12b effector protein can encode for additional components, such as a reporter protein. In some embodiments, the engineered Cas12b effector protein comprises a fluorescent protein, e.g., GFP.

[0209] CRISPR-Cas12b editing system

[0210] In another aspect, the present disclosure also provides an engineered CRISPR-Cas12b system, comprising: (a) an engineered Cas12b nuclease or a variant or derivative thereof or an engineered Cas12b effector protein (e.g., an engineered Cas12b nuclease, a nickase, a nicking Cas12b protein, a transcriptional repressor, a transcriptional activator, a base editor, or a prime editor), or a nucleic acid encoding the above-mentioned enzyme and / or effector protein; and (b) a guide RNA comprising a guide sequence that is complementary to a target sequence of a target nucleic acid, or one or more nucleic acids encoding the guide RNA. For example, the engineered Cas12b nuclease or the engineered Cas12b effector protein can form a CRISPR complex with the guide RNA, which specifically binds to the target nucleic acid comprising the target sequence and induces editing of the target nucleic acid.

[0211] In some embodiments, the engineered CRISPR-Cas12b system comprises one or more nucleic acids encoding the engineered Cas12b nuclease or a variant or derivative thereof or the engineered Cas12b effector protein, and / or the guide RNA. In some embodiments, the engineered CRISPR-Cas12b system comprises one or more gene expression vectors encoding the engineered Cas12b nuclease or a variant or derivative thereof or the engineered Cas12b effector protein, and / or the guide RNA. In some embodiments, the engineered Cas12b nuclease or a variant or derivative thereof or the engineered Cas12b effector protein, and / or the guide RNA are encoded by one or more vectors (e.g., adeno-associated virus (AAV) vectors). In some embodiments, the engineered CRISPR-Cas12b system comprises a ribonucleoprotein (RNP) complex comprising the engineered Cas12b nuclease or a variant or derivative thereof or the engineered Cas12b effector protein bound to the guide RNA, and / or nucleic acids encoding the above-mentioned substances. In some embodiments, the engineered CRISPR-Cas12b system comprises an LNP vector comprising the engineered Cas12b nuclease or a variant or derivative thereof or the engineered Cas12b effector protein bound to the guide RNA, and / or nucleic acids encoding the above-mentioned substances.

[0212] For example, the present disclosure provides protospacer adjacent motifs (PAMs) for Cas12b. In some embodiments, the engineered Cas12b nuclease or variant or derivative thereof, the engineered Cas12b effector protein, the Cas12b nuclease, or the Cas12b effector protein recognizes a PAM comprising (or consisting of) a 5’-TTN-3’ sequence, where N is A, T, G, or C. In some embodiments, the PAM comprises or consists of 5’-TTC-3’, 5’-TTA-3’, 5’-TTT-3’, or 5’-TTG-3’. For example, a target nucleic acid can be associated with a short sequence recognized by the PAM, i.e., the CRISPR complex. The target sequence is selected according to the nature of the CRISPR-Cas protein, such that its complementary sequence in the DNA double strand (the complement of the target sequence) is located upstream or downstream of the PAM. In one embodiment of the present disclosure, the complement of the target sequence is located downstream or 3’ of the PAM (i.e., the PAM sequence is located 5’ of the guide sequence). The exact sequence and length of the PAM can be adjusted as desired.

[0213] Guide RNA

[0214] For example, the present disclosure provides any suitable guide RNA. A guide RNA (gRNA) can comprise a guide sequence capable of hybridizing to a target sequence in a target nucleic acid of interest. For example, a gRNA can comprise a scaffold sequence and / or a guide sequence. In some embodiments, a gRNA comprises a non-cognate gRNA that does not naturally occur in the CRISPR locus of a reference Cas12b protein. For example, a cognate gRNA for AaCas12b, AkCas12b, AmCas12b, BhCas12b, BsCas12b, Bs3Cas12b, LsCas12b, and SbCas12b. In some embodiments, a CRISPR-Cas12b system described herein comprises one or more gRNAs (e.g., 1, 2, 3, 4, 5, 10, 15, or more), or a nucleic acid encoding the same. In some embodiments, two or more gRNAs target different target sites, e.g., 2 target sites of the same target DNA or gene, or 2 target sites of 2 different target DNAs or genes.

[0215] For example, a guide sequence can have a suitable length. In some embodiments, a guide sequence or spacer is about 10 nucleotides to about 100 nucleotides in length. In some embodiments, a guide sequence or spacer is at least about 16 nucleotides in length, preferably about 16 to about 100 nucleotides, more preferably about 16 to about 50 nucleotides (e.g., any of about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides). In some embodiments, the spacer is about 16 to about 27 nucleotides, such as any of about 17 to about 24 nucleotides, about 18 to about 24 nucleotides, about 18 to about 23 nucleotides, or about 18 to about 22 nucleotides. In some embodiments, a guide sequence is about 18 to about 35 nucleotides, including, for example, any of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0216] In some embodiments, a guide sequence is at least about 60% (e.g., at least about any of 70%, 75%, 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementary to a target sequence. In some embodiments, there are at least about 15 (e.g., at least about any of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more) base pairs between a guide sequence and a target sequence of a target nucleic acid (e.g., DNA).

[0217] In some embodiments, the nucleic acid of the application comprises a base modification. In some embodiments, the gRNA comprises a base modification. In some embodiments, the scaffold sequence and / or the guide sequence comprises a base modification. In some embodiments, the nucleic acid is or comprises a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); a chemically modified base; a biologically modified base (e.g., a methylated base); an intervening base; a modified sugar (2’- e.g., fluoro ribose, ribose, 2’-deoxyribose, arabinose, and hexose); and / or a modified phosphate group (e.g., phosphorothioate and 5’-N-phosphoramidite linkages).

[0218] In some embodiments, the sgRNA (single guide RNA) comprises a scaffold sequence (Frame) comprising a stem-loop structure (e.g., 1, 2, 3, 4, or more stem loops) located near the 5’ end of the guide sequence. In some embodiments, the stem comprises a stem of at least about 4 bp (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) or fewer (e.g., 3, 2) base pairs. In some embodiments, the stem comprised in the scaffold sequence comprises (e.g., consists of) 5 pairs of complementary bases that hybridize to each other, and the loop length is 6, 7, 8, or 9 nucleotides. In some embodiments, the stem-loop structure comprises a first stem nucleotide chain of 5 nucleotides in length; a second stem nucleotide chain of 5 nucleotides in length, wherein the first and second stem nucleotide chains can hybridize to each other; and a loop nucleotide chain arranged between the first and second stem nucleotide chains, wherein the loop nucleotide chain comprises 6, 7, or 8 nucleotides. In some embodiments, the scaffold sequence of a gRNA that can guide any of the engineered Cas12b effector proteins of the application to a target site can comprise one or more nucleotide changes selected from the group consisting of nucleotide additions, insertions, deletions, and substitutions that do not result in a substantial difference in secondary structure compared to the scaffold sequence set forth in SEQ ID NO: 5109 or a functionally truncated version thereof. In some embodiments, the gRNA scaffold comprises the sequence of SEQ ID NO: 5109, or a variant thereof comprising up to about 10 nucleotide (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide) differences.

[0219] For example, a scaffold sequence can have base modifications. For example, one or more base modifications can be selected from: inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A “nucleotide” consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. For example, a base modification of the present invention can comprise a 2’-O-methyl modification and / or a 3’ thiophosphate internucleoside linkage. For example, a scaffold sequence of the present invention can have a base modification at 1 to 93 nucleotides, for example, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 93 nucleotides. For example, a scaffold sequence of the present invention can have a base modification at a nucleotide position of SEQ ID NO: 5109 selected from: positions 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 33, 34, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 81, 83, 84, 85. For example, a scaffold sequence of the present invention can have a 2’-O-methyl modification and / or a 3’ thiophosphate internucleoside linkage at nucleotides 1, 2, 3, a 2’-O-methyl modification at nucleotides 26 to 34, and a 2’-O-methyl modification at nucleotides 70 to 85 of SEQ ID NO: 5109. For example, a scaffold sequence of the present invention can have nucleotide modifications of SEQ ID NO: 5109, for example, can be:

[0220] mC*mC*mA*GGUCGUCUAUAGGACGGCGAGUmUmUmUmUmCmAmAmCmGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCmCmGmUmUmGmAmGmCmUmUmCmAmAmAmGmAAGUGGCAC (wherein m represents: 2’-O-methyl modification, * represents: 3’ thiophosphate internucleoside linkage).

[0221] For example, a guide sequence can have base modifications. For example, one or more base modifications can be selected from: inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. For example, a base modification of the present application can comprise a 2'-O-methyl modification and / or a 3' phosphorothioate internucleoside linkage. For example, a 3' end and / or a 5' end of a guide sequence of the present application can be linked to 1 to 10 nucleotides with base modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides with base modifications added to the 3' end of the guide sequence. For example, a guide sequence of the present application can have 1, 2, or 3 nucleotides with 2'-O-methyl modification and / or 3' phosphorothioate internucleoside linkage added to the 3' end. For example, nucleotides with base modifications added to the 3' end of a guide sequence of the present application can have any sequence, for example, UUU, GCA, AGC, GGC, UGA, UGG, AGG, GAG, GGG. For example, nucleotides with base modifications added to the 3' end of a guide sequence of the present application can comprise: *mG*mC*mA, *mA*mG*mC, *mG*mG*mC, *mU*mG*mA, *mU*mG*mG, *mA*mG*mG, *mG*mA*mG, *mG*mG*mG (where m represents: 2'-O-methyl modification, * represents: 3' phosphorothioate internucleoside linkage).

[0222] For example, when a target gene of the present application needs to be edited, the present application provides an exemplary target sequence corresponding to the target gene, a target segment comprising the target sequence, and / or a guide sequence capable of binding to the target sequence, a guide nucleic acid (such as a guide RNA) comprising the guide sequence. In some embodiments, the guide RNA (gRNA) is a single-stranded guide RNA (sgRNA). For example, the sgRNA can comprise a scaffold sequence and a guide sequence.

[0223] Knockout of a target gene or a combination thereof

[0224] The present application provides a method of reducing expression and / or attenuating activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family and / or functionally active fragments thereof in a cell. For example, the present application uses a Cas12 editing system to reduce expression and / or attenuate activity of the one or more family members and / or functionally active fragments thereof. For example, the Cas12 editing system of the present application comprises a Cas12 nuclease, a guide nucleic acid, and / or a nucleic acid encoding the same. The present application provides a target sequence corresponding to a target gene or a combination thereof, a target segment comprising the target sequence, and a guide sequence capable of binding to the target sequence, a guide nucleic acid comprising the guide sequence.

[0225] In one aspect, the present application provides a method of culturing a cell to reduce expression and / or attenuate activity of a Peptidase C64 family member and / or a functionally active fragment thereof in the cell. For example, the Peptidase C64 family member can comprise a ubiquitin binding domain. For example, the Peptidase C64 family member can comprise TNFAIP3.

[0226] For example, the target gene of the present application can be a gene encoding a Peptidase C64 family member and / or a functionally active fragment thereof. For example, a cell obtained by reducing expression and / or attenuating activity of at least one target gene in the cell can exhibit improved cell properties as compared to a cell in which expression and / or activity of the target gene is not changed. In one embodiment, the cell in which expression and / or activity of the target gene is not changed can refer to a cell derived from the same donor and in which expression and / or activity of at least one target gene in the cell has not been reduced and / or attenuated. In one embodiment, the cell in which expression and / or activity of the target gene is not changed can refer to a cell derived from the same donor and in which expression of another gene other than the target gene in the cell has been reduced and / or attenuated (e.g., the other gene is knocked out, which has substantially no effect on cell function).

[0227] In one aspect, the present application provides a method of culturing a cell to reduce expression and / or attenuate activity of a ZC3H12 family member and / or a functionally active fragment thereof in the cell. For example, the ZC3H12 family member can comprise a C3H1-type zinc finger domain. For example, the ZC3H12 family member can comprise ZC3H12A.

[0228] For example, the target gene of the present application can be a gene encoding a member of the ZC3H12 family and / or a functionally active fragment thereof. For example, a cell obtained by reducing expression and / or attenuating activity of at least one target gene of the cell compared to a cell in which expression and / or activity of the target gene is not changed can exhibit improved cell properties. In one embodiment, the cell in which expression and / or activity of the target gene is not changed can mean a cell derived from the same donor and in which expression and / or activity of at least one target gene of the cell has not been reduced and / or attenuated. In one embodiment, the cell in which expression and / or activity of the target gene is not changed can mean a cell derived from the same donor and in which expression of another gene other than the target gene of the cell (e.g., the other gene is knocked out, which has substantially no effect on cell function) has not been reduced and / or attenuated.

[0229] In one aspect, the present application provides a method of culturing a cell, in which expression and / or activity of a member of the STAT-induced STAT suppressor (SSI) family and / or a functionally active fragment thereof of the cell is reduced and / or attenuated. For example, the member of the STAT-induced STAT suppressor (SSI) family can comprise an SH2 domain. For example, the member of the STAT-induced STAT suppressor (SSI) family can comprise SOCS1.

[0230] For example, the target gene of the present application can be a gene encoding a member of the STAT-induced STAT suppressor (SSI) family and / or a functionally active fragment thereof. For example, a cell obtained by reducing expression and / or attenuating activity of at least one target gene of the cell compared to a cell in which expression and / or activity of the target gene is not changed can exhibit improved cell properties. In one embodiment, the cell in which expression and / or activity of the target gene is not changed can mean a cell derived from the same donor and in which expression and / or activity of at least one target gene of the cell has not been reduced and / or attenuated. In one embodiment, the cell in which expression and / or activity of the target gene is not changed can mean a cell derived from the same donor and in which expression of another gene other than the target gene of the cell (e.g., the other gene is knocked out, which has substantially no effect on cell function) has not been reduced and / or attenuated.

[0231] In one aspect, the present application provides a method of culturing a cell, in which expression and / or activity of a member of at least two families selected from the group consisting of the peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT suppressor (SSI) family and / or a functionally active fragment thereof of the cell is reduced and / or attenuated.

[0232] For example, the peptidase C64 family member can comprise a ubiquitin binding domain. For example, the peptidase C64 family member can comprise TNFAIP3. For example, the ZC3H12 family member can comprise a C3H1 type zinc finger domain. For example, the ZC3H12 family member can comprise ZC3H12A. For example, the STAT-induced STAT inhibitor (SSI) family member can comprise an SH2 domain. For example, the STAT-induced STAT inhibitor (SSI) family member can comprise SOCS1.

[0233] For example, the cell further can comprise reduced expression and / or attenuated activity of optionally selected from CBLB, BRD4, FAS, FIBP, IKZF1, LAG3, MED12, PD1, RASA2, TIGIT, TIM3, ADNP, NFKBIA, PTPN6, and TNIP1.

[0234] For example, in the cell of the application, expression of a peptidase C64 family and a ZC3H12 family member and / or a functionally active fragment thereof is reduced and / or attenuated. For example, expression of TNFAIP3 and ZC3H12A is reduced and / or attenuated.

[0235] For example, in the cell of the application, expression of a peptidase C64 family and a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof is reduced and / or attenuated. For example, expression of TNFAIP3 and SOCS1 is reduced and / or attenuated.

[0236] For example, in the cell of the application, expression of a ZC3H12 family and a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof is reduced and / or attenuated. For example, expression of ZC3H12A and SOCS1 is reduced and / or attenuated.

[0237] For example, in the cell of the application, expression of a peptidase C64 family, a ZC3H12 family and a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof is reduced and / or attenuated. For example, expression of TNFAIP3, ZC3H12A and SOCS1 is reduced and / or attenuated.

[0238] For example, the gene of interest can be a gene encoding a member of at least 1-3 (e.g., at least 2) families selected from the group consisting of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof. For example, a cell having reduced expression and / or attenuated activity of at least 1-3 (e.g., at least 2) genes of interest can exhibit improved cell properties as compared to a cell having unaltered expression and / or activity of the genes of interest. In one embodiment, a cell having unaltered expression and / or activity of the genes of interest can refer to a cell derived from the same donor and which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell. In one embodiment, a cell having unaltered expression and / or activity of the genes of interest can refer to a cell derived from the same donor and which has not been subjected to reduction of expression and / or attenuation of activity of a gene other than the genes of interest (e.g., the other gene is knocked out without substantially affecting cell function) of the cell.

[0239] In one embodiment, a corresponding cell which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell can refer to a cell derived from the same donor and isolated by the same manner and which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell. In one embodiment, a corresponding cell which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell can refer to a cell derived from the same donor and from the same tumor source and which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell. In one embodiment, a corresponding cell which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell can refer to a cell derived from the same donor and from the same tumor source and which has been divided into two groups, wherein the group of cells which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell can be the corresponding cell which has not been subjected to reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest of the cell. For example, reduction of expression and / or attenuation of activity of at least 1-3 (e.g., at least 2) genes of interest can refer to a certain level of expression of the gene of interest in a native cell, and the treatment according to the present application can result in a reduction of the expression of the gene of interest in the cell, i.e., the reduction of expression of the gene of interest can be a change from expressing the gene of interest to substantially not expressing the gene of interest or a reduction in the amount of expression of the gene of interest in the cell.

[0240] For example, the cells comprise immune cells. For example, the cells comprise immune effector cells. For example, the cells comprise immune effector T cells, immune effector NK cells, immune effector NKT cells. For example, the cells comprise phagocytes, lymphocytes, neutrophils, eosinophils, and / or basophils.

[0241] For example, the cells comprise monocytes, macrophages, and / or dendritic cells.

[0242] For example, the cells of the application further comprise cells derived from differentiation of stem cells. For example, the cells of the application further comprise cells derived from differentiation of pluripotent stem cells. For example, the stem cells of the application can be obtained by induction. For example, the above-mentioned stem cells of the application can comprise induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells, and / or peripheral blood stem cells.

[0243] For example, the "stem cells" of the application also include multipotent cells, pluripotent 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 germinal tissue of a fetus. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs".

[0244] For example, the cells comprise B cells, T cells, natural killer cells, and / or natural killer T cells (NKT). For example, "unmodified cells" or "unengineered cells" can refer to cells or cell populations in which the genome has not been modified and do not comprise a gene regulation system or comprise a control gene regulation system (e.g., empty vector control, non-targeting gRNA, interfering siRNA, etc.). For example, the cells comprise αβ T cells and / or γδ T cells. For example, the cells comprise tumor infiltrating lymphocytes (TILs). For example, the TILs are TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion, and / or peritoneal effusion and / or derived from TILs recovered after cryopreservation.

[0245] For example, the TILs of the application can be TILs derived from tumor tissue, tumor associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of cancer adjacent tissue, pleural effusion and / or peritoneal effusion and / or derived from TILs recovered after cryopreservation. For example, the TILs of the application can be obtained by processing tumor tissue into tumor fragments. For example, the tumor fragments of the application have a volume of about 1-27 cubic millimeters. For example, the tumor fragments of the application 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.

[0246] For example, the cells comprise an engineered immune receptor displayed on the surface of the cell. For example, the engineered immune receptor specifically binds to an antigen expressed on a target cell. For example, the cells comprise a chimeric antigen receptor and / or a T cell receptor.

[0247] In one aspect, the application provides a method of culturing tumor infiltrating lymphocytes (TILs), which can comprise reducing expression and / or activity in the TILs of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof.

[0248] For example, TILs derived from tumor tissue, pleural effusion and / or peritoneal effusion and not subjected to in vitro expansion can be subjected to at least one stage of in vitro expansion, wherein in at least one stage of the in vitro expansion, expression and / or activity in the TILs of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof can be reduced.

[0249] For example, TILs derived from tumor tissue, pleural effusion and / or peritoneal effusion and not subjected to in vitro expansion of the application 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 application, expression and / or activity in the TILs of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof can be reduced.

[0250] For example, TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not subjected to ex vivo expansion according to the present application can be subjected to a first stage of ex vivo expansion and a second stage of ex vivo expansion, and in the first stage of ex vivo expansion according to the present application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced.

[0251] For example, TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not subjected to ex vivo expansion according to the present application can be subjected to a first stage of ex vivo expansion and a second stage of ex vivo expansion, and in the first stage of ex vivo expansion according to the present application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced, and in the second stage of ex vivo expansion according to the present application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced.

[0252] For example, TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not subjected to ex vivo expansion according to the present application can be subjected to a first stage of ex vivo expansion, a second stage of ex vivo expansion and a third stage of ex vivo expansion, and in the first stage of ex vivo expansion according to the present application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced.

[0253] For example, TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not subjected to ex vivo expansion according to the present application can be subjected to a first stage of ex vivo expansion, a second stage of ex vivo expansion and a third stage of ex vivo expansion, and in the second stage of ex vivo expansion according to the present application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced.

[0254] For example, TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not subjected to ex vivo expansion according to the present application can be subjected to a first stage of ex vivo expansion, a second stage of ex vivo expansion and a third stage of ex vivo expansion, and in the third stage of ex vivo expansion according to the present application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced.

[0255] For example, the TILs derived from tumor tissue, pleural effusion, and / or ascites of the present invention, which have not been expanded in vitro, can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion. In the first-stage in vitro expansion of the present invention, the expression and / or activity of one or more members of the peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family and / or their functionally active fragments in the TILs can be reduced. In the second-stage in vitro expansion of the present invention, the expression and / or activity of one or more members of the peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family and / or their functionally active fragments in the TILs can be reduced.

[0256] For example, the TILs derived from tumor tissue, pleural effusion, and / or ascites of the present invention, which have not been expanded in vitro, can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion. In the first-stage in vitro expansion of the present invention, the expression and / or activity of one or more members of the peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family and / or their functionally active fragments in the TILs can be reduced. In the third-stage in vitro expansion of the present invention, the expression and / or activity of one or more members of the peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family and / or their functionally active fragments in the TILs can be reduced.

[0257] For example, the TILs derived from tumor tissue, pleural effusion, and / or ascites of the present invention, which have not been amplified in vitro, can be subjected to a first-stage in vitro amplification, a second-stage in vitro amplification, and a third-stage in vitro amplification. In the second-stage in vitro amplification of the present invention, the expression and / or activity of one or more members of the peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family and / or their functionally active fragments in the TILs can be reduced. In the third-stage in vitro amplification of the present invention, the expression and / or activity of one or more members of the peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family and / or their functionally active fragments in the TILs can be reduced.

[0258] For example, TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not expanded in vitro of the application 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 application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced, and in the second stage of in vitro expansion of the application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced, and in the third stage of in vitro expansion of the application, the expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the TILs can be reduced.

[0259] For example, each stage of in vitro expansion can be divided by a change in the number of TIL cells, e.g., when the number of TIL cells increases by at least about 1-fold, the TIL cells can be considered to have entered the next stage of in vitro expansion. In some embodiments, when the number of TIL cells increases by at least about 1-1000-fold, e.g., 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, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold, the TIL cells can be considered to have entered the next stage of in vitro expansion. For example, each stage of in vitro expansion can also be divided by a change in the conditions of TIL cell culture. For example, when cell activators and / or cell growth factors are added to or supplemented in the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion. For example, when IL-2 is added to or supplemented in the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion. For example, when one or more gene regulation systems are added to or supplemented in the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion. For example, when feeder cells are added to or supplemented in the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion. For example, when TIL cells are subjected to centrifugation and / or cell washing, the TIL cells can be considered to have entered the next stage of in vitro expansion. For example, each stage can also be divided by the number of days of TIL cell culture. For example, when TIL cells are cultured in vitro for about 1-100 days, e.g., 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.

[0260] For example, wherein reducing expression and / or activity of a member of the C64 family of peptidases in the cell comprises inhibiting the function of a deubiquitinating enzyme. For example, wherein reducing expression and / or activity of a member of the ZC3H12 family in the cell comprises inhibiting the function of a nuclease. For example, wherein reducing expression and / or activity of a member of the STAT-induced STAT inhibitor (SSI) family in the cell comprises inhibiting the negative regulation of cytokine signaling.

[0261] For example, the improved cell quantity of the present application refers to at least about 1-50 fold, e.g., 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, increase in the number of cells of the present application in which expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is reduced and / or activity is attenuated in at least one in vitro expansion phase as compared to cells in which expression of and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is not altered.

[0262] For example, the improved cell quantity of the present application refers to at least about 1-50 fold, e.g., 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, increase in the number of cells of the present application in which expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is reduced and / or activity is attenuated in at least one in vitro expansion phase as compared to cells in which expression of and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is not altered.

[0263] For example, the improved cell quantity of the present application refers to at least about 1-50 fold, e.g., 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, increase in the number of cells of the present application in which expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is reduced and / or activity is attenuated in at least one in vitro expansion phase as compared to cells in which expression of and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is not altered.

[0264] For example, the increased cytokine secretion capacity of the present application can refer to an increase in the proportion of cells secreting cytokines in the present cells in which the expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is reduced and / or the activity is attenuated in at least one in vitro expansion phase, by at least about 1-50 fold, e.g., 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 of and / or activity of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is not changed. For example, the increased cytokine secretion capacity of the present application can refer to an increase in the proportion of cells secreting cytokines in the present cells in which the expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is reduced and / or the activity is attenuated in at least one in vitro expansion phase, by at least about 100%-0.1%, e.g., 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%, compared to cells in which the expression of and / or activity of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is not changed.

[0265] For example, the improved tumor cell killing ability of the present application can mean that the tumor cell killing rate of the cells of the present application in which the expression and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is reduced and / or attenuated in at least one in vitro expansion phase can be increased by at least about 1-50 fold, such as 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 one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is not changed. For example, the improved tumor cell killing ability of the present application can mean that the tumor cell killing rate of the cells of the present application in which the expression and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is reduced and / or attenuated in at least one in vitro expansion phase can be increased by at least about 100%-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, compared to cells in which the expression and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family is not changed. For example, the tumor cell killing rate of the cells of the present application can be measured by IncuCyte system or CFSE and DAPI staining method. For example, the tumor cell killing of the cells of the present application can mean the ability of the cells to kill solid tumor cells.

[0266] For example, the improved ratio of cell subpopulation of the present application can comprise one or more selected from the group consisting of: increased CD8 + cell ratio, increased central memory cell and / or naive cell ratio, decreased regulatory cell ratio, increased activated cell ratio, increased tumor specific cell ratio, and increased stem cell like cell ratio.

[0267] For example, the reduced proportion of exhausted cells of the application can be a reduction in the proportion of CD8 + The proportion of cells, central memory cells, and / or naive cells, activated cells, tumor-specific cells, and / or stem cell-like cells can be increased by at least about 100%-0.1%, e.g., 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%.

[0268] For example, the reduced proportion of exhausted cells of the application can be a reduction in the proportion of CD8 + , LAG-3 + , TIM-3 + , and / or CD39 + cells in the cells. For example, the reduced proportion of regulatory cells of the application can be a reduction in the proportion of CD4 + CD25 + Foxp3 + cells in the cells. For example, the reduced proportion of apoptotic cells of the application can be a reduction in the proportion of CD95 + caspas3 + cells and / or CD95 + DR5 + cells in the cells.

[0269] For example, depleting the proportion of cells, regulatory cells, and / or apoptotic cells in the cells can be reduced by at least about 100%-0.1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, 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%, or can be reduced by at least about 1-50 fold, such as 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.

[0270] For example, the culture method of the present application can comprise a gene editing step for the cells. For example, it comprises subjecting the cells to at least one stage of in vitro expansion, wherein in at least one stage of the in vitro expansion, a gene regulation system can be introduced into the cells.

[0271] For example, the gene regulation system can disrupt the target gene at the DNA level. For example, the gene regulation system can disrupt a region of the target gene or a fragment thereof in the genome of the cells. For example, after using the gene regulation system, the DNA region or a fragment thereof in the cells where the target gene is located is cleaved, and the expression ability of the target gene is reduced or the activity of the target gene is inhibited. For example, the editing effect of the gene regulation system on the target gene can be long-term and continuous. Wherein the genomic region of the present application is determined according to the human reference genome hg38 version.

[0272] For example, the gene regulation system can comprise a guide nucleic acid molecule and an enzyme protein. For example, the enzyme protein can have a nucleic acid cleavage enzyme activity, and the guide nucleic acid molecule can guide the enzyme protein to specifically cleave the region or fragment of the target gene. For example, the guide nucleic acid molecule and the enzyme protein can exist in the form of a ribonucleoprotein complex (RNP), or each independently exist alone. For example, the enzyme protein can comprise a Cas protein. For example, a polynucleotide encoding gRNA and Cas protein can be introduced, or each independently introduced into the target cells.

[0273] For example, the present application can reduce and / or attenuate the expression of at least one target gene of a cell can comprise introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the cell. For example, the enzyme protein can comprise a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the guide nucleic acid molecule can comprise a guide RNA (gRNA). For example, a complex comprising a polynucleotide encoding a gRNA and a Cas protein can be introduced into the cell. For example, a complex comprising a gRNA and a Cas protein can be introduced into the cell.

[0274] For example, the gRNA can be used to bind to a sequence of the target gene. For example, the binding of the gRNA to the sequence of the target gene can be fully complementary, can be partially complementary, can also be moderately stringent or stringent condition hybridized to the sequence of the target gene. 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.

[0275] For example, the target region for editing of the present application can be a region in front of a promoter. For example, the target region for editing of the present application can be a region with high transcription factor binding capacity. For example, the target region for editing of the present application can be a region with a specific number of transcription factor binding number. For example, the target region for editing of the present application can be a continuous region with a number of transcription factor binding number of about 3 or more.

[0276] For example, when the gene editing system comprises CRISPR / Cas9, there can be a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application, which can be AGG, TGG, GGG or CGG. For example, when the PAM region of the target gene is determined, one skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides upstream of the 5' end of the PAM of the target gene, while a suitable gRNA can be designed 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.

[0277] For example, when the gene editing system comprises CRISPR / Cas12, there can be a protospacer adjacent motif (PAM) upstream of the region targeted by the guide nucleic acid molecule of the present application, which can 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 gene of interest is determined, one of skill in the art can readily 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 gene of interest, and a suitable gRNA can be designed against the target sequence. For example, the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides downstream of the 3’ end of a protospacer adjacent motif (PAM) selected from the group consisting of NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, 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.

[0278] For example, when the gene editing system of the present application comprises a wild-type Cas12a (which can also be referred to as Cpf1, such as AsCas12a, FnCas12a, LbCas12a, BbCas12a, CMaCas12a and OsCas12a), there can be a PAM sequence selected from the group consisting of NTTN, wherein N can be A, T, C or G, upstream of the region targeted by the guide nucleic acid molecule of the present application. For example, when the PAM region of the gene of interest is determined, one of skill in the art can readily 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 gene of interest, and a suitable gRNA can be designed against the target sequence.

[0279] For example, when the gene editing system of the present application comprises a mutant Cas12a, such as enAsCas12a (mutant sites E174R, S542R and K548R), the region targeted by the guide nucleic acid molecule of the present application can have a PAM sequence selected from the group consisting of TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV) upstream, 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 a gene of interest is determined, one skilled in the art can readily 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 gene of interest, and a suitable gRNA can be designed for the target sequence.

[0280] For example, when the gene editing system of the present application comprises a mutant Cas12a, such as opAsCas12a (mutant sites: E174R and S542R), the region targeted by the guide nucleic acid molecule of the present application can have a PAM sequence selected from the group consisting of TTTV (TTTA, TTTC, or TTTG) upstream. For example, when the PAM region of a gene of interest is determined, one skilled in the art can readily 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 gene of interest, and a suitable gRNA can be designed for the target sequence.

[0281] For example, when the gene editing system of the present application comprises a mutant Cas12a, such as AsCas12aUltra (mutant sites: M537R and F870L), the region targeted by the guide nucleic acid molecule of the present application can have a PAM sequence selected from the group consisting of TTTV, TATV, or TYCV upstream, wherein V can be A, C or G, and Y can be T or C. For example, when the PAM region of a gene of interest is determined, one skilled in the art can readily 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 gene of interest, and a suitable gRNA can be designed for the target sequence.

[0282] For example, when the gene editing system of the present application comprises a mutant Cas12a, such as hfCas12Max (mutant sites: N243R / E336R / D892R) and Cas12Max (mutant sites: N243R), there can be a PAM sequence selected from the group consisting of TNN, or NTN upstream of the region targeted by the guide nucleic acid molecule of the present application, wherein N can be A, T, C or G. For example, when the PAM region of the gene of interest is determined, one skilled in the art can readily 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 gene of interest, while a suitable gRNA can be designed for the target sequence.

[0283] Guide region and guide nucleic acid of TNFAIP3

[0284] For example, the guide nucleic acid molecule can comprise a target sequence consisting of about 10 to about 30 nucleotides downstream of the PAM region of TTA, TTT, TTG and / or TTC in the DNA where the gene encoding the Peptidase C64 family member and / or a functionally active fragment thereof is located. For example, the guide nucleic acid molecule can comprise a target sequence 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 downstream of the PAM region of TTA, TTT, TTG and / or TTC in the DNA where the gene encoding the Peptidase C64 family member and / or a functionally active fragment thereof is located.

[0285] For example, the target sequence can be a region defined by the genomic coordinates selected from Table 7A or a fragment thereof.

[0286] For example, the target sequence of the present application can be the OUT structure functional domain of TNFAIP3. For example, the target sequence of the present application can be the zinc finger structure functional domain of TNFAIP3. For example, the target sequence of the present application can be chr6: 137871134-137871624, chr6: 137874719-137875081, chr6: 137875117-137875158, chr6: 137875561-137876088, chr6: 137876104-137876212, chr6: 137879235-137879272, chr6: 137879275-137879452, chr6: 137879947-137880049, chr6: 137880060-137880249.

[0287] For example, the guide nucleic acid molecule can comprise a guide sequence which is complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1709-3416.

[0288] For example, the guide nucleic acid molecule can comprise a guide sequence (targeting domain) which can comprise a sequence as shown in SEQ ID NOs: 1-1708, 5115-5132.

[0289] For example, the guide nucleic acid molecule can comprise a guide sequence which can comprise a sequence as shown in SEQ ID NOs: 1-154, 5115-5132. For example, the guide nucleic acid molecule can comprise the following sequences: SEQ ID NOs: 5115, 5116, 5119, 5129, 5132. For example, the guide sequence of the present application with base-modified nucleotides added at the 3' end can comprise: *mG*mG*mC, *mU*mG*mA, *mU*mG*mG, *mA*mG*mG, *mG*mA*mG (wherein m represents: 2'-O-methyl modification, * represents: 3' thio-phosphate internucleoside linkage).

[0290] Guide region and guide nucleic acid of ZC3H12A

[0291] For example, the guide nucleic acid molecule can comprise a target sequence that is capable of binding to a region of DNA about 10 to about 30 nucleotides downstream of a TTA, TTT, TTG, and / or TTC indicated PAM region in which a gene encoding a ZC3H12 family member and / or a functionally active fragment thereof is located. For example, the guide nucleic acid molecule can comprise a target sequence that is capable of binding to a region of DNA 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 downstream of a TTA, TTT, TTG, and / or TTC indicated PAM region in which a gene encoding a ZC3H12 family member and / or a functionally active fragment thereof is located.

[0292] For example, the target sequence can be a region defined by genomic coordinates selected from Table 7B or a fragment thereof.

[0293] For example, the target sequence of the present application can be a C3H1-type zinc finger domain of ZC3H12A. For example, the target sequence of the present application can be chr1:37482415-37482548, chr1:37482586-37482761, chr1:37482763-37482799.

[0294] For example, the guide nucleic acid molecule can comprise a guide sequence that is complementary to a target sequence selected from the group consisting of SEQ ID NOs: 4058-4698.

[0295] For example, the guide nucleic acid molecule can comprise a guide sequence that can comprise a sequence as set forth in SEQ ID NOs: 3417-4057, 5133-5136.

[0296] For example, the guide nucleic acid molecule can comprise a guide sequence that can comprise a sequence as set forth in SEQ ID NOs: 3417-3474, 5133-5136. For example, the guide nucleic acid molecule can comprise the following sequence: SEQ ID NO: 5133. For example, the base-modified nucleotide added at the 3’ end of the guide sequence of the present application can comprise: *mG*mG*mG (wherein m represents: 2’-O-methyl modification, * represents: 3’ phosphorothioate internucleoside linkage).

[0297] Guide regions and guide nucleic acids for SOCS1

[0298] For example, the guide nucleic acid molecule can comprise a target sequence capable of binding about 10 to about 30 nucleotides downstream of a PAM region of TTA, TTT, TTG, and / or TTC in DNA where a gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof is located. For example, the guide nucleic acid molecule can comprise a target sequence capable of binding 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 downstream of a PAM region of TTA, TTT, TTG, and / or TTC in DNA where a gene encoding a STAT-induced STAT inhibitor (SSI) family member and / or a functionally active fragment thereof is located.

[0299] For example, the target sequence can be a region defined by genomic coordinates selected from Table 7C or a fragment thereof.

[0300] For example, the guide nucleic acid molecule can comprise a guide sequence that is complementary to a target sequence selected from the group consisting of SEQ ID NOs: 4904-5108.

[0301] For example, the guide nucleic acid molecule can comprise a guide sequence that can comprise a sequence as set forth in SEQ ID NOs: 4699-4903, 5110-5114.

[0302] For example, the guide nucleic acid molecule can comprise a guide sequence that can comprise a sequence as set forth in SEQ ID NOs: 4699-4753, 5110-5114. For example, the guide nucleic acid molecule can comprise the following sequences: SEQ ID NOs: 5113, 5114. For example, the guide sequence of the present application with base-modified nucleotides added at the 3’ end can comprise: *mG*mC*mA, *mA*mG*mC (wherein m represents: 2’-O-methyl modification, * represents: 3’ thio-phosphate internucleoside linkage).

[0303] Guide regions and guide nucleic acids of gene combinations

[0304] For example, the activity of TNFAIP3 and ZC3H12A is inhibited in the cells of the present application. For example, the preferred sub-regions of TNFAIP3 and ZC3H12A shown in Tables 7A and 7B, respectively, of the present application are knocked out and / or inhibited in the cells. For example, the combinations of preferred targeting sub-regions shown in TNFAIP3 R_1 to 67 in Table 7A and ZC3H12AR_1 to 84 in Table 7B of the present application are combined two by two, constituting combinations numbered 1 to 5628 of the combinations are knocked out and / or inhibited.

[0305] For example, the activity of TNFAIP3 and SOCS1 is inhibited in the cells of the present application. For example, the preferred sub-regions of TNFAIP3 and SOCS1 shown in Tables 7A and 7C, respectively, of the present application are knocked out and / or inhibited in the cells. For example, the combinations of preferred targeting sub-regions shown in TNFAIP3 R_1 to 67 in Table 7A and SOCS1 R_1 to 33 in Table 7C of the present application are combined two by two, constituting combinations numbered 5629 to 7839 of the combinations are knocked out and / or inhibited.

[0306] For example, the activity of ZC3H12A and SOCS1 is inhibited in the cells of the present application. For example, the preferred sub-regions of ZC3H12A and SOCS1 shown in Tables 7B and 7C, respectively, of the present application are knocked out and / or inhibited in the cells. For example, the combinations of preferred targeting sub-regions shown in ZC3H12A R_1 to 84 in Table 7B and SOCS1 R_1 to 33 in Table 7C of the present application are combined two by two, constituting combinations numbered 7840 to 10611 of the combinations are knocked out and / or inhibited.

[0307] For example, the guide nucleic acid molecule can comprise a target sequence capable of binding to a region of about 15 to about 25 nucleotides downstream of the PAM region of TTA, TTT, TTG and / or TTC in DNA where a gene encoding a member selected from the group consisting of at least 2 families of Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and / or a functionally active fragment thereof is located. For example, the guide nucleic acid molecule can comprise a target sequence capable of binding to a region 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 22 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 20, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides downstream of the PAM region of TTA, TTT, TTG and / or TTC in DNA where a gene encoding a member selected from the group consisting of at least 2 families of Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family and / or a functionally active fragment thereof is located. For example, the target sequence can be a region defined by genomic coordinates selected from Tables 8A-8C or a fragment thereof. In the present application, only a part of the combination of species is shown for the convenience of display and is limited by the limited display space; however, the target sequences of the target points selected from two or more of Tables 8A-8C in the present application can be combined with each other, and can also be used for double or multiple knock of the target points.

[0308] For example, the guide nucleic acid molecule can comprise a guide sequence of an sgRNA targeting TNFAIP3 as shown in any one of SEQ ID NOs: 1-1708, 5115-5132, a guide sequence of an sgRNA targeting ZC3H12A as shown in any one of SEQ ID NOs: 3417-4057, 5133-5136, a guide sequence of an sgRNA targeting SOCS1 as shown in any one of SEQ ID NOs: 4699-4903, 5110-5114.

[0309] For example, the activity of TNFAIP3 and ZC3H12A in the cell of the present application is inhibited. For example, the guide sequences shown in SEQ ID NOs: 1-154 and 3417-3474 in the present application are combined in pairs to form the knockout and / or inhibition of the combinations of editing combination numbers 1 to 8932.

[0310] For example, the activity of TNFAIP3 and SOCS1 is inhibited in the cells of the application. For example, the combinations of SEQ ID NOs: 1-154 and guide sequences set forth in SEQ ID NOs: 4699-4753, two by two, forming the combinations of edit combination numbers 8933 to 17402, are knocked out and / or inhibited.

[0311] For example, the activity of ZC3H12A and SOCS1 is inhibited in the cells of the application. For example, the combinations of SEQ ID NOs: 3417-3474 and guide sequences set forth in SEQ ID NOs: 4699-4753, two by two, forming the combinations of edit combination numbers 17403 to 20592, are knocked out and / or inhibited.

[0312] For example, the activity of TNFAIP3 and ZC3H12A is inhibited in the cells of the application. For example, the first guide sequence of the application can be selected from the group comprising the sequences set forth in SEQ ID NOs: 5115-5132 and the second guide sequence of the application can be selected from the group comprising the sequences set forth in SEQ ID NOs: 5133-5136.

[0313] For example, the activity of TNFAIP3 and SOCS1 is inhibited in the cells of the application. For example, the first guide sequence of the application can be selected from the group comprising the sequences set forth in SEQ ID NOs: 5115-5132 and the second guide sequence of the application can be selected from the group comprising the sequences set forth in SEQ ID NOs: 5110-5114.

[0314] For example, the activity of ZC3H12A and SOCS1 is inhibited in the cells of the application. For example, the first guide sequence of the application can be selected from the group comprising the sequences set forth in SEQ ID NOs: 5133-5136 and the second guide sequence of the application can be selected from the group comprising the sequences set forth in SEQ ID NOs: 5110-5114.

[0315] For example, the combination of guide sequences of the application comprises the combination of the sequences set forth in SC-015 and TP-005. For example, the combination of guide sequences of the application comprises the combination of the sequences set forth in SC-015 and TP-233. For example, the combination of guide sequences of the application comprises the combination of the sequences set forth in SC-015 and TP-268. For example, the combination of guide sequences of the application comprises the combination of the sequences set forth in SC-027 and TP-005. For example, the combination of guide sequences of the application comprises the combination of the sequences set forth in SC-027 and TP-233. For example, the combination of guide sequences of the application comprises the combination of the sequences set forth in SC-027 and TP-268.

[0316] For example, one or more guide sequences of the application can be located on different guide nucleic acid molecules. For example, one or more guide sequences of the application can be located on the same guide nucleic acid molecule. For example, one or more guide sequences of the application can be contacted with a cell to be edited simultaneously. For example, one or more guide sequences of the application can be contacted with a cell to be edited sequentially.

[0317] For example, the proportion of cells expressing a product of a target gene can be reduced and / or the amount of expression of a target gene per cell can be reduced in a cell obtained by reducing expression and / or activity of at least one target gene in a cell compared to a cell in which expression and / or activity of the target gene is unchanged.

[0318] For example, the proportion of cells expressing a product of a gene of interest is reduced by at least about 5% in cells obtained by reducing expression and / or activity of at least one gene of interest in the method of the application compared to cells in which expression and / or activity of the gene of interest is unchanged. For example, the proportion of cells expressing a product of a gene encoding one or more members of the Peptidase C64 family, the ZC3H12 family, STAT-induced STAT Inhibitor (SSI) family, and / or a functionally active fragment thereof is reduced by at least about 100%-5%, e.g., 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 a product of a gene encoding one or more members of the Peptidase C64 family, the ZC3H12 family, STAT-induced STAT Inhibitor (SSI) family, and / or a functionally active fragment thereof is reduced from the proportion of cells that can be observed to 0%. For example, the proportion of cells expressing a product of a gene encoding one or more members of the Peptidase C64 family, the ZC3H12 family, STAT-induced STAT Inhibitor (SSI) family, and / or a functionally active fragment thereof can be reduced by at least about 100%-1%, e.g., 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 a product of a gene encoding one or more members of the Peptidase C64 family, the ZC3H12 family, STAT-induced STAT Inhibitor (SSI) family, and / or a functionally active fragment thereof can be detected by flow cytometry.

[0319] For example, the proportion of cells expressing the product of the gene encoding one or more of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof in a cell obtained by reducing expression and / or activity of at least one target gene of the cell in the methods of the application can be at most about 95%. For example, the proportion of cells expressing the product of the gene encoding one or more of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof can be at most about 95%-5%, such as at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%. For example, the proportion of cells expressing the product of the gene encoding one or more of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof can be detected by a cell flow cytometer.

[0320] For example, the expression of a target gene in a single cell in a cell obtained by reducing the expression and / or activity of at least one target gene in a cell compared to a cell in which the expression and / or activity of the target gene is unchanged in the methods of the application can be reduced by at least about 5%. For example, the expression of a target gene in a single cell can be reduced by at least about 100% to 5%, e.g., 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 a target gene in a single cell can be reduced from an observable amount to 0%. For example, the expression of a target gene in a single cell can be reduced by at least about 100% to 1%, e.g., 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%.

[0321] For example, the amount of expression of the gene of interest in a single cell in a cell obtained by reducing expression and / or attenuating activity of at least one gene of interest in the cell in the methods of the application can be at most about 95% of the amount of expression and / or activity of the gene of interest in a cell in which expression and / or activity of the gene of interest has not been altered. For example, the amount of expression of the gene encoding one or more of a member of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof (e.g., a gene encoding TNFAIP3, ZC3H12A, SOCS1) in a single cell can be at most about 95%-5%, e.g., at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 19%, at most about 18%, at 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 amount of expression and / or activity of the gene encoding one or more of a member of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof in a cell in which expression and / or activity of the gene encoding one or more of a member of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and / or a functionally active fragment thereof has not been altered.

[0322] For example, the methods of the application comprise subjecting the cells to at least one stage of in vitro expansion, wherein expression and / or activity of one or more of a member of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family is reduced and / or attenuated in at least one stage of the in vitro expansion.

[0323] For example, the fragments derived from tumor tissue, tumor-associated lymph node with or without tumor metastasis, tumor metastasis lesion, peritumoral tissue, pleural effusion, and / or peritoneal effusion that have not been subjected to in vitro expansion are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and expression and / or activity of one or more of a member of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family is reduced and / or attenuated in the second stage of in vitro expansion in the TILs subjected to the first stage of in vitro expansion.

[0324] For example, the first stage of in vitro expansion is performed for at least about 7 days. For example, the second stage of in vitro expansion is performed for at least about 7 days.

[0325] For example, the cells can be contacted with the one or more cell activators and the expression and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof in the cells can be reduced in a single stage of the in vitro expansion of the application. For example, the cell activators 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, the expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or the activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family in the cells of the application can be reduced and / or attenuated and contacted with one or more cell activators of the application in a single stage of the in vitro expansion. For example, the expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or the activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family in the TILs of the application can be reduced and / or attenuated and contacted with one or more cell activators of the application in the first stage of the in vitro expansion. For example, the expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or the activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family in the TILs of the application can be reduced and / or attenuated and contacted with one or more cell activators of the application in the second stage of the in vitro expansion. For example, the expression of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and / or the activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family in the TILs of the application can be reduced and / or attenuated and contacted with one or more cell activators of the application in the third stage of the in vitro expansion.

[0326] For example, in a single stage of in vitro expansion of the application, the cells of the application substantially simultaneously reduce expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and contact with one or more cell activators of the application. For example, in a single stage of in vitro expansion of the application, the cells of the application can first reduce expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, e.g., 2-48 hours in advance, e.g., 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell activators of the application. For example, in a single stage of in vitro expansion of the application, the cells of the application can first contact one or more cell activators of the application, e.g., 2-48 hours in advance, e.g., 2 hours in advance, 4 hours in advance, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family.

[0327] For example, in a first stage of in vitro expansion of the application, the TILs of the application substantially simultaneously reduce expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and contact with one or more cell activators of the application. For example, in a second stage of in vitro expansion of the application, the TILs of the application substantially simultaneously reduce expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and contact with one or more cell activators of the application. For example, in a third stage of in vitro expansion of the application, the TILs of the application substantially simultaneously reduce expression and / or activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, the STAT-induced STAT inhibitor (SSI) family, and contact with one or more cell activators of the application.

[0328] In another aspect, the present application provides a method of culturing tumor infiltrating lymphocytes (TILs), which can comprise: (A) contacting a first population of TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not expanded in vitro with one or more cell growth factors; wherein a second population of TILs is obtained after step (A); (B) reducing expression and / or activity of one or more family members of the C64 family of peptidases, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family in the second population of TILs; wherein a third population of TILs is obtained after step (B).

[0329] In one embodiment, the first phase of in vitro expansion of the present application can be used in place of step (A) in the methods of the above aspects. In one embodiment, the second phase of in vitro expansion of the present application can be used in place of step (B) in the methods of the above aspects. In one embodiment, the first phase of in vitro expanded TILs of the present application can be used in place of the second population of TILs obtained after step (A) in the methods of the above aspects. In one embodiment, the second phase of in vitro expanded TILs of the present application can be used in place of the third population of TILs obtained after step (B) in the methods of the above aspects. In one embodiment, the third phase of in vitro expansion of the present application can be used in place of any additional step (C) in the methods of the above aspects, if desired. In one embodiment, the third phase of in vitro expanded TILs of the present application can be used in place of the fourth population of TILs obtained after any additional step (C) in the methods of the above aspects, if desired.

[0330] In another aspect, the present application provides a method of culturing tumor infiltrating lymphocytes (TILs), which can comprise: (A) contacting a first population of TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not expanded in vitro with one or more cell growth factors; wherein a second population of TILs is obtained after step (A); (B) contacting the second population of TILs with a plurality of cell growth factors, contacting the TILs with a plurality of cell activators, reducing expression and / or activity of one or more family members of the C64 family of peptidases, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family, and co-culturing the TILs with feeder cells; wherein a third population of TILs is obtained after step (B).

[0331] In another aspect, the present application provides a method of culturing tumor infiltrating lymphocytes (TILs), which can comprise: (A) contacting a first population of TILs derived from tumor tissue, pleural effusion and / or ascites fluid and not expanded in vitro with a cell growth factor; wherein a second population of TILs is obtained after the step (A); (B) contacting the second population of TILs with a cell growth factor, contacting the second population of TILs with a cell activating agent, reducing expression and / or activity of one or more of the following family members: peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, and co-culturing the TILs with feeder cells, wherein the one or more of the following family members: peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family, can comprise TNFAIP3, ZC3H12, SOCS1, respectively; wherein a third population of TILs is obtained after the step (B).

[0332] In another aspect, the present application provides a method of culturing tumor infiltrating lymphocytes (TILs). The method of obtaining TIL cells from a subject tissue sample can be from a patient surgery to obtain a primary tumor sample or a metastatic tumor sample, which can weigh at least about 1 g, and can be from multiple pieces of tissue combined. The tumor tissue, pleural effusion, and / or peritoneal effusion can be transported at about 2-8 °C in a sample transport solution, which can be a commercially available tumor tissue transport solution, tumor tissue preservation solution, or tumor tissue transport solution, and processed within 48 hours. The tissue pieces can be mechanically disrupted to about 1-27 cubic millimeters per piece, transferred into a gas permeable culture bag or Grex, and cultured in cell serum-free medium and IL-2 at a concentration of 300-9000 IU / mL (e.g., can be 1000-9000 IU / mL, e.g., can be 6000 IU / mL) for about 3-14 days. The cells in the culture medium can be collected, transferred into a gas permeable culture bag, or Grex, or Xuri device, and the cell serum-free medium can be supplemented with a CD28 antibody of the present application, a CD3 antibody, and magnetic beads (e.g., Dynabeads) comprising the CD3 antibody and the CD28 antibody and / or a nanomatrix (e.g., transACT) comprising the CD3 antibody and the CD28 antibody, IL-2 at a concentration of 300-9000 IU / mL (e.g., can be 1000-9000 IU / mL, e.g., can be 6000 IU / mL), and a STAT-induced STAT inhibitor (SSI) family member (e.g., TNFAIP3, ZC3H12A, SOCS1) expression-reducing and / or activity-reducing agent (e.g., by transducing the cells in the TILs with a ribonucleoprotein complex (RNP) comprising a gRNA of the present application and a Cas protein to reduce the proportion of cells in the TILs encoding a STAT-induced STAT inhibitor (SSI) family member to about 95% or less), and the TILs are activated for a certain period of time, irradiated PBMCs (TILs and PBMCs at a ratio of about 1:40 to about 1:400) are added, and the culture is expanded for about 3-14 days. The cells in the culture medium can be collected using a cell processing system, washed, cryopreserved, and tested. The final product can have a CD3 proportion greater than 80%, a cell survival rate (or cell viability) greater than 50%, and greater than 80% of the cells can be memory effector cells and effector cells. The cells can secrete IFN-γ after stimulation, and / or can have an upregulated proportion of activated cells.

[0333] TIL cell culture

[0334] For example, the first stage in vitro expansion of the present application is performed for at least about 3 days. For example, the first stage in vitro expansion of the present application can be performed for at least about 7-14 days, e.g., at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days. For example, the first stage in vitro expansion of the present application can be performed for about 9 days to about 14 days, e.g., the first stage in vitro expansion of the present application can be performed for about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days. For example, the first stage in vitro expansion of the present application can be considered a pre-REP stage.

[0335] For example, the second stage in vitro expansion of the present application can be performed for at least about 7-14 days, e.g., at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days. For example, the second stage in vitro expansion of the present application can be performed for about 9 days to about 14 days, e.g., the second stage in vitro expansion of the present application can be performed for about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days. For example, the second stage in vitro expansion of the present application can be considered a REP (rapid expansion protocol) stage.

[0336] For example, the number of days the second stage in vitro expansion of the present application is performed can be calculated from the time the second stage in vitro expansion is initiated. For example, at the time the second stage in vitro expansion is initiated, it can be considered that the second stage in vitro expansion has been performed for about 0 hours. For example, about 24 hours after the second stage in vitro expansion is initiated, it can be considered that the second stage in vitro expansion has been performed for about 1 day. For example, on the day the second stage in vitro expansion is initiated, it can be considered that the second stage in vitro expansion has been performed for about 0 days. For example, on the day after the second stage in vitro expansion is initiated, it can be considered that the second stage in vitro expansion has been performed for about 1 day.

[0337] For example, the cell activator of the application can comprise one or more selected from the group of: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and functionally active fragments thereof. For example, the cell activator of the application can comprise an agonist of one or more targets selected from the group of: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB. For example, the cell activator of the application can comprise an antibody and antigen binding fragments thereof selected from the group of: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB. For example, the cell activator of the application can comprise a CD3 agonist. For example, the cell activator of the application can comprise an antibody and / or antigen binding fragments thereof against CD3, for example OKT3 by Miltenyi Biotech, for example SP34 by BD. For example, the cell activator of the application can comprise a CD28 agonist. For example, the cell activator of the application can comprise an antibody and / or antigen binding fragments thereof against CD28, for example 15E8 by Merck.

[0338] For example, a cell activator of the application can comprise an antibody and / or antigen binding fragment thereof against CD3, for example can comprise the light chain VL and heavy chain VH of OKT3 of Miltenyi Biotech, can comprise the light chain VL and heavy chain VH of SP34 of BD. For example, a cell activator of the application can comprise a CD28 agonist. For example, a cell activator of the application can comprise an antibody and / or antigen binding fragment thereof against CD28, for example can comprise the light chain VL and heavy chain VH of 15E8 of Merck. For example, a cell activator of the application can comprise an antibody and / or antigen binding fragment thereof against CD3, for example can comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of OKT3 of Miltenyi Biotech, can comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of SP34 of BD, an antibody and / or antigen binding fragment thereof against CD3 of the application can have CD3 binding ability. For example, a cell activator of the application can comprise a CD28 agonist. For example, a cell activator of the application can comprise an antibody and / or antigen binding fragment thereof against CD28, for example can comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of 15E8 of Merck, an antibody and / or antigen binding fragment thereof against CD28 of the application can have CD28 binding ability. In the present application, an antibody or antigen binding fragment thereof of the application comprises at least one CDR in the antibody heavy chain variable region VH and / or at least one CDR in the antibody light chain variable region VL. A CDR of the present application can be defined according to IMGT nomenclature, a CDR of the present application can be defined according to Chothia, or a CDR of the present application can be defined according to Kabat.

[0339] For example, contacting a cell of the application with one or more cell activators of the application can comprise one or more modes selected from the group consisting of: (1) adding a cell activator of the application to a cell culture medium of a cell of the application; (2) adding an engineered cell expressing a cell activator of the application to a cell culture medium of a cell of the application; (3) adding a solid phase medium comprising a cell activator of the application to a cell culture medium of a cell of the application. For example, contacting a cell of the application with one or more cell activators of the application can comprise adding a solid phase medium comprising a cell activator of the application to a cell culture medium of a cell of the application. For example, contacting a cell of the application with one or more cell activators of the application can comprise adding a solid phase medium comprising a CD28 antibody and a CD3 antibody of the application to a cell culture medium of a cell of the application.

[0340] For example, the initial concentration of the cell activator in the cell culture medium of the cells of the application can be at least about 30 ng / mL. For example, the initial concentration of the CD28 antibody of the application in the cell culture medium of the cells of the application can be at least about 30 ng / mL; for example, the initial concentration of the CD3 antibody of the application in the cell culture medium of the cells of the application can be at least about 30 ng / mL. For example, the selection of the initial concentration of the CD28 antibody of the application can be independent of the selection of the initial concentration of the CD3 antibody of the application; for example, the initial concentration of the CD28 antibody of the application and the initial concentration of the CD3 antibody of the application in the cell culture medium of the cells of the application can be any combination. For example, the initial concentration of the CD28 antibody of the application in the cell culture medium of the cells of the application can be any selected from the group consisting of about 30 ng / mL to about 300 ng / mL. For example, the initial concentration of the CD3 antibody of the application in the cell culture medium of the cells of the application can be any selected from the group consisting of about 30 ng / mL to about 300 ng / mL. For example, the initial concentration of the CD28 antibody of the application in the cell culture medium of the cells of the application can be any selected from the group consisting of about 30 ng / mL to about 300 ng / mL, and the initial concentration of the CD3 antibody of the application in the cell culture medium of the cells of the application can be any selected from the group consisting of about 30 ng / mL to about 300 ng / mL, and the selection of the initial concentration of the CD28 antibody of the application can be independent of the selection of the initial concentration of the CD3 antibody of the application. For example, the diameter of the solid phase media of the application can be about 500 nanometers to about 10 micrometers. For example, the diameter of the solid phase media of the application can be about 1 micrometer to about 10 micrometers. For example, the diameter of the solid phase media of the application can be measured by transmission electron microscopy. For example, the diameter of the solid phase media of the application can be about 1 nanometer to about 500 nanometers. For example, the diameter of the solid phase media of the application can be about 100 nanometers to about 500 nanometers. For example, the diameter of the solid phase media of the application can be about 200 nanometers to about 500 nanometers. For example, the diameter of the solid phase media of the application can be measured by transmission electron microscopy.

[0341] For example, the solid phase media of the application can comprise a polymer. For example, the solid phase media of the application can comprise dextran.

[0342] For example, the solid phase media of the application can comprise at least about 25 μg of the cell activator of the application per mg of the solid phase media of the application.

[0343] For example, the solid phase media comprising the cell activator of the application can be added to the cell culture medium of the cells of the application at a ratio of the solid phase media of the application to the cells of the application of about 100: 1 to about 1 :2000, preferably about 1 : 100 to about 1 :2000. For example, the solid phase media comprising the cell activator of the application can be added to the cell culture medium of the cells of the application at a ratio of the solid phase media of the application to the cells of the application of about 2: 1 to about 1 :2.

[0344] For example, when the diameter of the solid phase media of the application is about 500 nanometers to about 10 micrometers, the solid phase media comprising a cell activator of the application can be added to the cell culture medium of the cells of the application at a ratio of the solid phase media of the application to the cells of the application of about 2: 1 to about 1 :2. For example, when the diameter of the solid phase media of the application is about 1 micrometer to about 10 micrometers, the solid phase media comprising a cell activator of the application, e.g., a CD3 agonist and / or a CD28 agonist, can be added to the cell culture medium of the cells of the application at a ratio of the solid phase media of the application to the cells of the application of about 2: 1 to about 1 :2, at about 2: 1 to about 1 : 1, or at about 1 : 1 to about 1 :2.

[0345] For example, when the diameter of the solid phase media of the application is about 100 nanometers to about 500 nanometers, the solid phase media comprising a cell activator of the application can be added to the cell culture medium of the cells of the application at a ratio of the solid phase media of the application to the cells of the application of about 1 : 100 to about 1 :2000. For example, when the diameter of the solid phase media of the application is about 100 nanometers to about 500 nanometers, the solid phase media comprising a CD28 agonist and a CD3 agonist of the application can be added to the cell culture medium of the cells of the application at a ratio of the solid phase media of the application to the cells of the application of about 1 : 100 to about 1 :2000, at about 1 :200 to about 1 :2000, at about 1 :300 to about 1 :2000, at about 1 :400 to about 1 :2000, at about 1 :500 to about 1 :2000, at about 1 :600 to about 1 :2000, at about 1 :700 to about 1 :2000, at about 1 :800 to about 1 :2000, at about 1 :900 to about 1 :2000, at about 1 : 1000 to about 1 :2000, at about 1 : 1200 to about 1 :2000, at about 1 : 1400 to about 1 :2000, at about 1 : 1600 to about 1 :2000, or at about 1 : 1800 to about 1 :2000.

[0346] For example, the methods of the application can further comprise contacting the cells of the application with one or more cell growth factors during at least one of the in vitro expansion phases of the application.

[0347] For example, in a single stage of in vitro expansion according to the application, the cells according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a first stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a second stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a third stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application.

[0348] For example, in a single stage of in vitro expansion according to the application, the cells according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a first stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a second stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a third stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application.

[0349] For example, in a single stage of in vitro expansion according to the application, the cells according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a first stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a second stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application. For example, in a third stage of in vitro expansion according to the application, the TILs according to the application can be contacted with a cell activator according to the application and with one or more cell growth factors according to the application.

[0350] For example, the cell growth factor of the application can be selected from one or more of the following group: IL-2, IL-7, IL-12, IL-15, IL-21, gamma interferon, and functionally active fragments thereof. For example, the cell growth factor of the application can comprise IL-2 and / or a functionally active fragment thereof. For example, the functionally active fragment of IL-2 can comprise a fragment of IL-2 known in the art to bind to the IL-2 receptor of a cell. For example, the cell growth factor of the application can comprise IL-2 and / or a functionally active fragment thereof, IL-7 and / or a functionally active fragment thereof, and IL-15 and / or a functionally active fragment thereof.

[0351] For example, contacting the cell of the application with one or more cell growth factors of the application can comprise adding the cell growth factor of the application to the cell culture medium of the cell of the application. For example, the initial concentration of the cell growth factor of the application in the cell culture medium of the cell of the application can be at least about 300 IU / mL. For example, the initial concentration of IL-2 of the application in the cell culture medium of the cell of the application can be at least about 300-9000 IU / mL, such as at least about 300 IU / mL, at least about 350 IU / mL, at least about 400 IU / mL, at least about 500 IU / mL, at least about 600 IU / mL, at least about 700 IU / mL, at least about 800 IU / mL, at least about 900 IU / mL, at least about 1000 IU / mL, at least about 1100 IU / mL, at least about 1200 IU / mL, at least about 1300 IU / mL, at least about 1400 IU / mL, at least about 1500 IU / mL, at least about 2000 IU / mL, at least about 2500 IU / mL, at least about 2600 IU / mL, at least about 2700 IU / mL, at least about 2800 IU / mL, at least about 2900 IU / mL, at least about 3000 IU / mL, at least about 3100 IU / mL, at least about 3200 IU / mL, at least about 3300 IU / mL, at least about 3400 IU / mL, at least about 3500 IU / mL, at least about 4000 IU / mL, at least about 4500 IU / mL, at least about 5000 IU / mL, at least about 5500 IU / mL, at least about 6000 IU / mL, at least about 6500 IU / mL, at least about 7000 IU / mL, at least about 7500 IU / mL, at least about 8000 IU / mL, at least about 8500 IU / mL, or at least about 9000 IU / mL.

[0352] For example, the cells of the application can be contacted with IL-2, IL-7, and IL-15 in reduced amounts relative to the amount of cytokine contacted when the cells are contacted with IL-2 alone. For example, the amount of IL-2 can be reduced in the presence of IL-7 and IL-15. For example, the concentration of IL-7 can be about 1 to 1000 ng / mL, preferably about 1-100 ng / mL. For example, the concentration of IL-15 can be about 1 to 1000 ng / mL, preferably about 1-100 ng / mL. For example, the amount of IL-2 can be reduced to the range commonly used in the art for the various immune cells, for example, to 50-10% of the range commonly used in the art, for example, 50%, 20%, or 10%. For example, the amount of IL-2 for TCR-Ts can be in the range commonly used in the art of 30-300 IU / mL. For example, the amount of IL-2 for TILs can be in the range commonly used in the art of 300-9000 IU / mL (e.g., 1000-9000 IU / mL).

[0353] For example, the methods of the application can further comprise co-culturing the cells of the application with feeder cells during at least one stage of the in vitro expansion of the application.

[0354] For example, the cells of the application can be contacted with one or more cell activators and / or one or more cell growth factors and co-cultured with feeder cells of the application during a single stage of the in vitro expansion of the application, for example, a single stage of the in vitro expansion of the application can refer to the in vitro expansion of the application during the same stage, for example, during the first stage of the in vitro expansion of the application, during the second stage of the in vitro expansion of the application, or during the third stage of the in vitro expansion of the application, etc.

[0355] For example, the TILs of the application can be contacted with one or more cell activators and / or one or more cell growth factors and co-cultured with feeder cells of the application during the first stage of the in vitro expansion of the application. For example, the TILs of the application can be contacted with one or more cell activators and / or one or more cell growth factors of the application and co-cultured with feeder cells of the application during the second stage of the in vitro expansion of the application. For example, the TILs of the application can be contacted with one or more cell activators and / or one or more cell growth factors of the application and co-cultured with feeder cells of the application during the third stage of the in vitro expansion of the application.

[0356] For example, in a single stage of in vitro expansion according to the present application, the cells of the present application can be contacted with one or more cell activators and / or one or more cell growth factors for a period of time prior to co-culture with feeder cells according to the present application. For example, in a first stage of in vitro expansion according to the present application, the TILs of the present application can be contacted with one or more cell activators and / or one or more cell growth factors for a period of time prior to co-culture with feeder cells according to the present application. For example, in a second stage of in vitro expansion according to the present application, the TILs of the present application can be contacted with one or more cell activators and / or one or more cell growth factors for a period of time prior to co-culture with feeder cells according to the present application. For example, in a third stage of in vitro expansion according to the present application, the TILs of the present application can be contacted with one or more cell activators and / or one or more cell growth factors for a period of time prior to co-culture with feeder cells according to the present application.

[0357] For example, in a single stage in vitro expansion of the application, the cells of the application can be contacted with one or more cell activators and / or one or more cell growth factors for a certain period of time prior to co-culture with the feeder cells of the application. For example, the certain period of time of the application can be at least about 1 hour. For example, the certain period of time of the application can be at least about 1-72 hours, such as at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours. For example, the certain period of time of the application can be about 6 hours to about 72 hours, or about 12 hours to 72 hours. For example, the certain period of time of the application can be about 6 hours to about 7 hours, about 6 hours to about 8 hours, about 6 hours to about 9 hours, about 6 hours to about 10 hours, about 6 hours to about 11 hours, about 6 hours to about 12 hours, about 6 hours to about 13 hours, about 6 hours to about 14 hours, about 6 hours to about 15 hours, about 6 hours to about 16 hours, about 6 hours to about 17 hours, about 6 hours to about 18 hours, about 6 hours to about 19 hours, about 6 hours to about 20 hours, about 6 hours to about 21 hours, about 6 hours to about 22 hours, about 6 hours to about 23 hours, about 6 hours to about 24 hours, about 6 hours to about 36 hours, about 6 hours to about 48 hours, about 6 hours to about 60 hours, or about 6 hours to about 72 hours. For example, the certain period of time of the application can be about 12 hours to about 13 hours, about 12 hours to about 14 hours, about 12 hours to about 15 hours, about 12 hours to about 16 hours, about 12 hours to about 17 hours, about 12 hours to about 18 hours, about 12 hours to about 19 hours, about 12 hours to about 20 hours, about 12 hours to about 21 hours, about 12 hours to about 22 hours, about 12 hours to about 23 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, or about 12 hours to about 72 hours.For example, a certain time of the application can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.

[0358] For example, a feeder cell of the application can comprise an antigen presenting cell. For example, a feeder cell of the application can comprise one or more selected from the group consisting of a peripheral mononuclear cell, a dendritic cell, and an artificial antigen presenting cell. For example, a feeder cell of the application can be a peripheral mononuclear cell. For example, a feeder cell of the application can be an irradiated feeder cell. For example, a feeder cell of the application can be an isolated artificial antigen presenting cell (aAPC), an artificial antigen presenting cell of the application can comprise a cell expressing HLA-A / B / C, CD64, CD80, ICOS-L, and / or CD58, and can be modified to express one or more cell activators of the application. For example, a feeder cell of the application can be irradiated, for example, can be gamma irradiated, or can be X-irradiated.

[0359] For example, co-culturing a cell of the application with a feeder cell of the application can comprise contacting a surface of the feeder cell of the application with a surface of the cell of the application. For example, co-culturing a cell of the application with a feeder cell of the application comprises adding a feeder cell of the application to a cell culture medium of a cell of the application.

[0360] For example, a feeder cell of the application can be added to a cell culture medium of a cell of the application at a ratio of about 40: 1 to about 400: 1 feeder cells of the application to cells of the application. For example, a feeder cell of the application can be added to a cell culture medium of a cell of the application at a ratio of about 40: 1 to about 400: 1, at a ratio of about 40: 1 to about 300: 1, at a ratio of about 40: 1 to about 200: 1, at a ratio of about 40: 1 to about 100: 1, at a ratio of about 40: 1 to about 90: 1, at a ratio of about 40: 1 to about 80: 1, at a ratio of about 40: 1 to about 70: 1, at a ratio of about 40: 1 to about 60: 1, at a ratio of about 40: 1 to about 50: 1, at a ratio of about 50: 1 to about 400: 1, at a ratio of about 60: 1 to about 400: 1, at a ratio of about 70: 1 to about 400: 1, at a ratio of about 80: 1 to about 400: 1, at a ratio of about 90: 1 to about 400: 1, at a ratio of about 100: 1 to about 400: 1, at a ratio of about 200: 1 to about 400: 1, or at a ratio of about 300: 1 to about 400: 1 feeder cells of the application to cells of the application.

[0361] In one aspect, the present application provides a cell, the cell of the present application can be cultured according to the culture method of the present application. In one embodiment, the cell provided by the present application can comprise one or one batch of cells cultured by the culture method of the present application. In one embodiment, the cell provided by the present application can comprise a plurality of or a plurality of batches of cells cultured by the culture method of the present application and combined in any ratio.

[0362] In some embodiments, the cells expanded using the methods of the present application can be administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition can be a suspension of the cells in a sterile buffer. The cells expanded using the PBMCs of the present application can be administered by any suitable route known in the art. In some embodiments, the cells can be administered in a single intra-arterial or intravenous infusion, which can last for about 30 to 60 minutes. Other suitable routes of administration can include intraperitoneal, intrathecal, and intralymphatic administration.

[0363] In some embodiments, any suitable dose of cells can be administered. In some embodiments, for example when the tumor is a melanoma, about 1 x 10 9 to about 13.7 x 10 10 , preferably about 2.3 x 10 9 to about 13.7 x 10 10 cells can be administered. In some embodiments, about 1 x 10 9 to about 12 x 10 10 cells can be administered. In some embodiments, about 1.2 x 10 10 to about 4.3 x 10 10 cells can be administered. In some embodiments, about 3 x 10 10 to about 12 x 10 10 cells can be administered. In some embodiments, about 4 x 10 10 to about 10 x 10 10 cells can be administered. In some embodiments, about 5 x 10 10 to about 8 x 10 10 cells can be administered. In some embodiments, about 6 x 10 10 to about 8 x 10 10 cells can be administered. In some embodiments, about 7 x 10 10 to about 8 x 10 10 cells can be administered. In some embodiments, the therapeutically effective dose can be about 1 x 10 9 to about 13.7 x 10 10 , preferably about 2.3 x 10 9 to about 13.7 x 10 10In some embodiments, the therapeutically effective dose can be about 1 x 10 9 to about 12 x 10 10 cells. In some embodiments, the therapeutically effective dose can be about 1.2 x 10 10 to about 4.3 x 10 10 cells. In some embodiments, the therapeutically effective dose can be about 3 x 10 10 to about 12 x 10 10 cells. In some embodiments, the therapeutically effective dose can be about 4 x 10 10 to about 10 x 10 10 cells. In some embodiments, the therapeutically effective dose can be about 5 x 10 10 to about 8 x 10 10 cells. In some embodiments, the therapeutically effective dose can be about 6 x 10 10 to about 8 x 10 10 cells. In some embodiments, the therapeutically effective dose can be about 7 x 10 10 to about 8 x 10 10 cells.

[0364] In some embodiments, the number of cells provided in the compositions of the present application can be about 1 x 10 6 - 9 x 10 13 , e.g., about 1 x 10 6 , about 2 x 10 6 , about 3 x 10 6 , about 4 x 10 6 , about 5 x 10 6 , about 6 x 10 6 , about 7 x 10 6 , about 8 x 10 6 , about 9 x 10 6 , about 1 x 10 7 , about 2 x 10 7 , about 3 x 10 7 , about 4 x 10 7 , about 5 x 10 7 , about 6 x 10 7 , about 7 x 10 7 , about 8 x 10 7 , about 9 x 10 7 , about 1 x 10 8 , about 2 x 10 8 , about 3 x 10 8 , about 4 x 10 8 , about 5 x 10 8 , about 6 x 10 8 , about 7 x 10 8 , about 8 x 108 about 9 x 105 8 about 1 x 105 9 about 2 x 105 9 about 3 x 105 9 about 4 x 105 9 about 5 x 105 9 about 6 x 105 9 about 7 x 105 9 about 8 x 105 9 about 9 x 105 9 about 1 x 105 1 0 about 2 x 105 10 about 3 x 105 10 about 4 x 105 10 about 5 x 105 10 about 6 x 105 10 about 7 x 105 10 about 8 x 105 10 about 9 x 105 10 about 1 x 105 11 about 2 x 105 11 about 3 x 105 11 about 4 x 105 11 about 5 x 105 11 about 6 x 105 11 about 7 x 105 11 about 8 x 105 11 about 9 x 105 11 about 1 x 105 12 about 2 x 105 12 about 3 x 105 12 about 4 x 105 12 about 5 x 105 12 about 6 x 105 12 about 7 x 105 12 about 8 x 105 12 about 9 x 105 12 about 1 x 105 13 about 2 x 105 13 about 3 x 105 13 about 4 x 105 13 about 5 x 105 13 about 6 x 105 13 about 7 x 105 13 about 8 x 105 13 about 9 x 105 13 In some embodiments, the number of cells provided in the compositions of the application can range from about 1 x 105 6 to 5 x 105 6 about 5 x 105 6 to 1 x 105 7about 1 x 10 7 about 5 x 10 7 about 1 x 10 7 about 5 x 10 8 about 1 x 10 8 about 5 x 10 8 about 1 x 10 8 about 5 x 10 9 about 1 x 10 9 about 5 x 10 9 about 1 x 10 9 about 5 x 10 10 about 1 x 10 10 about 5 x 10 10 about 1 x 10 10 about 5 x 10 11 about 1 x 10 11 about 5 x 10 12 about 1 x 10 12 about 5 x 10 12 about 1 x 10 12 about 5 x 10 13 about 1 x 10 13 about 5 x 10 13 about 1 x 10 13 about 5 x 10 13 .

[0365] In some embodiments, the concentration of cells provided in the compositions of the application can be less than about 100-0.0001% w / w, w / v, or v / v of the composition, e.g., about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, about 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%, about 0.0009%, about 0.0008%, about 0.0007%, about 0.0006%, about 0.0005%, about 0.0004%, about 0.0003%, about 0.0002%, or about 0.0001% w / w, w / v, or v / v.

[0366] In some embodiments, the concentration of cells provided in the compositions of the application can be greater than about 90-0.0001% w / w, w / v, or v / v of the composition, e.g., about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17%, about 16.75%, about 16.50%, about 16.25%, about 16%, about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14%, about 13.75%, about 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75%, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%, about 9%, about 8.75%, about 8.50%, about 8.25%, about 8%, about 7.75%, about 7.50%, about 7.25%, about 7%, about 6.75%, about 6.50%, about 6.25%, about 6%, about 5.75%, about 5.50%, about 5.25%, about 5%, about 4.75%, about 4.50%, about 4.25%, about 4%, about 3.75%, about 3.50%, about 3.25%, about 3%, about 2.75%, about 2.50%, about 2.25%, about 2%, about 1.75%, about 1.50%, about 1.25%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, about 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%, about 0.0009%, about 0.0008%, about 0.0007%, about 0.0006%, about 0.0005%, about 0.0004%, about 0.0003%, about, or 0.0002%, or about 0.0001% w / w, w / v, or v / v.

[0367] In some embodiments, the concentration of cells provided in the compositions of the present application can range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v, or v / v of the composition.

[0368] In some embodiments, the concentration of cells provided in the compositions of the present application can range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w / w, w / v, or v / v of the composition.

[0369] In some embodiments, the amount of cells provided in the compositions of the present application can be equal to or less than about 10-0.0001 g, for example, about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g, about 6.5 g, about 6.0 g, about 5.5 g, about 5.0 g, about 4.5 g, about 4.0 g, about 3.5 g, about 3.0 g, about 2.5 g, about 2.0 g, about 1.5 g, about 1.0 g, about 0.95 g, about 0.9 g, about 0.85 g, about 0.8 g, about 0.75 g, about 0.7 g, about 0.65 g, about 0.6 g, about 0.55 g, about 0.5 g, about 0.45 g, about 0.4 g, about 0.35 g, about 0.3 g, about 0.25 g, about 0.2 g, about 0.15 g, about 0.1 g, about 0.09 g, about 0.08 g, about 0.07 g, about 0.06 g, about 0.05 g, about 0.04 g, about 0.03 g, about 0.02 g, about 0.01 g, about 0.009 g, about 0.008 g, about 0.007 g, about 0.006 g, about 0.005 g, about 0.004 g, about 0.003 g, about 0.002 g, about 0.001 g, about 0.0009 g, about 0.0008 g, about 0.0007 g, about 0.0006 g, about 0.0005 g, about 0.0004 g, about 0.0003 g, about 0.0002 g, or about 0.0001 g.

[0370] In some embodiments, the amount of cells provided in the compositions of the present application can be greater than about 0.0001-10 g, such as about 0.0001 g, about 0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006 g, about 0.0007 g, about 0.0008 g, about 0.0009 g, about 0.001 g, about 0.0015 g, about 0.002 g, about 0.0025 g, about 0.003 g, about 0.0035 g, about 0.004 g, about 0.0045 g, about 0.005 g, about 0.0055 g, about 0.006 g, about 0.0065 g, about 0.007 g, about 0.0075 g, about 0.008 g, about 0.0085 g, about 0.009 g, about 0.0095 g, about 0.01 g, about 0.015 g, about 0.02 g, about 0.025 g, about 0.03 g, about 0.035 g, about 0.04 g, about 0.045 g, about 0.05 g, about 0.055 g, about 0.06 g, about 0.065 g, about 0.07 g, about 0.075 g, about 0.08 g, about 0.085 g, about 0.09 g, about 0.095 g, about 0.1 g, about 0.15 g, about 0.2 g, about 0.25 g, about 0.3 g, about 0.35 g, about 0.4 g, about 0.45 g, about 0.5 g, about 0.55 g, about 0.6 g, about 0.65 g, about 0.7 g, about 0.75 g, about 0.8 g, about 0.85 g, about 0.9 g, about 0.95 g, about 1 g, about 1.5 g, about 2 g, about 2.5 g, about 3 g, about 3.5 g, about 4 g, about 4.5 g, about 5 g, about 5.5 g, about 6 g, about 6.5 g, about 7 g, about 7.5 g, about 8 g, about 8.5 g, about 9 g, about 9.5 g, or about 10 g.

[0371] In some embodiments, the cells can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, the cells can be administered in multiple doses. The doses can be once a year, twice, three times, four times, five times, six times, or more than six times a year. The doses can be once a month, once every two weeks, once a week, or once every 2 days. In some embodiments, the administration of the cells can be continuous administration.

[0372] In one aspect, the present application provides a pharmaceutical composition. In some embodiments, it can comprise the cells of the present application, with a pharmaceutically acceptable carrier.

[0373] In one aspect, the present application provides a kit, the kit of the present application can comprise the cell activator, the cell growth factor and / or the feeder cell of the cell culturing method of the present application and the instruction describing the steps of the cell culturing method of the present application. In one aspect, the present application provides a kit, the kit of the present application can comprise the cell of the present application and / or the pharmaceutical composition of the present application.

[0374] In one aspect, the present application provides a method of affecting the growth of a cell, e.g., a tumor cell, which can comprise administering to a subject the cell of the present application and / or the pharmaceutical composition of the present application. In some embodiments, affecting the growth of a tumor can comprise reducing the volume of a tumor by about 99-0.1%, e.g., about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% from the pre-administration.

[0375] In one aspect, the present application provides the use of the cell of the present application and / or the pharmaceutical composition of the present application in the manufacture of a medicament, the medicament of the present application can be used for preventing and / or treating a disease and / or a symptom. For example, the disease and / or the symptom of the present application can comprise a tumor. In some embodiments, the tumor of the present application is selected from a solid tumor. In some embodiments, the tumor of the present application can be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0376] In one aspect, the present application provides a method of preventing and / or treating a disease and / or a symptom, which can comprise administering to a subject the cell of the present application and / or the pharmaceutical composition of the present application. For example, the disease and / or the symptom of the present application can comprise a tumor. In some embodiments, the tumor of the present application is selected from a solid tumor. In some embodiments, the tumor of the present application can be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0377] In one aspect, the present application provides a TIL of the present application and / or a pharmaceutical composition of the present application, which can be used for preventing and / or treating a disease and / or a symptom. For example, the disease and / or the symptom of the present application can comprise a tumor. In some embodiments, the tumor of the present application is selected from a solid tumor. In some embodiments, the tumor of the present application can be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0378] Without wishing to be bound by any theory, the examples below are merely to illustrate the methods and uses of the present application, and are not intended to limit the scope of the present application.

[0379] Examples

[0380] Example 1: Experimental materials and methods

[0381] (I) TIL cell culture

[0382] 1.1 Tumor tissue reception and processing

[0383] 1.1.1 Tissue reception

[0384] Receive the tumor tissue and blood sample of the donor, check the sample information and record, and print the corresponding sample label.

[0385] 1.1.2 Tissue processing and culture

[0386] Using 75% alcohol to disinfect sample tubes and blood collection tubes, transfer to the biosafety cabinet. According to the above-mentioned PBMC manual separation and cryopreservation operation procedure, separate and cryopreserve the PBMC cells in the blood sample. Take a culture bottle or culture bag with a breathable surface, such as a culture bag (Origen), and add 300 mL of warmed complete medium. The complete medium can be arbitrarily selected from X-vivo 15 medium or other commercial T cell culture medium, such as T cell culture medium of Stem Cell, Lonza, Thermo, Meitianyi, etc., and can be added with essential amino acids and antibiotics, and can be added with IL-2 at a concentration of 300-9000 IU / mL (such as 1000-9000 IU / mL, for example, 6000 IU / mL). Take several 10 cm culture dishes, add an appropriate amount of culture medium, and use sterile ophthalmic forceps to remove the tumor tissue from the sample tube into the 10 cm culture dish, wash the tissue and change the culture dish. Use ophthalmic scissors and ophthalmic forceps for preliminary cutting, remove fat tissue and necrotic tissue, and continue to cut each tissue block to about 27 cubic millimeters in size. Take the non-suspended tumor tissue block, remove the inner piston using a 20 mL syringe, connect it to the culture bag, and use a pipette to transfer about 1 g of tissue block into the culture bag through the syringe. Place the culture bag in a carbon dioxide incubator for culture. Clean the scissors and forceps, and sterilize them after preliminary disinfection with 75% alcohol, ultrasonic cleaning, and sterilization to obtain the first TIL population.

[0387] 1.2 Step (A) in vitro expansion and harvest

[0388] 1.2.1 Step (A) in vitro expansion

[0389] According to the cell growth state, supplement or half-volume exchange every 3-7 days to ensure cell nutrition. Use complete medium, which can be arbitrarily selected from X-vivo 15 medium or other commercial T cell culture medium, such as T cell culture medium of Stem Cell, Lonza, Thermo, Meitianyi, etc., and can be added with essential amino acids and antibiotics, and can be added with IL-2 (Shuanglu and / or Sihuan) at a concentration of 300-9000 IU / mL (such as 1000-9000 IU / mL, for example, 6000 IU / mL). During step (A), 3-14 days, for example, sample counting can be taken on the 13th or 14th day, and if the cell number is between 5x10 5 and 5x10 8 , enter the harvesting step of step (A).

[0390] 1.2.2 Harvesting of step (A)

[0391] The cells at the end of step (A) in vitro expansion are collected, centrifuged, the culture medium is discarded, the cells are washed once with PBS or physiological saline, the TILs at the end of step (A) in vitro expansion (second TIL population) are obtained, and about 5 x 10 5 to 2 x 10 8 cells are sampled and counted for about 5 x 10 5 cells to be taken for subsequent step of in vitro expansion; about 5 x 10 5 cells can be taken for quality control testing; the rest of the cells are frozen in cryopreservation solution as cryopreserved preREP TIL in vitro cells.

[0392] 1.3 Step (B) TIL activation

[0393] The TILs at the end of step (A) in vitro expansion (second TIL population) are continued to be cultured, or the cryopreserved preREP TIL in vitro cells are thawed for cell recovery, and step (B) TIL activation is performed.

[0394] The complete culture medium can be optionally selected from X-vivo 15 culture medium or other commercially available T cell culture medium, such as T cell culture medium of StemCell, Lonza, Thermo, Meitiandi, etc., and necessary amino acids and antibiotics can be added, and the cell density is adjusted to 5 x 10 5 to 2 x 10 6 cells / mL, 1 mL / well in a suspension 24-well culture plate, and IL-2 is added at a concentration of 300-9000 IU / mL (for example, 1000-9000 IU / mL, for example, 6000 IU / mL). T cell activators can also be added to the culture medium of each TIL cell population, for example, CD3 agonists and / or CD28 agonists are added, for example, about 30 ng / mL of CD3 antibody (Miltenyi Biotech, OKT3), about 30 ng / mL of CD28 antibody (Merck, 15E8), magnetic beads (diameter about 1 to 10 pm Dynabeads, Thermo Fisher) are added at a ratio of about 1:2-2:1 of magnetic beads to TILs, and / or transACT (diameter about 100 to 500 nm, Miltenyi) is added at a ratio of about 1:100-1:2000 of transACT to TILs. The culture is continued for about 0-4 days to obtain a third TIL population.

[0395] 1.4 Step (C) TIL cell gene editing

[0396] 1.4.1 TIL cell gene editing based on Crispr Cas9 system

[0397] sgRNAs targeting each target site selected from the present application are synthesized, thawed and added to nuclease-free water, and brought to a concentration of about 100 mM. About 2 pL of gRNA (50 mM) is annealed by incubation at 95 °C for 2 min, added to P3 buffer, and 0.3-1 pL of Cas9 (e.g., Prime-Cas, CasX, or Acro, 10 mg / mL) is added, incubated at 25 °C for 10 min to form ribonucleoprotein complex (RNP). The above RNP is electroporated with about 1 x 10 6 cells of the third TIL population in P3 buffer (Lonza) by Lonza electroporator. For example, the electroporation program can be human T cell stim (EO115). The electroporated TILs are cultured for about 0-4 days after gene editing to obtain the fourth TIL population.

[0398] 1.4.2 TIL cell gene editing based on Crispr Cas12 system

[0399] sgRNAs targeting each target site selected from the present application are synthesized, thawed and added to nuclease-free water, and brought to a concentration of about 200 mM. About 2 pL of gRNA (100 mM) is annealed by incubation at 95 °C for 2 min, added to P3 buffer, and 1-2 pL of Cas12, such as Cas12b (e.g., Prime-Cas or CasX, 10 mg / mL) is added, incubated at 25 °C for 10 min to form ribonucleoprotein complex (RNP). The above RNP is electroporated with about 1 x 10 6 cells of the third TIL population in P3 buffer (Lonza) by Lonza electroporator. For example, the electroporation program can be human T cell stim (EO115). The electroporated TILs are cultured for about 0-4 days after gene editing to obtain the fourth TIL population.

[0400] 1.5 Step (D) Post TIL cell gene editing culture

[0401] Feeder cells (irradiated healthy donor PBMC T cells) are added to the fourth TIL cell population for culture. The time of TILs in contact with feeder cells needs to be after a certain time T n after the TILs in step (B) are in contact with IL-2 and T cell activators (e.g., T n may be taken from 0 hours to 12 days, for example, 24 hours or 48 hours). First, the feeder cells of 1-5 donors are recovered; the activated TIL cells, feeder cells are mixed at a ratio of TIL cells: feeder cells of about 1:200, transferred into G-Rex 100 culture flask or gas-permeable bag, supplemented with complete medium, sampled and counted every 1-3 days, and supplemented or half-changed according to the cell state until the total cell number is greater than 1 x 10 9or step (D) in vitro expansion culture for about 5 days to about 14 days, and terminating the step (D) in vitro expansion culture.

[0402] 1.6 Harvesting of tumor infiltrating lymphocytes

[0403] Take the step (D) expanded cells, centrifuge and discard the supernatant of the culture medium, and wash with PBS or normal saline or compound electrolyte solution for three times to obtain the step (D) expanded TILs (the fifth TIL group). Take a sample for counting at the third time of washing, and according to the counting result, discard the supernatant after the last centrifugation, and take 3x10 6 The cells are sent for quality control detection, and the rest of the cells are added into the cryopreservation solution to adjust the cell density to 1-3x10 8 cells / mL for freezing.

[0404] (B) TCR-T cell culture

[0405] T cell activation: thaw the T cells stored in liquid nitrogen, resuspend to 5E5 / ml by centrifugation using T cell culture medium RPMI 1640 (Gibco) + 10% FBS (Bovogen), add T cell TransAct (Miltenyi) at 1:100, add recombinant human IL-2 at a concentration of 30 IU / ml, and culture for about 72 hr.

[0406] TCR transduction: 1 day before transduction, coat a 24-well suspension culture plate with recombinant human fibronectin fragment (Retronectin, Takara) at a final concentration of 15 μg / mL, 250 μL per well of the 24-well plate. Avoid light, 4°C overnight for standby. Take out the coated 24-well plate, aspirate the coating solution, add 500 μL of 2% BSA blocking solution, and block at room temperature for 30 minutes. Aspirate the blocking solution, wash the plate twice with 500 μL / well of washing buffer containing 2.5% HEPES, and aspirate the washing buffer. The experimental group is transduced with a specific TCR nucleic acid fragment carrying the NY-ESO-1 antigen peptide. Add 0.1-1 mL of retrovirus solution per well, centrifuge at 2000 g for 2 hours at 32°C. Discard the supernatant of the 24-well plate, add the resuscitated and activated T cells to the 24-well plate, with a volume of 500-1000 μL and a cell concentration of about 5x10 5 6 / mL per well. Centrifuge at 1000 g for 10 minutes at 30-32°C. After centrifugation, place the culture plate in a 37°C, 5% CO2 incubator for culture to obtain the transduced cells. Culture the transduced cells for about 0-4 days to obtain the TCR-T cell population. According to the cell counting density and viability of the cells every 1-3 days, add T cell culture medium according to the counting result, add recombinant human IL-2 at a concentration of 30-100 IU / ml, adjust the initial culture cell density to 0.5-2x10 6 6 / ml, and continue to culture.

[0407] TCR-T gene editing: sgRNAs selected from the present invention targeting various sites are synthesized, thawed, and diluted with nuclease-free water to a concentration of approximately 100-200 μM. Approximately 2 μL of gRNA (50-100 μM) is annealed at 95°C for 2 minutes and then added to P3 buffer. 0.3-1 μL of Cas9 (e.g., Kex, Crizo, Acro, 10 mg / mL) or 1-2 μL of Cas12b (e.g., Nearshore or Sino, 10 mg / mL) is added, and the mixture is incubated at 25°C for 10 minutes to form a ribonucleoprotein complex (RNP). In P3 buffer (Lonza), the RNP is electroporated with approximately 1 × 10⁻⁶ sgRNAs using a Lonza electroporator. 6 TCR-T cells were electroporated. For example, the electroporation program could be human T cell stim (EO115). The electroporated T cells were then supplemented with recombinant human IL-2 at a concentration of 100–300 IU / ml and cultured for a further period to obtain the TCR-T cells with the target gene edited according to this invention.

[0408] (III) Knockout Efficiency Testing

[0409] Reagents and materials used to detect cell knockout efficiency: QuickExtract DNA extraction solution (Lucigen, QE09050), RNase / DNase free water (Tiangen), EDTA (Sangon Biotech, 0.5M), and Recombinant DNase I (RNase-free, Takara).

[0410] Genomic DNA extraction: Approximately 2-7 days after T cell knockout (refer to the gene editing method in step 1.4 of Example 1(I) or the TCR-T gene editing method in Example 1(II)), approximately 1×10⁻⁶ DNA samples are extracted. 5 Approximately 2 × 10 5 Cells were washed once with PBS, then resuspended in 44 μL PBS. 6 μL of a prepared nuclease mixture (containing 1 μL DNase I and 5 μL 10×DNase I buffer) was added, and the cells were incubated at 37°C for 5 minutes. 2.5 μL of 0.5 M EDTA was added to the sample, and the cells were incubated at 80°C for 10 minutes. After centrifugation and discarding the supernatant, 50 μL of DNA extraction buffer was added to the cell pellet. After brief centrifugation, the following program was run: 75°C for 10 minutes; 95°C for 5 minutes; 4°C for maintenance. A spectrophotometer (NanoDrop) can be used for further analysis. TM ) Detect the concentration of DNA samples.

[0411] Sequencing: PCR primers can be designed in the region about 100 to about 200 nucleotides upstream and downstream of the PAM site. The PCR reaction system is designed as follows:

[0412] Table 1 PCR reaction system

[0413] and amplified according to the following PCR program:

[0414] Table 2 PCR program amplification

[0415] The PCR product is subjected to Sanger sequencing analysis based on the Crispr Cas9 editing system. The PCR product is subjected to NGS sequencing analysis based on the Crispr Cas12b editing system.

[0416] Analysis of Crispr Cas9 knockout efficiency

[0417] The Crispr Cas9 knockout efficiency is analyzed according to the Sanger sequencing data by using the Tracking of Indels by DEcomposition (Tide) method. The specific method can be referred to (Brinkman et al, Nucl. Acids Res. (2014) or shinyapps.datacurators.nl / tide / ). By inputting the corresponding sgRNA sequence of the application, the control sequence before knockout, and the test sequence after Crispr Cas9 knockout, the P-value threshold is set to 0.001, and the knockout efficiency analysis is performed.

[0418] Analysis of Crispr Cas12b knockout efficiency

[0419] The genomic crude product is subjected to PCR amplification similar to this example to construct an NGS library. The size of the PCR amplification region can be about 250 bp, and the distance between the amplification primers and the editing site can be greater than about 70 bp.

[0420] The constructed library is subjected to second-generation sequencing, library detection and second-generation sequencing are performed using Illumina Miseq Reagent Kit (v2), and sequencing data is returned. The editing efficiency analysis is performed using the BAE lab tool (rgenome.net / cas-analyzer / #!) or other tools in the art for calculating editing efficiency. The sequencing data is selected as the corresponding compressed sequencing data, the DNA sequence of the PCR amplification region is input in the full reference sequence, the nucleic acid type is selected as a single nucleic acid, the nuclease is selected as the type of PAM sequence of TT N, the binding sequence of the gRNA is input in the target DNA; the analysis parameter is set as 70 in the comparison region (R) and 20 in the WT marker (r), and the editing efficiency is analyzed. The optional editing efficiency analysis method can also be performed using the CRISPResso software, the parameters -r1 and -r2 are selected as the corresponding compressed sequencing data, -a is selected as the DNA sequence of the PCR amplification region, -g is selected as the binding sequence of the gRNA, --quantification_window_size and --quantification_window_center are set as 20 and 0, respectively, and the rest of the parameters can be referred to the tool recommendation, and the editing efficiency is analyzed.

[0421] (iv) amplification detection

[0422] Test preparation

[0423] The proliferation of TIL cells is detected 7-10 days after gene editing in Example 1 (I) (IL-2 is removed), and the TIL cells in each group are harvested; or the TCR-transduced T cells obtained in Example 1 (II) are used.

[0424] The cells are washed once with PBS, resuspended with T cell culture medium (without IL-2), counted, and adjusted to a cell density of 1e5 to 2e6 / mL, and then added to a flat-bottom 96-well plate at 100 μL / well. In the unstimulated medium group, no cell activator is added to the medium; in the CD3 antibody stimulation group, CD3 antibody (OKT3) 30 ng / ml is added for stimulation; in the transACT stimulation group, transACT (about 100 to 500 nm in diameter, Miltenyi) is added to make the working solution concentration of transACT 1:1000 (v / v); and in the medium group, only the same volume of cell culture medium is added. The amount of fluorescence of T cells when plated is analyzed using the CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega), the amount of fluorescence of T cells is analyzed after 3 days using the CTG kit, and the expansion efficiency of T cells is characterized by the amount of fluorescence on the third day / the amount of fluorescence when plated.

[0425] (v) Cell killing ability detection

[0426] From the 6th day after gene editing, tumor target cells were plated in 96-well flat-bottom plates, and the next day, TIL cells in each group were co-cultured with target cells at different effector-to-target ratios (T cells: target cells, E: T). Alternatively, TCR-transduced T cells obtained in Example 1 (ii) were used.

[0427] 100 μL of target cells and T cells were set up in triplicate for each group, with a control group containing only target cells. The target cells can be selected from A375 melanoma cells.

[0428] According to the instructions of the apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL / well of apoptosis detection reagent was added, and 25 μL / well of culture medium was added to dilute the Caspase 3 / 7 Green Dye. The Incucyte recorder (Sartorius) was used to record the activity of Caspase 3 / 7 to analyze the killing ability of TIL cells to target cells, recording once every 3 hours, with a total recording time of about 5 days.

[0429] (vi) Flow cytometry detection of T cell subsets and function-related markers

[0430] The expression of T cell exhaustion, stemness, and other related molecules in TIL cells obtained on the 8th day after gene editing was detected by flow cytometry. Alternatively, TCR-transduced T cells obtained in Example 1 (ii) were detected.

[0431] V-bottom 96-well plates, manufacturer Corning, item number 3894; flow cytometry tubes, manufacturer Corning, item number 352052; flow cytometry antibodies purchased from BD or Biolegend.

[0432] Cell surface molecule detection: 1 x 10 5 to 5 x 10 5Cell surface staining: Add 100-500 μL of antibody working solution to each well of a 96-well plate or to each tube of a flow tube. Incubate at 2-8°C for 30 minutes in the dark. Wash the cells once with PBS (200 μL / well for a 96-well plate or 1 mL / tube for a flow tube). Centrifuge at 600g for 3 minutes at room temperature. Discard the supernatant. Resuspend the cells in 100-500 μL of PBS. Perform flow cytometry detection.

[0433] Intracellular molecule detection: Prepare an antibody mixture working solution for cell surface staining CD3 / CD4 / CD8. The antibody concentration is 1:100, and the cell viability detection dye concentration is 1:10,000. Incubate at 2-8°C for 30 minutes in the dark. Wash the cells once with PBS (200 μL / well for a 96-well plate or 1 mL / tube for a flow tube). Centrifuge at 600g for 3 minutes at room temperature. Discard the supernatant. Add 100 μL of fixation and permeabilization solution (BD) to each well. Incubate at 2-8°C for 20-40 minutes in the dark. After fixation and permeabilization, wash the cells twice with 1x Perm / Wash Buffer (200 μL / well for a 96-well plate or 1 mL / tube for a flow tube). Centrifuge at 600g for 3 minutes at room temperature. Discard the supernatant. Prepare an intracellular molecule antibody (e.g., TCF1) solution in 1x Perm / Wash Buffer. Resuspend the TIL cells (50 μL / well for a 96-well plate or 100 μL / tube for a flow tube). Incubate at 2-8°C for 30 minutes in the dark. After intracellular molecule staining, wash the cells once or twice with 1x Perm / Wash Buffer (200 μL / well for a 96-well plate or 1 mL / tube for a flow tube). Centrifuge at 600g for 3 minutes at room temperature. Discard the supernatant. Resuspend the cells in 100-500 μL of PBS. Perform flow cytometry detection.

[0434] (Seven) Cytokine expression flow cytometry detection

[0435] For each test group, obtain the TIL cell population on day 7 or day 8 after gene editing and perform flow cytometry detection of cytokine expression; or obtain the TCR-transduced T cells according to Example 1 (ii) and perform detection.

[0436] Preparation of culture medium for intracellular factor expression detection: take TIL cell culture medium, add Golgistop 0.7:1000, Golgiplug 1:1000, CD107a antibody 1:500 (2 μL / mL) according to volume ratio. No interleukin is added.

[0437] Detection step

[0438] After centrifugation of T cells of each test group, the T cells of each group are resuspended using the above-mentioned culture medium for intracellular factor expression detection, and after counting, the cell density is adjusted to 1×10 6 After centrifugation of T cells of each test group, the T cells of each group are resuspended using the above-mentioned culture medium for intracellular factor expression detection, and after counting, the cell density is adjusted to 1×10

[0439] Sources of main reagents and materials for cytokine flow detection test:

[0440] V-bottom 96-well plates, manufacturer: Corning, item number: 3894; flow tubes, manufacturer: Corning, item number: 352052; flow antibodies are purchased from BD or Biolegend.

[0441] After incubation, wash once with 200 μL / well PBS, centrifuge at 600g for 3 minutes, and discard the supernatant. Prepare the antibody mixture working solution for cell surface staining CD3 / CD4 / CD8, with an antibody concentration of 1:100, cell viability detection dye concentration (1:10000), 50 μL / well of a 96-well plate, 100 μL / tube of a flow tube, and incubation at 2-8°C for 30 minutes in the dark. Wash the cells once with PBS (200 μL / each time for a 96-well plate, and 1 mL / each time for a flow tube), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant. Add 100 μL of fixation and permeabilization solution (BD) to each well, and incubate at 2-8°C for 20-40 minutes in the dark. After fixation and permeabilization, wash twice with 1x Perm / Wash Buffer (200 μL / each time for a 96-well plate, and 1 mL / each time for a flow tube), centrifuge at 600g for 3 minutes, and discard the supernatant. Prepare the cytokine detection antibody (e.g., GZMB, TNF-a, IFN-g) using 1x Perm / Wash Buffer, and resuspend the TIL cells (50 μL / well of a 96-well plate, and 100 μL / tube of a flow tube), and incubate at 2-8°C for 30 minutes in the dark. After cytokine staining, wash once or twice with 1x Perm / Wash Buffer (200 μL / each time for a 96-well plate, and 1 mL / each time for a flow tube), centrifuge at 600g for 3 minutes, and discard the supernatant. Resuspend the cells using 100-500 μL of PBS, and perform flow cytometry detection.

[0442] (Eight) Apoptosis detection

[0443] Apoptosis detection is performed on the TIL population obtained on day 7 or day 8 after gene editing for each test group in Example 1, or on the TCR-transduced T cells obtained in Example 1 (ii).

[0444] The TILs in the gene knockout group or the control group (NT, no treatment) are detected for T cell apoptosis level using an apoptosis detection kit (BD 559763 Annexin V PE Apoptosis kit).

[0445] (Nine) PDO model culture

[0446] The tumor tissue is obtained from surgery, transported and stored at 2-8°C after excision, mechanically dissociated within 24 hours, and cut into pieces of about 0.5 mm 3Size, after using special tissue digestion solution (bioGenous) at 37°C digestion treatment, using 10% FBS to terminate the reaction, the tissue suspension is filtered using a 100 μm filter, washed, placed on ice and mixed with Martrigel, then the mixture is spotted on the bottom of the cell culture plate, and the plate is placed in a 37°C incubator. After the Martrigel is fully solidified, carefully add complete medium for culture. When the organoids grow to a sufficient size and density, they are passaged or cryopreserved to obtain a PDO (tumor organoid, Patient-Derived Organoid) model.

[0447] The Matrigel and organoid mixture is scraped off with a pipette tip, transferred to a centrifuge tube containing the base medium, and mixed by pipetting to separate the organoids and Matrigel. After centrifugation, add the tissue digestion solution for passaging, mix well, and place in a 37°C incubator for digestion. After adding the base medium to dilute the digestion solution, centrifuge and wash, take a small amount of cell suspension and add 8 times the volume of digestion solution to make it single cell. After using FBS to terminate the reaction, count, and according to the counting results, take the required volume of organoids, centrifuge, and resuspend in T cell medium; count the cells to be tested (such as TIL cells or TCR-T cells), resuspend the cells to be tested to the required density according to different effector target ratios; add 100 μl of PDO target cells and cells to be tested in a flat-bottom 96-well plate, set three replicates for each group, and use for PDO co-culture detection. Set several different effector target ratios, at the same time, set a group containing only PDO target cells, and set a control group (NT) for co-culturing unedited cells to be tested in PDO target cells only.

[0448] Killing ability detection in PDO model

[0449] Referring to the preparation of the PDO model according to the embodiments of the present application and co-culturing with TIL cells or TCR-T cells, Caspase3 / 7 substrate is added for apoptosis signal labeling, and the experimental plate is placed in Incucyte for observation and recording.

[0450] (X) Cell multi-round killing ability detection

[0451] IncuCyte to monitor the change of fluorescence intensity of target cells A375-GFP in multiple rounds of killing. After the A375-GFP cells were uniformly plated in a multi-well plate and incubated at 37°C for 4 hours, the cells without editing or knocking out the target point of the application were taken and added to the corresponding wells, and then the IncuCyte was used to record the GFP fluorescence signal. Each group had 3 replicate wells. After 24 hours of killing, the co-culture supernatant (20 μL / well) was taken for CBA detection; after 3 days of killing, a new multi-well plate was uniformly plated with A375-GFP, and after incubation at 37°C for 4 hours, the mixture of cells without editing or knocking out the target point of the application and A375-GFP from the previous round of killing was transferred to a new 96-well plate, and the IncuCyte was used to record the change curve of the GFP fluorescence signal in the new round of killing.

[0452] (XI) CBA method for detecting cytokine release

[0453] For co-culture of gene-edited cells with autologous tumor cells, the co-culture supernatant was collected, and the CBA kit (BD) was used to detect the cytokine release of cells without editing or knocking out the target point of the application.

[0454] (XII) In vivo efficacy detection of gene-edited cells

[0455] Surgical tumor tissue samples were transferred to the laboratory under sterile conditions, and the necrotic tissue was removed in a sterile clean bench. The tumor tissue was cut into 1-2 mm 3 in size, and the cut tumor tissue was inoculated subcutaneously in NOG mice (Vivian, strain code 408) in SPF animal rooms with a little Matrigel glue, 4-5 pieces of tissue were inoculated in each mouse, and the batch of tumor tissue was F0 generation. When the F0 tumor tissue grew to 800 mm 3 in size subcutaneously in mice, the F0 tumor tissue was surgically removed, and the tumor tissue was again cut into 1-2 mm 3 in size, part was frozen for later use, and part was inoculated subcutaneously in the second batch of NOG mice, and the tumor tissue was continuously passaged and expanded in mice, and the batch of tumor tissue was F1 generation. In turn, until the tumor tissue was transmitted to F3 generation in mice, the tumor tissue size was measured regularly, and after the tumor volume grew to about 100 mm 3 , the mice were randomly divided into groups. The NT group of mice was injected intravenously with cells that were not gene-edited, and the test group of mice was injected intravenously with gene-edited cells of the application.

[0456] (XIII) HEK193 cell culture and transfection

[0457] HEK293 cells were cultured and passaged according to the conventional standard method, and the culture medium was DMEM + 10% FBS. One day before transfection, the cells were inoculated in a 96-well plate. The next day, the transfection reagent mixture was prepared: 1. Opti-MEM (ThermoFisher) 5 μL, mRNA encoding Cas12b enzyme 0.1 μL (100 ng), sgRNA 1 μL (3 pmol, about 114 ng); 2. Opti-MEM (ThermoFisher) 5 μL, Lipofectamine 3000 (ThermoFisher) 0.3 μL. After mixing and incubating at room temperature for 10 minutes, the mixture was added to the 293 cells in the incubator for 72 hours.

[0458] The cell lysis solution was prepared according to the number of samples. One portion of the lysis solution was 96 μL DNA Extraction Solution + 4 μL Enzyme Mix (ZYMO), mixed thoroughly, and the lysis solution was added to the cells, which were incubated in a 55°C water bath for 15 min. Then the sample was incubated in a 95°C water bath for 5 min. 100 μL of Stop Solution was added to the above sample and mixed to obtain the lysis product.

[0459] (Twelfth) PMBC cell culture and transfection

[0460] The Cas12b enzyme and gRNA were mixed at a ratio of 1:3 (molar ratio). The amount of Cas12b protein for electroporation was 30, 60, 90, 120 and 180 pmol, and the corresponding control groups (Cas protein only, gRNA only, no protein and gRNA, no electroporation) were set. After mixing the Cas12b protein and gRNA, incubate at room temperature for 15 minutes.

[0461] RNP or control sample was added to each resuspended PBMC cell, mixed and transferred to a 16-well Lonza electroporation cup, and the instrument was started for electroporation. After electroporation, the cells were completely transferred to the culture plate by adding culture medium and placed in the incubator for culture. After 24 hours of culture, the cell state was observed and the cell activity (countable) was recorded. The cells were collected after further culture, and the genomic crude product was prepared.

[0462] The sequences of sgRNAs (single guide RNAs, or simply guides) targeting each target are synthesized according to the sequences provided in the present application. The region targeted by each target can be selected from the following: an exon region of the target gene, an intron region about 100 bp or about 20 bp away from the exon of the target gene, and a region about 1500 bp before the start codon of the target gene. When the region targeted by each target is selected from the region about 1500 bp before the start codon of the target gene, FIGS. 1A-1C show a continuous region (e.g., the region shown by the black line segment with a vertical coordinate of 3 or more) having a number of transcription factor bindings of about 3 or more before the start codon, according to the present application.

[0463] According to bioinformatics analysis, 34, 193 and 41 editing sites were screened for SOCS1, TNFAIP3 and ZC3H12A, respectively, according to the standard of mismatch 1 = 0 and mismatch 2 < 4 (for sequences, see the sgRNA excel files of SOCS1, TNFAIP3 and ZC3H12A in the appendix). The specific distribution is shown in Table 3. The screened sites were IVT synthesized to synthesize corresponding sgRNAs.

[0464] Table 3 Bioinformatics screening sites of SOCS1, TNFAIP3 and ZC3H12A

[0465] IVT preparation of sgRNA adopts HiScribe TM T7 Quick High Yield RNA Synthesis Kit (New England Biolab) kit, for details, see: sgRNA Synthesis Using the HiScribe TM Quick T7 High Yield RNA Synthesis Kit (NEB#E2050) | NEB, sgRNA IVT products are purified by Oligo Clean & Concentrator (Zymo Research) kit according to the method provided by the kit, and the sgRNA concentration is determined by Nanodrop.

[0466] FIG. 1D shows the electrophoresis results of the T7E1 enzyme digestion of the amplification products of the SOCS1 candidate sites. M is 1 kb DNA ladder, and 01-34 is the candidate site. The arrow indicates that the PCR product is digested, indicating that the site is edited. The boxed site is selected for further verification in PBMC after modification by guide RNA.

[0467] Figure 1E shows the results of electrophoresis of the amplification products of the TNFAIP3 candidate sites (mismatch 2 = 0) after T7E1 enzyme digestion. M is 1 kb DNA ladder, 1-120 is the candidate site. The sites marked by arrows are significantly edited. The boxed sites are further verified in PBMC after modification by selected guide RNA.

[0468] Figure 1F shows the results of electrophoresis of the amplification products of the TNFAIP3 candidate sites (mismatch 2 < 4) after T7E1 enzyme digestion. M is 1 kb DNA ladder, 01-73 is the candidate site. The sites marked by arrows are significantly edited. The boxed sites are further verified in PBMC after modification by selected guide RNA.

[0469] Figure 1G shows the results of electrophoresis of the amplification products of the ZC3H12A candidate sites after T7E1 enzyme digestion. M is 1 kb DNA ladder, 1-41 is the candidate site. The sites marked by arrows are significantly edited. The boxed sites are further verified in PBMC after modification by selected guide RNA.

[0470] Figure 1H shows the verification of candidate sgRNA in PBMC. The RNP formed by the candidate sgRNA of SOCS1, TNFAIP3 and ZC3H12A is electroporated into PBMC, and the electropherogram of T7E1 enzyme digestion of the amplified corresponding fragments is shown. The boxed sites are further verified in PBMC after modification by selected guide RNA.

[0471] Figure 1I shows the electropherogram of T7E1 enzyme digestion of the amplified sites after electroporating PBMC with modified sgRNA. M: DNA 1 kb ladder; 1: SOCS1-015-b16, 2: SOCS1-027-b16, 3: TNFAIP3-005-b16, 4: TNFAIP3-mm2-33-b16, 5: TNFAIP3-mm2-68-b16, 6: ZC3H12A-003-b16.

[0472] Screening experiments were performed on three targets, SOCS1, TNFAIP3 and ZC3H12A. 34, 193 and 41 editing sites of AaCas12bMax with low off-target probability were selected by bioinformatics methods, and preliminary screening was performed on HEK293 cell lines using T7E1 enzyme digestion analysis method. 15 (identified as SC-015) and 27 (identified as SC-027) of SOCS1, 001 (identified as TP-001), 005 (identified as TP-005), 007, 029, 082 (identified as TP-082), 111, 114, 117, mm-2-33 (identified as TP-033) and mm2-68 (identified as TP-068) of TNFAIP3, and 003 (identified as ZC-003), 012, 023 and 033 of ZC3H12A were selected for further verification in PBMC cells. The sgRNAs of the sites with higher editing efficiency, including 15 and 27 of SOCS1, 005, mm2-33 and mm2-66 of TNFAIP3, and 003 of ZC3H12A, were modified and further tested on PBMC. The results showed that the editing efficiency of each editing site was greatly improved, and the editing efficiency of RNP at a dose of 180 pmol was basically above 80%, and the highest was close to 90%. These editing sites can be used as candidate sites for further experiments such as optimization of electroporation conditions, functional verification and off-target analysis of pure guide RNA on TIL cells.

[0473] Table 4 Editing efficiency determined by NGS

[0474] The present application also provides sgRNAs targeting the exon region of each target, the intron region about 100 bp or about 20 bp away from the exon of the target gene, and enhancing the function of cells after knockout. The sgRNA for Cas12b is divided into two parts, a universal scaffold part (Frame or Scaffold), and a part for binding DNA. In the examples, the scaffold sequence and the part for binding DNA in each sgRNA number contain the sequences shown as follows:

[0475] Table 5 Exemplary sgRNA sequences

[0476] Example 2: Knockout effect of sgRNA

[0477] According to the test method in the examples, the knockout effect of sgRNA was detected.

[0478] The mRNA of Cas12b, about 30 to about 200 sgRNAs of each target point, were co-transfected into HEK293 cells by Lipofectamine 3000, and the cells were lysed after 3 days of culture. The genomic crude extract was amplified by PCR to obtain the corresponding editing site, and the editing efficiency was preliminarily detected by T7E1 enzyme digestion.

[0479] The electrophoresis results after enzyme digestion showed that the sgRNAs corresponding to SC-005, SC-006, SC-007, SC-015, SC-027 targeting SOCS1 all showed obvious site editing; the sgRNAs corresponding to TP-001, TP-005, TP-007, TP-029, TP-082, TP-111, TP-114, TP-117, TP-201, TP-203, TP-205, TP-208, TP-214, TP-228, TP-233, TP-257, TP-259, TP-268 targeting TNFAIP3 all showed obvious site editing; the sgRNAs corresponding to ZC-003, ZC-012, ZC-023, ZC-033 targeting ZC3H12A all showed obvious site editing.

[0480] Further, in the RNP editing system, the molar ratio of Cas12b:sgRNA was 1:3, and the amount of Cas12b was about 30, 60, 90, 120, and 180 pmol, all of which showed editing effect. In addition, the 3' end of sgRNA was modified with any nucleotide, which could further improve the editing efficiency. For example, the 3' end of sgRNA could be added with one or more modified nucleotides, which could include 2'-O-methyl modification and / or 3' thio-phosphoryl internucleotide linkage.

[0481] Based on the NGS-based editing efficiency analysis, the above SC-015, SC-027, TP-005, TP-233, TP-268, when used for single knockout or combined for double target knockout, the knockout efficiency of the target point of the present application for Cas12 gene editing all reached about 50% or more.

[0482] Example 3: Killing ability of edited cells

[0483] Among them, donor 505 is a lung cancer patient, donor 804 is a melanoma patient, and donor 709 is a cervical cancer patient. The killing ability of the edited cells was detected according to the example.

[0484] Figure 2A shows the target cell killing ability of TIL cells edited by a single target point.

[0485] Figure 2B shows the target cell killing ability of TIL cells edited by a combination of genes.

[0486] Wherein the significant difference is relative to the unedited NT group, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0487] The results show that the target gene edited TIL cells of the application can have significantly improved target cell killing ability relative to the control group (NT).

[0488] FIG. 2C shows the target cell killing ability of single target gene edited TIL cells.

[0489] Wherein the significant difference is relative to the SC-C9 group of the Cas9 gene editing system, ** represents P < 0.01.

[0490] The results show that the target gene edited TIL cells of the application can have significantly improved target cell killing ability relative to the control group (NT).

[0491] Example 4: Proliferation ability of edited cells

[0492] The proliferation ability of edited cells was detected according to the example.

[0493] FIG. 3A shows the TIL cell expansion fold of single target gene edited TIL cells.

[0494] FIG. 3B shows the TIL cell expansion fold of combined gene edited TIL cells in two batches of tests. The results show that the target gene edited TIL cells of the application can have significantly improved cell expansion ability relative to the Cas9 gene editing.

[0495] FIG. 3C shows the TIL cell expansion fold of single target gene edited and combined edited TIL cells when IL-2 is removed from the culture medium.

[0496] Wherein the significant difference is relative to the unedited NT group, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0497] The results show that the target gene edited TIL cells of the application can have significantly improved expansion ability relative to the control group (NT).

[0498] Example 5: Apoptosis detection of edited cells

[0499] The apoptosis level of edited cells was detected according to the example.

[0500] Figure 4A shows the apoptosis level of TIL cells with single target gene editing.

[0501] Figure 4B shows the apoptosis level of TIL cells with combined gene editing.

[0502] The results show that the TIL cells with the targets of the present application for Cas12 (e.g. Cas12b) gene editing can have significantly improved anti-apoptosis ability relative to Cas9 gene editing.

[0503] Example 6: Cell flow cytometry detection of edited cells

[0504] The cell phenotype features of the edited cells are detected according to the embodiments.

[0505] Figure 5A shows that TIL cells with single target gene editing have a higher proportion of stem cells.

[0506] Figure 5B shows that TIL cells with combined gene editing have a higher proportion of stem cells.

[0507] Figure 5C shows that TIL cells with single target gene editing have a higher proportion of central memory T cells.

[0508] Figures 5D, 5E show that TIL cells with combined gene editing have a higher proportion of central memory T cells.

[0509] Figure 5F shows that TIL cells with single target gene editing have a lower proportion of exhausted T cells.

[0510] Figures 5G, 5H show that TIL cells with combined gene editing have a lower proportion of exhausted T cells.

[0511] For example, the exhausted T cells can be PD-1 positive, LAG-3 positive, TIM-3 positive, CD38 positive and / or CD101 positive cells. For example, the central memory T cells can be CD45RO positive CD62L positive cells. For example, the stem cells can be CD39 negative CD69 negative cells.

[0512] The results show that the TIL cells with the targets of the present application for Cas12 (e.g. Cas12b) gene editing can have more favorable cell phenotype features relative to Cas9 gene editing.

[0513] Example 7: Cytokine expression and release detection of edited cells

[0514] The cytokine expression levels of the edited cells are detected according to the embodiments.

[0515] FIG. 6A shows that the TIL cells with various combination gene editing after stimulation of the No Stim Medium group have higher cytokine expression ratio.

[0516] FIG. 6B, FIG. 6C shows that the TIL cells with various combination gene editing after stimulation of the TransAct (cell stimulator with CD3 antibody and CD28 antibody) stimulation group have higher cytokine expression ratio.

[0517] For example, the cytokine expression ability includes higher IFN-γ expression ability, higher TNF-α expression ability, higher CD107a expression ability, or higher GZMB expression ability.

[0518] The results show that the TIL cells with target gene editing of the application have higher functional cytokine expression ability.

[0519] According to the embodiment, the cytokine release level of the edited cells is detected.

[0520] FIG. 6D shows that the TIL cells with combination gene editing have higher cytokine release ability when co-cultured with A375 tumor cells.

[0521] FIG. 6E shows that the TIL cells with combination gene editing have higher cytokine release ability when co-cultured with autologous tumor cells of the TIL cell donor.

[0522] FIG. 6F shows that the TIL cells with combination gene editing have higher cytokine release ability when co-cultured with A375 tumor cells, relative to Cas9 gene editing, the target of the application is used for Cas12 (for example, Cas12b) gene editing.

[0523] For example, the cytokine release ability includes higher IFN-γ release ability, higher IL-2 release ability, or higher GZMB release ability.

[0524] The results show that the TIL cells with target gene editing of the application have higher functional cytokine release ability.

[0525] Example 8: In vivo efficacy detection of gene edited cells

[0526] The immunodeficient mice (NOG mice, Vantianhua, strain code 408) are subcutaneously inoculated with A375 cells, and the inoculation amount is about 1×10 6 cells per mouse. After 7 days of inoculation, when the tumor volume reaches 50-80mm 3Mice were randomly divided into groups based on tumor size. Each group received a tail vein injection of the sgRNA (as specified in this invention) designated for use in Cas12 gene-edited TCR-T cells or TIL cells, with the day of injection recorded as day 0. Simultaneously, IL-2 was administered intraperitoneally every 12 hours for six consecutive weeks. Tumor volume was measured twice weekly, and the tumor growth inhibition rate at the experimental endpoint was calculated.

[0527] The results showed that the experimental group mice exhibited better tumor control and / or in vivo functional cytokine release compared to the NT group, demonstrating that the sgRNA provided by this invention, when used for Cas12 (e.g., Cas12b) gene editing, resulted in knockout cells with significantly stronger tumor-suppressive effects than the NT group. The results also showed that, compared to Cas9 gene editing, TIL cells targeting Cas12 (e.g., Cas12b) gene editing according to this invention could exhibit stronger tumor-suppressive effects.

[0528] Example 9: Functional Detection of Cas12b Gene-Edited Cells

[0529] This embodiment compares the advantages of Cas12b in editing immune cells. Donor information for TILs is shown in Table 6:

[0530] Table 6 TIL Supplier Information

[0531] The method for preparing TIL edited by Cas12b is as described in the foregoing embodiments of this disclosure.

[0532] Figure 7A shows that TILs edited with Cas12b have a stronger killing effect on target cells compared to TILs edited with Cas9.

[0533] Figure 7B shows that TILs edited by Cas12b have a stronger proliferative capacity compared to TILs edited by Cas9.

[0534] Figure 7C shows that Cas12b-edited TILs have enhanced cytokine-independent T cell survival compared to Cas9-edited TILs.

[0535] Figure 7D shows that Cas12b-edited TILs have a lower level of apoptosis compared to Cas9-edited TILs.

[0536] Figure 7E shows that Cas12b-edited TILs, compared to Cas9-edited TILs, have reduced expression of T cell exhaustion markers.

[0537] Figure 7F shows that Cas12b-edited TILs have an increased proportion of central memory T cells compared to Cas9-edited TILs.

[0538] FIG. 7G shows that Cas12b edited TILs have increased expression of T cell activation markers compared to Cas9 edited TILs.

[0539] FIG. 7H shows that Cas12b edited TILs have increased proportion of stem-like T cells (CD39 CD69 in CD8+ TILs) compared to Cas9 edited TILs.

[0540] Example 10: Multi-gene edited TILs

[0541] This example demonstrates the advantage of Cas12b in editing multiple targets within immune cells. The method of preparing Cas12b edited TILs is as described in the previous examples of the present disclosure. In this example, Cas12b edits two target genes, TNFAIP3 and ZC3H12A, in TIL cells.

[0542] FIG. 8A shows that Cas12b edited TILs have stronger killing ability against target cells compared to unedited TILs.

[0543] FIG. 8B shows that Cas12b edited TILs have stronger ability of continuous killing of target cells compared to unedited TILs.

[0544] FIG. 8C-8D show that Cas12b edited TILs have stronger proliferative ability compared to unedited TILs.

[0545] FIG. 8E-8H show that Cas12b edited TILs have increased expression of T cell activation markers compared to unedited TILs.

[0546] FIG. 81 shows that Cas12b edited TILs have increased expression of T cell cytokine IFN-γ compared to unedited TILs.

[0547] FIG. 8J shows that Cas12b edited TILs have increased expression of T cell cytokine TNF-a compared to unedited TILs.

[0548] FIG. 8K shows that Cas12b edited TILs have increased expression of T cell cytokine GZMB compared to unedited TILs.

[0549] FIG. 8L shows that Cas12b edited TILs have increased expression of T cell cytokine IFN-γ upon TranAct activation compared to unedited TILs.

[0550] FIG. 8M shows that Casl2b edited TILs have elevated T cell cytokine TNF-a expression levels when activated by TranAct compared to unedited TILs.

[0551] FIG. 8N shows that Casl2b edited TILs have elevated T cell cytokine GZMB expression levels when activated by TranAct compared to unedited TILs.

[0552] FIG. 8O shows that Casl2b edited TILs have an increased proportion of central memory T cells compared to unedited TILs.

[0553] FIG. 8P shows that Casl2b edited TILs have elevated T cell stemness molecule TCF-1 expression compared to unedited TILs.

[0554] The foregoing detailed description has been presented for purposes of illustrations and description. It is not intended to be exhaustive or to limit the claims to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the subj ect matter claimed be defined by the claims appended hereto and their equivalents.

[0555] Table 7A Genomic coordinates of preferred targeting sub-regions of human TNFAIP3

[0556] Table 7B Genomic coordinates of preferred targeting sub-regions of human ZC3H12A

[0557] Table 7C Genomic coordinates of preferred targeting sub-regions of human SOCS1

[0558] Table 8A Genomic coordinates of human TNFAIP3 according to the present application

[0559] Table 8B Inventor ZC3H12A genomic coordinates

[0560] Table 8C Inventor SOCS1 genomic coordinates

[0561] Table 9 Inventor sequence listing

Claims

1. A method of culturing a cell, the method comprising: reducing expression and / or attenuating activity of one or more family members of the Peptidase C64 family, the ZC3H12 family, and the STAT-induced STAT inhibitor (SSI) family, and / or functionally active fragments thereof, in the cell by a Casl2 nuclease.

2. The method of claim 1, wherein the cell comprises an immune cell.

3. The method of claim 2, wherein the immune cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil, and / or a basophil.

4. The method of any one of claims 2-3, wherein the immune cell comprises a monocyte, a macrophage, and / or a dendritic cell.

5. The method of any one of claims 2-4, wherein the immune cell is derived from a stem cell differentiated immune cell.

6. The method of claim 5, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).

7. The method of any one of claims 2-6, wherein the immune cell comprises a B cell, a T cell, a natural killer cell, and / or a natural killer T cell (NKT).

8. The method of any one of claims 2-7, wherein the immune cell comprises an alpha beta T cell and / or a gamma delta T cell.

9. The method of any one of claims 2-8, wherein the immune cell comprises a tumor infiltrating lymphocyte (TIL).

10. The method of claim 9, wherein the TIL is a TIL derived from a fragment of tumor tissue, a pleural effusion, and / or an ascites fluid and / or a TIL derived from a post- cryopreservation thawed TIL.

11. The method of claim 10, wherein the fragment has 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 immune receptor 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 of any one of claims 1-14, wherein reducing expression and / or attenuating activity of a Peptidase C64 family member in the cell comprises inhibiting the function of a deubiquitinase, reducing expression and / or attenuating activity of a ZC3H12 family member in the cell comprises inhibiting the function of a nuclease, and / or reducing expression and / or attenuating activity of a STAT-induced STAT inhibitor (SSI) family member in the cell comprises inhibiting the function of a negative regulator of cytokine signaling.

16. The method of any one of claims 1-15, wherein the cell having reduced expression and / or attenuated activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family exhibits improved cellular properties compared to a cell having unaltered expression and / or activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family.

17. The method of claim 16, wherein the improved cellular properties comprise one or more selected from the group consisting of: improved cell proliferation capacity, increased proportion of viable cells, improved proportion of cell subpopulations, increased cytokine secretion capacity, and increased tumor cell killing capacity.

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

19. The method of any one of claims 1-18, wherein the Peptidase C64 family member, ZC3H12 family member, STAT-induced STAT inhibitor (SSI) family member comprises a ubiquitin binding domain, a C3H1-type zinc finger domain, a SH2 domain, respectively.

20. The method of any one of claims 1-19, wherein the Peptidase C64 family member, ZC3H12 family member, STAT-induced STAT inhibitor (SSI) family member comprises TNFAIP3, ZC3H12A, SOCS1, respectively.

21. The method of any one of claims 1-20, wherein reducing expression and / or attenuating activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family in the cell comprises introducing a gene regulation system into the cell.

22. The method of claim 21, wherein the gene regulation system is capable of disrupting one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family at the DNA level.

23. The method of any one of claims 21-22, wherein the gene regulation system comprises a guide nucleic acid molecule and a Cas12 nuclease.

24. The method of claim 23, wherein reducing expression and / or attenuating activity of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family comprises introducing into the cell a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the Cas12 nuclease, an LNP comprising a gRNA and a Cas12 nuclease, or an LNP comprising a nucleic acid encoding a gRNA and a nucleic acid encoding a Cas12 nuclease.

25. The method of any one of claims 1-24, wherein the Cas12 nuclease comprises an AaCas12b nuclease or a variant or functional derivative thereof.

26. The method of any one of claims 23-25, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA) capable of binding to a Cas12 nuclease.

27. The method of any one of claims 23-26, wherein the guide nucleic acid molecule is capable of binding to a sequence of one or more family members of the Peptidase C64 family, ZC3H12 family, STAT-induced STAT inhibitor (SSI) family.

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 genomic coordinates selected from Tables 7A-7C 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 selected from the group consisting of: SEQ ID NOs: 1709-3416, 4058-4698, 4904-5108 or a fragment thereof.

30. The method of any one of claims 23-29, wherein the guide nucleic acid molecule comprises a guide sequence comprising a sequence as set forth in any one of SEQ ID NOs: 1-1708, 3417-4057, 4699-4903, 5110-5136.

31. The method of claim 30, the guide nucleic acid molecule further comprises a scaffold sequence linked at its 3’ end to the 5’ end of the guide sequence, the scaffold sequence for binding to the Cas12 nuclease, optionally the scaffold sequence comprises a base modification.

32. The method of any one of claims 23-31, the guide nucleic acid molecule comprises a base modification.

33. The method of any one of claims 30-32, the guide nucleic acid molecule comprises one or more nucleotides with 2’-O-methyl modification, and / or 3’ thio-phosphate internucleosidic linkage added at the 3’ end of the guide sequence.

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 affecting 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 of claim 34, the composition of claim 35, and / or the pharmaceutical composition of claim 36 in the manufacture of a medicament, wherein the medicament is for the prevention and / or treatment of a disease and / or a condition.

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

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

41. The use of any one of claims 38-40, wherein the disease and / or condition comprises one or more selected from the group consisting of melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

42. A composition comprising one or more guide nucleic acid molecules comprising a guide sequence selected from the group consisting of the guide sequences set forth in SEQ ID NOs: 1-1708, 3417-4057, 4699-4903, and 5110-5136.

43. The composition of claim 42, further comprising a Cas12 nuclease.

44. An LNP comprising the composition of claim 42 or 43.

45. A cell comprising the composition of claim 42 or 43, or the LNP of claim 44.

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