Method for identifying target and use thereof

Abstract

The present invention relates to the field of biomedicine, and specifically provided is a method for identifying a target. The method specifically comprises determining the effect of a candidate target on a cell to be tested after leaving the cell to be tested in a hypofunction state. Further provided in the present invention is a system for identifying the target.

Description

A method for identifying a target and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for identifying a target and an application thereof. Background Art

[0002] In the biomedical field, screening and determining gene targets that can regulate cell function has important application prospects. Existing target gene screening platforms usually use freshly obtained primary immune cells as test cells to identify target genes that affect cell function. However, the target genes identified by the above-mentioned existing platforms usually do not show improved therapeutic effects after gene editing in cell products. Therefore, this field is in urgent need of a method for sensitive, rapid and specific screening of gene targets. Summary of the Invention

[0003] On the one hand, the present invention provides a method for identifying a target, which comprises determining the effect of a candidate target on the activation level of the cells to be tested after the cells to be tested have basically lost their killing ability, and before and / or simultaneously with the determination, the expression or activity of the candidate target in the cells to be tested is regulated.

[0004] On the other hand, the present invention provides a method for identifying a target, comprising the following steps: (1) regulating the expression or activity of a candidate target in a test cell; (2) substantially losing the killing ability of the test cell; (3) measuring the activation level of the regulated test cell; and (4) determining the effect of regulating the candidate target on the cell through the reading result obtained by the determination.

[0005] On the other hand, the present invention also provides a system for identifying targets, which comprises: a measurement module for measuring the effect of a candidate target on the activation level of the cells to be tested after the cells to be tested have basically lost their killing ability, and before and / or simultaneously with the measurement, the expression or activity of the candidate target in the cells to be tested is regulated.

[0006] In order to solve the existing technical problems, the present invention designs a target gene screening process, which adopts an optimized culture method and obtains test cells in a functionally impaired state, which is used to identify target genes that can affect cell function in actual application environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The features and advantages of the present invention can be better understood by referring to the accompanying drawings, which are briefly described as follows:

[0008] FIG1 shows the ratio of tumor cells after 3 days and 7 days of co-culture of effector cells (immune cells to be tested, E) and target cells (A375 cells, T) at one stage.

[0009] FIG2 shows the number of tumor cells after 4 and 7 days of co-culture of effector cells (immune cells to be tested, E) and target cells (A375 cells, T) at two stages.

[0010] FIG3 shows the release capacity of IFN-γ and TNF by the immune cells to be tested after two-stage culture.

[0011] FIG4 shows the number of tumor cells after two-stage culture.

[0012] FIG5 shows the difference in expression ratio of apoptotic cells in the tested immune cells on day 7 relative to day 3 after two-stage culture.

[0013] FIG6 shows the difference in expression ratio of exhaustion markers in the tested immune cells on day 7 relative to day 3 after two-stage culture.

[0014] Figure 7 shows the gRNA distribution results analyzed by this screening platform.

[0015] Figure 8A shows that gene-edited T cells in the no-stimulation group can have significant expansion capacity.

[0016] Figure 8B shows that the gene-edited T cells in the CD3 antibody stimulation group can have significant expansion capacity.

[0017] Figure 8C shows the target cell killing ability of gene-edited T cells.

[0018] Figure 8D shows that gene-edited T cells have a lower proportion of T cells expressing exhaustion markers.

[0019] Figure 8E shows that the gene-edited T cells in the unstimulated group had a higher cytokine expression ratio.

[0020] Figure 8F shows that the gene-edited T cells in the stimulation group had a higher cytokine expression ratio.

[0021] Figure 9A shows the expansion fold of TILs with BCL2L11 single-target gene editing in the non-stimulation culture medium group.

[0022] Figure 9B shows the expansion fold of TILs with BCL2L11 single-target gene editing in the TransACT stimulation group.

[0023] Figure 9C shows the central memory T cell proportion of TIL cells after BCL2L11 editing.

[0024] FIG9D shows the proportion of naive T cells in TIL cells after BCL2L11 editing.

[0025] Figure 9E shows the proportion of cells expressing depletion markers in TIL cells after BCL2L11 editing.

[0026] Figure 9F shows the stem-like T cell proportion of TIL cells after BCL2L11 editing.

[0027] Figure 9G shows that the BCL2L11-edited TIL cells in the non-stimulation culture medium group had a higher cytokine expression ratio.

[0028] Figure 9H shows that the BCL2L11-edited TIL cells in the non-stimulation culture medium group had a higher cytokine expression ratio.

[0029] Figure 9I and Figure 9J show that the BCL2L11-edited TIL cells in the TransACT stimulation group had a higher cytokine expression ratio.

[0030] Figure 9K shows that BCL2L11-edited cells in TCR-T cells have higher cytokine release levels.

[0031] Figure 10 shows the gRNA distribution results analyzed after expanding the sample size of this screening platform. DETAILED DESCRIPTION

[0032] The following describes the embodiments of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0033] Definition of terms

[0034] In the present invention, the term "target" generally refers to an objective. For example, a target gene is obtained from candidate genes through screening or identification. Depending on the results of the screening or identification, the "candidate gene" or the protein encoded by the "candidate gene" is identified as a potential target suitable for regulating cells. In the present invention, the term "gene" generally refers to a nucleic acid molecule that, when placed under the control of appropriate regulatory or control sequences, is transcribed into RNA or translated into a polypeptide in vitro or in vivo.

[0035] In the present invention, the term "cytokine" generally refers to a compound or component produced by a cell and affecting the physiological state of the cell (itself) or other cells that produces the cytokine. For example, cytokines also include any compound or component produced by recombinant or synthetic processing, and the products of these processing have similar structures and / or biological activities as the naturally occurring form. For example, the cytokine also encompasses its truncated forms, functionally active fragments, homologues, analogs and variants.

[0036] In the present invention, the term "IFN-γ" generally refers to a member of the interferon family. For example, the UniProt number of IFN-γ may be P01579. The IFN-γ of the present invention may also encompass functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or variants comprising the functionally active fragments thereof produced after processing and / or modification in cells. For example, the IFN-γ of the present invention may comprise functionally active fragments thereof as well as any other domains.

[0037] In the present invention, the term "TNF-α" generally refers to a member of the tumor necrosis factor family. For example, the UniProt number of TNF-α may be P01375. The TNF-α of the present invention may also encompass functionally active fragments thereof, including but not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants thereof, active fragments thereof, or variants thereof containing such functionally active fragments after processing and / or modification in cells. For example, the TNF-α of the present invention may include functionally active fragments thereof as well as any other domains.

[0038] In the present invention, the term "CD107a" generally refers to a lysosome-associated membrane protein (Lysosome-Associated Membrane Protein) family member. For example, the UniProt number of CD107a can be P11279. The CD107a of the present invention can also encompass its functionally active fragments, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, its active fragments, or variants containing the functionally active fragments produced after processing and / or modification thereof in cells. For example, the CD107a of the present invention can include its functionally active fragments and any other domains.

[0039] In the present invention, the term "co-culture" generally refers to culturing two or more different populations of substances with a certain degree of contact between them. The "contact" of the two or more different populations of cells can be direct contact, i.e., direct physical contact between cells of one population and cells of another population, in one embodiment. Alternatively, in one embodiment, the contact can be indirect contact mediated by a shared culture medium. The shared culture medium can contain metabolites produced and released by at least one population of co-cultured cells and be used to culture the cells of the other population.

[0040] As used herein, the term "contact" generally refers to the process of being brought into close proximity. For example, a cell is brought into close proximity with a substance such that the cell can interact with the substance and / or be affected by the presence of the substance. The contact may be temporary or transient. The contact may also be continuous for a period of time or permanent.

[0041] In the present invention, the term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence. In some embodiments, expression includes translation of mRNA introduced into the cell.

[0042] In the present invention, the term "expression vector" generally refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system.

[0043] In the present invention, the term "nucleic acid" or "polynucleotide" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known natural and / or non-natural nucleotide analogs that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions, e.g., conservative substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0044] As used herein, the terms "transfected," "transformed," or "transduced" generally refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary host cells and their progeny.

[0045] In the present invention, the terms "about" and "approximately" generally refer to a statistically significant numerical range. Such a range can be within an order of magnitude of a given value or range, can be included within 50%, preferably included within 20%, more preferably included within 10%, and most preferably included within 5%. The permissible variations encompassed by the terms "about" or "approximately" may depend on the specific system under study and can be readily understood by those of ordinary skill in the art.

[0046] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0047] In the present invention, "include", "comprise" or "contain" are open expressions, while "consisting of" is a closed expression. The former covers the latter, and the latter is a special form of the former.

[0048] Detailed Description of the Invention

[0049] The present invention designs a target gene screening process, in which an optimized culture method is adopted and test cells in a hypofunctional state are obtained, which is used to identify target genes that can affect cell function in actual application environments.

[0050] On the one hand, the present invention provides a method for identifying a target, which comprises determining the effect of a candidate target on the activation level of the cells to be tested after the cells to be tested have basically lost their killing ability, and before and / or simultaneously with the determination, the expression or activity of the candidate target in the cells to be tested is regulated.

[0051] In one aspect, the present invention provides a method for identifying a target, comprising the following steps: (1) regulating the expression or activity of a candidate target in a test cell; (2) substantially eliminating the killing ability of the test cell; (3) determining the activation level of the regulated test cell; and (4) determining the effect of regulating the candidate target on the cell by reading the results obtained by the determination.

[0052] For example, the substantially losing killing ability of the cells to be tested comprises co-culturing the cells to be tested with tumor antigens.

[0053] For example, compared to cells that have not been co-cultured with tumor antigens or cells expressing tumor antigens, the hypofunctional cells obtained by the method of the present invention do not substantially kill tumor cells. For example, compared to cells that are functionally normal, the hypofunctional cells obtained by the method of the present invention do not substantially kill all tumor cells. For example, compared to cells that are functionally normal, when the hypofunctional cells obtained by the method of the present invention are co-cultured with tumor cells, the number of tumor cells that remain unkilled is greater than 1×10 5 More than 2×10 5 More than 3×10 5 , greater than 4×10 5 More than 5×10 5 , greater than 1×10 6 or greater than 2×10 6For example, the amount of tumor cell killing is measured by flow cytometry. For example, when the hypofunctional cells obtained by the method of the present invention are co-cultured with tumor cells, the ratio of the number of tumor cells remaining unkilled to the number of tumor cells added at the beginning of the co-culture is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99%. For example, the amount of tumor cell killing is measured by flow cytometry.

[0054] For example, the test cells express a receptor that recognizes the tumor antigen, and the tumor antigen is expressed on tumor cells, and the co-culture comprises co-culturing the test cells with the tumor cells. For example, in the present invention, prior to or concurrently with the assay, the cells are caused to express an exogenous cellular receptor or a functional fragment thereof. For example, in the present invention, the exogenous cellular receptor comprises a T cell receptor or an antigen-binding fragment thereof. For example, in the present invention, prior to or concurrently with the assay, the cells are contacted with an antigen specific for the exogenous cellular receptor or a functional fragment thereof. For example, cells expressing a specific antigen include any cell expressing the NYESO1 antigen. For example, cells expressing a specific antigen include A375 cells expressing the NYESO1 antigen. For example, in the present invention, prior to or concurrently with the assay, the test cells expressing the exogenous cellular receptor or a functional fragment thereof are contacted with target cells expressing the antigen specific for the exogenous cellular receptor or a functional fragment thereof. For example, in the co-culture of the present invention, the test cells expressing the exogenous cellular receptor or a functional fragment thereof are co-cultured with target cells expressing the antigen specific for the exogenous cellular receptor or a functional fragment thereof.

[0055] For example, the method of substantially depriving the cells to be tested of their killing ability comprises: in a first stage, co-culturing the cells to be tested and the tumor cells at a ratio of 1:1 to 1:30, preferably 1:3 to 1:30; for example, at the beginning of the first stage, the ratio of the cells to be tested and the tumor cells in the culture environment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, or 1:30.

[0056] For example, the method of substantially eliminating the killing ability of the test cells may optionally include: a second stage, co-culturing the test cells to the tumor cells at a ratio of 1:1 to 1:30, preferably 1:3 to 1:30; for example, at the beginning of the second stage, the ratio of the test cells to the tumor cells in the culture environment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, or 1:30. For example, the number of test cells and tumor cells in the culture environment after the first stage can be measured, and a certain amount of tumor cells can be added to increase the ratio of test cells to tumor cells to the predetermined number of the present invention.

[0057] For example, wherein the first stage lasts for about 2 to 6 days. For example, wherein the second stage lasts for about 2 to 6 days. For example, the method of substantially losing the killing ability of the cells to be tested of the present invention may only comprise two stages of culture.

[0058] For example, the method of substantially losing the killing ability of the cells to be tested may optionally include a third stage, a fourth stage, a fifth stage, or at least a tenth stage of co-culturing with tumor cells to substantially lose the killing ability of the cells to be tested.

[0059] For example, in the present invention, before or simultaneously with the assay, the first or second stage of co-culturing the cells to be tested with the specific antigen of the exogenous cell receptor or its functional fragment is about 0 hours to about 72 hours. For example, the first or second stage of co-culturing the cells to be tested with the specific antigen of the exogenous cell receptor or its functional fragment is 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 28 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours. For example, in the present invention, the first or second stage of co-culturing the cells expressing the exogenous cell receptor or its functional fragment with the target cells expressing the specific antigen of the exogenous cell receptor or its functional fragment is about 6 hours, about 12 hours, about 16 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or 96 hours. For example, in the present invention, the first or second stage of co-culturing the cells expressing the exogenous cell receptor or its functional fragment with the target cells expressing the specific antigen of the exogenous cell receptor or its functional fragment is 48 hours, 72 hours or 96 hours before or simultaneously with the assay.

[0060] For example, at least one stage (e.g., the first stage) of the present invention is cultured to allow the cells to reach a state of hypofunction. For example, at least two stages of culture of the present invention are cultured to allow the cells to reach a state of hypofunction. For example, relative to the functionally normal cells to be tested, the hypofunctional cells obtained by the method of the present invention substantially do not release cytokines. For example, relative to the functionally normal cells to be tested, the amount of TNF released by the hypofunctional cells obtained by the method of the present invention is less than 10 pg / ml, less than 9 pg / ml, less than 8 pg / ml, less than 7 pg / ml, less than 6 pg / ml, less than 5 pg / ml, less than 4 pg / ml, less than 3 pg / ml, less than 2 pg / ml, less than 1 pg / ml, less than 0.1 pg / ml, or less than 0.01 pg / ml. For example, relative to the amount of IFN-γ released by the dysfunctional cells obtained by the method of the present invention, the amount of IFN-γ released is less than 150 pg / ml, less than 100 pg / ml, less than 50 pg / ml, less than 20 pg / ml, less than 10 pg / ml, less than 9 pg / ml, less than 8 pg / ml, less than 7 pg / ml, less than 6 pg / ml, less than 5 pg / ml, less than 4 pg / ml, less than 3 pg / ml, less than 2 pg / ml, less than 1 pg / ml, less than 0.1 pg / ml, or less than 0.01 pg / ml. For example, the amount of TNF released by the dysfunctional cells obtained by the method of the present invention relative to the amount of TNF released by the normal functioning test cells is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, or less than 1%. For example, the amount of IFN-γ released by the hypofunctional cells obtained by the method of the present invention is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, or less than 1% relative to the amount of IFN-γ released by the functionally normal test cells. For example, the amount of cytokine release is measured using a CBA kit.

[0061] For example, relative to the test cells with normal function, the expression of apoptosis signals (e.g., Annexin V) in the functionally impaired cells obtained by the method of the present invention is increased by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more. For example, the amount of apoptosis signals in functionally impaired cells is measured by flow cytometry. For example, wherein after being cultured in the functionally impaired state, the expression of PD-1, CD38, and / or CD101 in the test cells relative to the functionally normal test cells is increased. For example, relative to the functionally normal test cells, the proportion of PD-1 positive cells in the functionally impaired cells obtained by the method of the present invention is 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more. For example, the expression of PD-1 is measured by flow cytometry.

[0062] For example, the present invention provides functionally impaired test cells, wherein the amount of TNF released by the functionally impaired cells obtained by the method of the present invention relative to the amount of TNF released by the functionally normal test cells is less than 50%, the amount of IFN-γ released by the functionally impaired cells obtained by the method of the present invention relative to the amount of IFN-γ released by the functionally normal test cells is less than 50%, after co-culture with tumor cells, the ratio of the number of remaining tumor cells that have not been killed to the number of tumor cells added at the beginning of co-culture is greater than 50%, the expression level of apoptosis signals (such as Annexin V) is increased by more than 20%, and the proportion of PD-1 positive cells is increased by more than 30%. For example, the method of identifying a target of the present invention comprises determining the effect of a candidate target on the activation level of the functionally impaired test cells provided by the present invention, and before and / or simultaneously with the determination, the expression or activity of the candidate target in the test cells is regulated.

[0063] For example, before or simultaneously with the determination of the present invention, the cells to be tested are contacted with a substance that inhibits cytokine secretion. For example, the substance that inhibits cytokine secretion can inhibit the cells from releasing the cytokine to the extracellular space. For example, the substance that inhibits cytokine secretion can have substantially no effect on the expression of the cytokine. For example, the substance that inhibits cytokine secretion in the present invention comprises Brefeldin A. For example, the substance that inhibits cytokine secretion in the present invention comprises Monensin. For example, before or simultaneously with the determination of the present invention, the cells to be tested are contacted with a substance that inhibits cytokine secretion for about 0 hours to about 72 hours.

[0064] For example, in the present invention, the cells to be tested are contacted with a substance that inhibits cytokine secretion for at least about 1 hour to at least about 72 hours, preferably about 4 hours to about 16 hours. For example, in the present invention, the cells to be tested are contacted with a substance that inhibits cytokine secretion for 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 28 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours. For example, in the present invention, the test cells are contacted with the substance that inhibits cytokine secretion for about 4 hours, about 12 hours, or about 16 hours.

[0065] For example, wherein the expression or activity of the candidate target in the cell to be tested is regulated, comprising introducing a target regulatory system into the cell to be tested, and the target regulatory system increases or decreases the expression or activity of the candidate target. For example, the regulation in the present invention comprises introducing a target regulatory system into the cell, and the target regulatory system increases or decreases the expression or activity of the candidate target. In some embodiments, a clustered regularly interspaced short palindromic repeat (CRISPR) system is used to regulate the candidate target. Alternatively, a transcription activator-like effector nuclease (TALEN) system, a zinc finger nuclease system, or a large range nuclease system can be used to regulate the candidate target. In some embodiments, an activation regulatory system regulates the candidate target. Alternatively, the expression or activity of the candidate target can be increased by overexpressing the candidate target.

[0066] For example, the regulation described in the present invention comprises increasing or decreasing the expression or activity of the candidate target at the genomic level. Alternatively, the expression or activity of the candidate target can be transiently increased or decreased at the RNA level. Alternatively, the expression or activity of the candidate target can be increased or decreased at the transcription level, translation level and / or post-translational modification level. In some embodiments, antisense RNA, siRNA, shRNA or short hairpin RNA are used to decrease the expression or activity of the candidate target. In some embodiments, activating RNA is used to increase the expression or activity of the candidate target. For example, the regulation described in the present invention comprises providing a substance that covalently binds and / or non-covalently binds to the candidate target to increase or decrease the expression or activity of the candidate target.

[0067] For example, the method of editing candidate targets of the present invention can be included in vivo, in vitro and / or in vitro. For example, the in vivo gene regulatory system can be delivered and edited to reduce the in vivo expression level of the candidate target in cells in vivo. For example, by targeting immune cells or their precursor cells, such as bone marrow stem cells, etc., LNPs containing gene regulatory systems or mRNA encoding gene regulatory systems are delivered, and editing of candidate targets in vivo can be performed. By adjusting the composition and / or ratio of the LNP components, or introducing components with targeting capabilities, the efficiency of the in vivo editing target of the present invention can be improved.

[0068] For example, the modulation of the present invention comprises targeting a specific nucleic acid sequence of the target, introducing single-strand breaks, double-strand breaks and / or mutations within, upstream and / or downstream of the nucleic acid sequence. For example, the modulation of the present invention comprises providing a CRISPR system, a zinc finger nuclease system, a meganuclease system and / or antisense RNA, siRNA, shRNA or short hairpin RNA.

[0069] For example, in the present invention, knockout or knockdown (together referred to as "silencing") candidate target genes can utilize chemically synthesized or in vitro transcribed small interfering RNA (siRNA), and PCR or DNA vector-based short hairpin RNA (shRNA). In addition, the shRNA molecules provided herein can be operably linked to a T cell-specific promoter to achieve T cell-specific targeting of the shRNA molecules to achieve silencing of the candidate target gene.

[0070] For example, the present invention may include a gene silencing method of a candidate target gene by RNA interference. The method involves creating a construct encoding an interfering (silencing) RNA and using a promoter active in a specific cell type (e.g., T cells) to drive the expression of the shRNA.

[0071] For example, the shRNA constructs used in the methods provided herein contain small nucleotide stretches directed against the candidate target gene, which are used to modulate the expression of a nucleic acid molecule encoding the candidate target gene. Inhibition of the candidate target gene is achieved by providing an oligomeric compound that hybridizes to one or more target nucleic acid molecules encoding the candidate target gene.

[0072] For example, in the present invention, candidate target genes can be silenced using a system based on transcription activator-like effectors and nucleases (TALENs). By assembling a sequence of repeat variable-di-residues (RVDs), transcription activator-like (TAL) effector sequences can be assembled to specifically bind to the target DNA sequence. The fusion protein of TAL effectors and nucleases (TALENs) can produce targeted double-strand breaks in the DNA of cells, which can be used to produce specific modifications to cells.

[0073] For example, the TAL effector domain that binds to a specific nucleotide sequence within a target DNA in the present invention can include 10 or more DNA binding repeats, or 15 or more DNA binding repeats. Each DNA binding repeat can include an RVD that determines the recognition of base pairs in the target DNA sequence, wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence.

[0074] For example, in the present invention, the substance that binds to the target nucleic acid can be linked to an effector domain, including but not limited to a transposase, an integrase, a recombinase, a resolvase, an invertase, a protease, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, a nuclease, a transcription repressor, a transcription activator, a transcription factor recruitment, a protein nuclear localization signal, or a cell uptake signal. For example, in the present invention, the effector domain includes but is not limited to transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, DNA methyltransferase activity, DNA demethylase activity, histone acetyltransferase activity, histone deacetylase activity, nuclease activity, nuclear localization signal activity, transcription repressor activity, transcription activator activity, transcription factor recruitment activity, or cell uptake signal activity.

[0075] For example, zinc fingers in the present invention are small protein structural motifs stabilized by one or more zinc ions. A zinc finger may comprise, for example, Cys2His2 and may recognize a sequence of approximately 3 bp. Various zinc fingers of known specificity may be combined to generate multi-zinc finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp. Zinc finger nuclease systems can generate double-strand breaks in DNA that, if not properly repaired, can produce frameshift mutations, leading to reduced expression of the target gene in the cell.

[0076] For example, the vector for integrating the sequence of interest in the present invention includes but is not limited to retroviral vectors, for example, lentiviral vectors or retroviral vectors, adenoviral vectors and baculoviral vectors. For example, an expression vector can be used for stable expression or transient expression of a polypeptide encoded by the nucleic acid sequence to be expressed, and the vector can be an extrachromosomal vector of self-replication, or a vector that is integrated into the host genome. In one embodiment, the vector is a genome-integrated vector, or an "integration vector", which can become integrated into the chromosomal DNA or RNA of a host cell, a cell system or a non-cellular system. In certain embodiments, non-viral methods include the use of a transposon (also referred to as a transposable element). In certain embodiments, a transposon is a DNA that can be inserted into a position in the genome, for example, a DNA that can self-replicate and insert its copy into the genome, or a DNA that can be spliced ​​out from a longer nucleic acid and inserted into another position in the genome. For example, a transposon comprises a DNA sequence consisting of an inverted repeat sequence for transposition of a flanking gene.

[0077] For example, the regulation described herein comprises introducing a target regulation system comprising a guide nucleic acid molecule and a nuclease into the cell. For example, the components of the target regulation system can be introduced into the cell separately and sequentially. For example, a viral or non-viral vector comprising a guide nucleic acid molecule is first introduced into the cell, and then a nucleic acid comprising a nuclease or encoding a nuclease is introduced into the cell. For example, a library of guide nucleic acid molecules for screening is packaged with a lentivirus or retrovirus and then introduced into the cell, and then the nuclease is introduced into the cell by electroporation. For example, a nucleic acid comprising a nuclease or encoding a nuclease is first introduced into the cell, and then a viral or non-viral vector comprising a guide nucleic acid molecule is introduced into the cell. For example, a nuclease is first introduced into the cell by electroporation, and then a library of guide nucleic acid molecules for screening is packaged with a lentivirus or retrovirus and then introduced into the cell. For example, the guide nucleic acid molecule described herein comprises a guide RNA (gRNA). For example, the nuclease described herein comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the nuclease described herein comprises Cas9 and / or Cas12. For example, the gRNA can be used to bind to the sequence of the candidate target. For example, the binding of the gRNA to the sequence of the candidate target can be fully complementary, partially complementary, or hybridize to the sequence of the candidate target under moderate or stringent conditions. For example, the binding of the gRNA to the sequence of the candidate target can cause the CRISPR system of the gRNA to specifically cleave the candidate target.

[0078] For example, when the gene editing system includes CRISPR / Cas9, the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) downstream, and the protospacer adjacent motif (PAM) may be AGG, TGG, GGG or CGG. For example, when the PAM region of the candidate target 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 candidate target PAM, and can design a suitable gRNA for the target sequence. For example, the guide nucleic acid molecule can bind to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG and CGG.

[0079] For example, when the gene editing system includes CRISPR / Cas12, the region targeted by the guide nucleic acid molecule of the present invention may have a protospacer adjacent motif (PAM) upstream, and the protospacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G. For example, when the PAM region of the candidate target is determined, those skilled in the art can easily determine a target sequence consisting of about 15 to about 25 (e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25) nucleotides downstream of the 3' end of the candidate target PAM, and can also design a suitable gRNA for the target sequence. For example, the guide nucleic acid molecule is capable of binding to a target sequence consisting of about 15 to about 25 nucleotides 3' downstream of the protospacer adjacent motif (PAM) selected from the group consisting of NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C or G, Y is T or C, V is A, C or G, and R is A or G.

[0080] For example, when the gene editing system of the present invention comprises wild-type Cas12a (also referred to as Cpf1, such as AsCas12a, FnCas12a, LbCas12a, BbCas12a, CMaCas12a and OsCas12a), the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following upstream: NTTN, wherein N can be A, T, C or G. For example, when the PAM region of the candidate target is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for the target sequence.

[0081] For example, when the gene editing system of the present invention includes a mutant Cas12a, such as enAsCas12a (mutation sites E174R, S542R and K548R), the upstream region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV), wherein N can be A, T, C or G, Y can be T or C, V can be A, C or G, and R can be A or G. For example, when the PAM region of the candidate target is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the candidate target PAM, and a suitable gRNA can be designed for the target sequence.

[0082] For example, when the gene editing system of the present invention includes a mutant Cas12a, such as opAsCas12a (mutation sites: E174R and S542R), the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTTV (TTTA, TTTC, or TTTG), wherein V may be A, C or G. For example, when the PAM region of the candidate target is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for the target sequence.

[0083] For example, when the gene editing system of the present invention includes a mutant Cas12a, such as AsCas12aUltra (mutation sites: M537R and F870L), the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TTTV, TATV, or TYCV, wherein V may be A, C or G, and Y may be T or C. For example, when the PAM region of the candidate target is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the candidate target PAM, and a suitable gRNA can be designed for the target sequence.

[0084] For example, when the gene editing system of the present invention includes mutant Cas12a, such as hfCas12Max (mutation site: N243R / E336R / D892R) and Cas12Max (mutation site: N243R), the upstream of the region targeted by the guide nucleic acid molecule of the present invention may have a PAM sequence selected from the following: TNN, or NTN, wherein N can be A, T, C or G. For example, when the PAM region of the candidate target is determined, those skilled in the art can easily determine a target sequence consisting of about 17 to about 25 (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) nucleotides downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for the target sequence.

[0085] For example, wherein the regulation comprises targeting a specific nucleic acid sequence of the target, introducing single-strand breaks, double-strand breaks and / or mutations within, upstream and / or downstream of the specific nucleic acid sequence. For example, wherein the regulation comprises increasing or decreasing the expression or activity of the candidate target at the genomic level. For example, wherein the regulation comprises introducing a target regulation system containing a guide nucleic acid molecule and a nuclease into the cell to be tested. For example, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA) targeting the target. For example, wherein the nuclease comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, wherein the nuclease comprises Cas 9 and / or Cas 12.

[0086] For example, the present invention can reduce the proportion of cells expressing the candidate target in the cell population obtained by the regulation to about 95% or less. For example, it can be reduced to at least about 95-1%, such as at least about 95%, 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%.

[0087] For example, the activation level of the cells to be tested comprises the level of substances expressed by the cells to be tested selected from the following groups: interleukin family members, interferon family members, tumor necrosis factor family members and lysosome-associated membrane protein family members. For example, the interferon family members of the present invention include IFN-γ. For example, the IFN-γ of the present invention may include its functionally active fragments and any of its structural domains. For example, the tumor necrosis factor family members of the present invention include TNF-α. For example, the TNF-α of the present invention may include its functionally active fragments and / or any of its structural domains. For example, the lysosome-associated membrane protein Lysosome-Associated Membrane Protein family members of the present invention include CD107a. For example, the CD107a of the present invention may include its functionally active fragments and / or any of its structural domains.

[0088] For example, wherein the activation level of the cells to be tested comprises the level of CD107a, GZMB, IFN-γ, TNF-α, IL2 and / or IL6 expressed by the cells to be tested. For example, wherein the determination comprises determining the level of expression of the cytokine by the regulated cells to be tested by flow cytometry, Western blotting or ELISA. For example, wherein the determination comprises determining by flow cytometry the increase in the level of the cytokine expressed by the cells to be tested in which the expression or activity of the candidate target is regulated, compared to the corresponding cells to be tested in which the expression or activity of the candidate target is not regulated.

[0089] For example, the present invention measures the level of IFN-γ expressed by the cells to be tested after conditioning. For example, the present invention measures the level of TNF-α expressed by the cells to be tested after conditioning. For example, the present invention measures the level of CD107a expressed by the cells to be tested after conditioning.

[0090] For example, the present invention measures the level of GZMB, IFN-γ, TNF-α, and / or CD107a expressed by the cells to be tested after adjustment. For example, the level of cytokine expression includes the expression amount of the cytokine, the secretion release amount, the level of the effect of the cytokine on the downstream signaling pathway, and / or the effect of the cytokine on cell function and / or cell characteristics. For example, the present invention measures the content of GZMB, IFN-γ, TNF-α, and / or CD107a in the cells to be tested after adjustment. For example, the present invention measures the release amount of GZMB, IFN-γ, TNF-α, and / or CD107a in the cells to be tested after adjustment. For example, the present invention measures the level of downstream pathway signals regulated by GZMB, IFN-γ, TNF-α, and / or CD107a in the cells to be tested after adjustment. For example, the present invention measures the cell function and / or cell characteristics affected by the regulation of GZMB, IFN-γ, TNF-α, and / or CD107a in the cells to be tested after adjustment. For example, the present invention can determine the expression level of the cytokine in the cells to be tested by flow cytometry, Western blotting or ELISA.

[0091] For example, based on the level of cytokines expressed by the cells, targets that can affect the cells can be determined. For example, the determined targets can affect cell proliferation ability, affect cell survival ability, affect cell subpopulation ratios, affect cytokine secretion ability, affect in vitro tumor cell killing ability and / or affect in vivo tumor killing ability. For example, the determined targets can improve cell proliferation ability, improve cell survival ability, improve cytokine secretion ability, improve in vitro tumor cell killing ability and / or improve in vivo tumor killing ability. For example, the determined targets can reduce cell proliferation ability, reduce cell survival ability, reduce cytokine secretion ability, reduce cell killing ability and / or reduce cell damage ability. For example, the determined targets can affect the proportion of activated cells in the cell population, affect the proportion of regulatory cells, affect the proportion of cells expressing exhaustion markers, affect the proportion of central memory cells and / or naive cells, affect the proportion of apoptotic cells and affect the proportion of stem cell-like cells.

[0092] For example, in the identification method of the present invention, in order to screen effective targets, the test cells are regulated against the candidate target. For example, in the test cells, the expression or activity of the candidate target is regulated. For example, the expression or activity of the candidate target in the test cells is modulated to be reduced to about 99-0.0001% of the original level, for example, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, 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 ... 5%, 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 0.5%, 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%.For example, in the test cells, the expression or activity of the candidate target is modulated to increase by about 0.1% to about 10,000 fold, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 1000-fold, or about 10000-fold. For example, the cells to be tested are divided into two groups, and the expression or activity of the candidate target is modulated to decrease or increase in the group of cells to be tested that has been regulated relative to the group of cells to be tested that has not been regulated. For example, the expression or activity of the candidate target is modulated to decrease or increase in the cells to be tested after regulation relative to the cells to be tested before regulation.

[0093] For example, depending on the effect on the level of cytokines expressed by cells, for example, an increase or decrease of about 0.1% to about 10,000 times, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 5 ... %, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 1000-fold, or about 10000-fold of the corresponding candidate target can be used as a target that can affect cells. For example, based on the confidence and / or significance of the level of cytokine expressed by the cell, the logarithm with base 10 is taken, and then the negative number is taken. The resulting value is greater than about 1, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10, which is the corresponding candidate target.

[0094] For example, statistical processing can be performed on the enriched guide RNA corresponding to the enriched cells after screening. For example, according to the amount of guide RNA enrichment, the corresponding candidate targets are ranked to determine the top 1 to top 1000 candidate targets, such as the top 1, top 2, top 3, top 4, top 5, top 6, top 7, top 8, top 9, top 10, top 15, top 20, top 25, top 30, top 35, top 40, top 45, top 50, top 60, top 70, top 80, top 90, top 100, top 200, top 300, top 400, top 500, top 600, top 700, top 800, top 900, or top 1000, as targets that can affect cells. For example, according to the number of guideRNA enrichments, the corresponding candidate targets are ranked from the bottom 1 to the top 1000, such as the top 1, top 2, top 3, top 4, top 5, top 6, top 7, top 8, top 9, top 10, top 15, top 20, top 25, top 30, top 35, top 40, top 45, top 50, top 60, top 70, top 80, top 90, top 100, top 200, top 300, top 400, top 500, top 600, top 700, top 800, top 900, or top 1000, as targets that can have the ability to resist dysfunction after cell knockout.

[0095] For example, the determination in the present invention comprises determining the level of the cytokine expressed by the test cells by flow cytometry, Western blotting, or ELISA. For example, the determination in the present invention comprises determining the amount and / or rate of increase in the level of the cytokine expressed by the cells in which the expression or activity of the candidate target is modulated, compared to corresponding cells in which the expression or activity of the candidate target is not modulated, by flow cytometry. For example, compared to the corresponding test cells in which the expression or activity of the candidate target is not modulated, the ratio of positive cells for the cytokine expressed by the test cells in which the expression or activity of the candidate target is modulated is increased (the ratio of positive cells in the test cells after modulation / the ratio of positive cells in the test cells not modulated = the ratio of increase) by about 1.0 times to about 10,000 times, for example, about 1.0 times, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 1000 times, or about 10,000 times. For example, compared to the corresponding test cells in which the expression or activity of the candidate target is not regulated, the increase in the proportion of positive cells expressing the cytokine in the test cells in which the expression or activity of the candidate target is regulated (the proportion of positive cells in the test cells after regulation - the proportion of positive cells in the test cells not regulated = the increase) is about 0.5% to about 100%, for example, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%. , about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.

[0096] For example, the cells to be tested in the present invention include immune cells. For example, the immune cells in the present invention include phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils. For example, the immune cells in the present invention include monocytes, macrophages and / or dendritic cells. For example, the immune cells in the present invention are derived from immune cells differentiated from stem cells. For example, the stem cells in the present invention include induced pluripotent stem cells (iPSC), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells, and / or peripheral blood stem cells. For example, the immune cells in the present invention include B cells, T cells, regulatory T cells, natural killer cells and / or natural killer-like T cells (NKT). For example, the immune cells in the present invention include αβT cells and / or γδT cells. For example, the immune cells in the present invention include tumor infiltrating lymphocytes (TIL). For example, TIL described in the present invention is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, paracancerous tissue fragments, pleural effusion and / or ascites TIL and / or derived from cryopreservation after recovery. Before amplification and genetic modification, the source of cells (e.g., immune effector cells (e.g., T cells or NK cells)) is obtained from a subject. The term "subject" is intended to include living organisms (e.g., mammals) that can induce an immune response therein. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue, and tumors.

[0097] On the one hand, the present invention provides a system for identifying targets, which comprises: a measurement module for measuring the effect of a candidate target on the activation level of the cells to be tested after the cells to be tested have essentially lost their killing ability (for example, after the cells to be tested are co-cultured with a tumor antigen), and before and / or simultaneously with the measurement, the expression or activity of the candidate target in the cells to be tested is regulated.

[0098] In one aspect, the present invention provides a method for identifying a target, the method comprising:

[0099] Step (1), simultaneously or sequentially introducing components of a target regulation system selected from a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system and / or an inhibitory RNA into the cells to be tested, wherein the target regulation system increases or decreases the expression or activity of the candidate target;

[0100] Step (1-a), causing the cells to be tested to express receptors that recognize the tumor antigen;

[0101] Step (2) co-culturing the cells to be tested (cells that have been or are about to be regulated with the candidate target) with the tumor antigen or tumor cells expressing the tumor antigen, so that the cells to be tested substantially lose their killing ability;

[0102] Step (3), determining the levels of GZMB, IFN-γ, TNF-α and / or CD107a expressed by the cells to be tested;

[0103] Step (4), determining the effect of regulating the candidate target on the cell by reading the results obtained by the assay;

[0104] There is no order restriction among step (1), step (1-a) and step (2), and step (1), step (1-a) and step (2) can optionally be performed substantially simultaneously.

[0105] In one aspect, the present invention provides a system for identifying a target, the system comprising the following modules for implementing the method provided by the present invention:

[0106] Module (1) is used to simultaneously or sequentially introduce components of a target regulation system selected from a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system and / or an inhibitory RNA into a cell to be tested, wherein the target regulation system increases or decreases the expression or activity of the candidate target;

[0107] Module (1-a) is used to make the cells to be tested express an exogenous cell receptor or a functional fragment thereof;

[0108] Module (2) is used to co-culture the cells to be tested (cells that have been or are about to be regulated by the candidate target) with the specific antigen of the exogenous cell receptor or its functional fragment or cells expressing the specific antigen, so that the cells to be tested basically lose their killing ability;

[0109] Module (3) is used to measure the levels of GZMB, IFN-γ, TNF-α and / or CD107a expressed by the cells to be tested;

[0110] Module (4) is used to determine the effect of regulating the candidate target on the cell by reading the results obtained by the assay;

[0111] There is no restriction on the order of modules (1), modules (1-a) and modules (2), and modules (1), modules (1-a) and modules (2) can be optionally arranged in parallel.

[0112] For example, the modules of the present invention and the systems incorporating them can be arranged and used in a variety of sequences and configurations. Furthermore, the systems and methods of the present invention can include additional components and steps not specifically described herein. For example, a portion of the systems described herein can be used in combination with a portion of the methods described herein, e.g., to form a semi-automated system. For example, the present invention can include partially disposable modules, tubing, etc., to form a complete system capable of isolating cells or removing cell products.

[0113] Without intending to be bound by any theory, the following examples are merely intended to illustrate the methods and uses of the present invention and are not intended to limit the scope of the present invention.

[0114] Example

[0115] Example 1 Screening Platform Screening Process

[0116] The present invention designs a target gene screening process, in which an optimized culture method is adopted and test cells in a hypofunctional state are obtained, which is used to identify target genes that can affect cell function in actual application environments.

[0117] Exemplary Cell Culture Methods

[0118] PBMC (peripheral blood mononuclear cells) were obtained from blood samples of healthy donors. PBMC T cells frozen in liquid nitrogen were revived and cultured, and resuspended to 5E5 / ml (5×10 5 T cell TransAct (Miltenyi) was added at a TransAct: cell ratio of 1:100, and recombinant human IL-2 at a concentration of 30 IU / ml was added, and cultured for about 72 hours.

[0119] High-throughput screening library construction

[0120] (1) Construction of whole genome screening library plasmid

[0121] The in vitro whole-genome screening library design is based on the Brunello library referenced by JG Doench et al. (DOI: 10.1038 / nbt.3437). It contains approximately 80,000 gRNAs targeting approximately 20,000 genes (each gene has four gRNA target sites), as well as 500 control gRNAs that are non-targeting or target non-functional regions as a quality control parameter. The data presented in this paper demonstrate that the Brunello gRNA library has high gene editing efficiency and low off-target effects, making it an ideal library for whole-genome screening.

[0122] First, the oligo sequence of the screening library is synthesized with 25nt homology arms at both ends. A round of PCR amplification is performed based on the homology arm sequence to obtain the dsDNA fragment of the screening library. The dsDNA fragment of the library is then assembled into the designed lentiviral expression vector using the Gibson kit. In addition, the vector also carries a surface marker gene that is easy to stain, which can be used to indicate the subsequent transduction and expression of the library in T cells, and can also be used to enrich transduction-positive cells. The assembled library plasmid is electroporated into competent cells (Endura), cultured overnight to amplify the library plasmid, and then the plasmid is extracted to prepare the library plasmid.

[0123] High-throughput NGS sequencing was used to perform quality control on the prepared library plasmids, and the coverage and distribution of gRNA were detected to determine that the library used in the screening platform of the present invention met the library construction standards. Among them, the sequencing data alignment rate of the plasmid library was greater than 70%, the coverage of gRNA was greater than 99%, the gene coverage was 100%, and the gini coefficient of gRNA was less than 0.3.

[0124] (2) Construction of whole genome knockout cell library

[0125] Lentivirus containing the whole-genome screening library was packaged in HEK293T cells and concentrated 20-fold. The viral titer of the screening library was tested on human PBMC T cells, reaching a biological titer of at least 2E7 / ml. Activated human PBMC T cells were co-transduced with the lentivirus carrying the screening library and a retrovirus expressing the NYESO1-TCR, according to an exemplary embodiment of the present invention. The transduction efficiencies were controlled at 30-40% (to limit the probability of two lentiviruses carrying different gRNAs entering the same cell to approximately 10% to minimize the impact on subsequent analysis) and above 70%, respectively.

[0126] Cell transduction with TCR

[0127] Exemplary pre-viral transduction treatment method for the embodiments of the present invention. One day before viral transduction, use recombinant human fibrin fragment (Retronectin, Takara) with a final concentration of 15μg / mL to coat 24 or 6-well suspension culture plates, 250μL per well of 24-well plates and 1000μL per well of 6-well plates. Protect from light and store at 4°C overnight. Remove the coated well plate, discard the coating solution, add 2% BSA blocking solution and block at room temperature for 30 minutes. Discard the blocking solution, wash the plate twice with a plate washing solution containing 2.5% HEPES, and discard the plate washing solution.

[0128] Methods for transducing exemplary specific receptors used in embodiments of the present invention. The experimental group underwent viral transduction using an appropriate amount of viral dilution, and the retroviral vector contained a nucleic acid fragment encoding NYESO1-TCR (the exemplary α chain variable region is shown in SEQ ID NO: 1, and the exemplary β chain variable region is shown in SEQ ID NO: 2).

[0129] The variable region of the TCR α chain is shown in SEQ ID NO: 1:

[0130] The variable region of the TCRβ chain is shown in SEQ ID NO: 2:

[0131] Optionally, the viral vector can contain the guide RNA of the CRISPR system (which can be used as a screening library). Centrifuge at 32°C, 2000g for 2 hours. Discard the supernatant from the well plate and add an appropriate volume of revived and activated T cells to each well, with a cell concentration of approximately 1×10 6 1000 g for 10 minutes at 30-32°C. After centrifugation, place the culture plate in a 37°C, 5% CO2 incubator to obtain transduced cells. After transduction, culture for approximately 3 days to enrich for virus-positive cell populations.

[0132] The marker protein of the screening library and magnetic beads carrying the corresponding antibodies are used to enrich T cells that are positive for virus transduction in the screening library, and then the Lonza electroporation system is used to electroporate the Cas9 protein for targeted gene editing.

[0133] Introducing the CRISPR Cas system into cells

[0134] Method for producing Cas9 cells in the embodiment of the present invention. Cas9 (Kactus Biosystem: Cas9 Nuclease) and cells were electroporated using a Lonza electroporator. After the Cas9 protein electroporation was completed, an appropriate amount of T cell culture medium was added according to the cell count density and viability according to the culture medium instructions, and recombinant human IL-2 at a concentration of 300 IU / ml was added. The initial culture cell density was adjusted to 1×10 6 After about a week, it was confirmed that the T cells had recovered and were able to proliferate normally.

[0135] The cell function-depleted state culture of the present invention

[0136] The present invention uses an optimized culture method to obtain cells in a hypofunctional state. Specifically, A375 cells expressing the NYESO1 antigen (exemplary amino acid sequence: SLLMWITQC) are co-cultured with edited T cells in a two-stage co-culture process.

[0137] At the beginning of the first stage (denoted as Day 0), edited T cells are co-cultured with A375 cells at a ratio of effector cells (immune cells to be tested, E): target cells (A375 cells, T) of 1:3 to 1:30. For example, the co-culture is carried out for about 4 days at an E:T ratio of about 1:3 to 1:30, for example, a ratio of live T cells to live A375 cells of 1:4.23.

[0138] At the beginning of the second stage (denoted as Day 4), A375 cells are further added to the above co-culture system; based on the number of T cells in the co-culture system, A375 cells are added to achieve a cell ratio of effector cells (test immune cells, E): target cells (A375 cells, T) of 1:3 to 1:30. For example, based on the number of viable T cells counted by flow cytometry, A375 cells are added to achieve an E:T ratio of approximately 1:3 to 1:30, such as 1:8.84 or 1:6.67, and the co-culture is continued for approximately 3 days.

[0139] Through the above optimized hypofunctional state culture method, TCR-T cells in a hypofunctional state are obtained.

[0140] T cell exhaustion markers and apoptosis flow cytometry

[0141] Take 100 μL of TCR-T cell suspension from each test group, about 1 to 2 × 10 5 Cells were washed once with 200 μL / well PBS per treatment group, centrifuged at 600 g for 3 minutes, and the supernatant discarded. Antibody working solution for cell surface depletion markers and apoptosis staining for CD3 / CD4 / CD8 / CD38 / CD101 / PD-1 / TCR / MCSP (BD Biolegend) was prepared at a 1:100 concentration. Cell surface depletion marker staining was performed using a cell viability assay dye (1:10,000). Apoptosis staining was performed using PBS, with 50 μL / well of a 96-well plate incubated at 2-8°C in the dark for 30 minutes. During the staining process, the reagents required for apoptosis staining were prepared (BD). After apoptosis surface staining, cells were washed twice with appropriate amounts of PBS (200 μL / well per 96-well plate), centrifuged at 600 g for 3 minutes, and the supernatant discarded. Apoptosis detection reagent (66 μL / well of a 96-well plate) was added and incubated at room temperature in the dark for 15 minutes. After staining, 1× Binding Buffer was added to terminate the staining (180 μL / time for 96-well plate), and the cells were resuspended for flow cytometry detection.

[0142] Functional screening model construction and screening process

[0143] Functional screening models can identify T cells that are resistant to hypofunction, thereby identifying target genes that can significantly enhance T cell resistance to hypofunction. The present invention tests the killing effect of NYESO1-TCR-T cells after two rounds of low-efficiency target ratio A375 stimulation, as well as T cell exhaustion markers and apoptosis detection, to identify corresponding T cell populations resistant to hypofunction. For example, after two rounds of low-efficiency target ratio stimulation, T cells with hypofunction resistance are collected.

[0144] Finally, the genome of the sorted cells is extracted, and the enriched gRNA sequences are captured through high-throughput sequencing and bioinformatics analysis, which can then screen out potential genes that can enhance T cell resistance to dysfunction.

[0145] Example 2 Effect of the present invention on the culture of cells in a hypofunctional state

[0146] Verification of the ratio of effector cells (immune cells to be tested, E): target cells (A375 cells, T) in one stage

[0147] Effector cells were generated by expressing a TCR targeting NYESO1 in PBMC T cells from two different donors; A375 cells expressing the NYESO1 antigen served as target cells. At the beginning of the first phase (Day 0), the edited T cells were co-cultured with A375 cells at a ratio of 1:3 to 1:30 for effector cells (immune cells to be tested): target cells (A375 cells, T). For example, each experimental group maintained co-culture for approximately four days at an E:T ratio of 1:30, 1:10, or 1:3.

[0148] FIG1 shows the tumor cell ratio after 3 days and 7 days of co-culture according to the effector cell (test immune cell, E): target cell (A375 cell, T) ratio of 1:30, 1:10 or 1:3 in one stage.

[0149] The results showed that at an E:T ratio of 1:3 to 1:30, the immune cells tested could still kill tumor cells after 3 days of co-culture. At 4 to 7 days of co-culture, the immune cells tested were still able to kill tumor cells, but the lower proportion of tumor cells at this time may have caused the immune cells tested to not reach a low functional state on day 7.

[0150] A two-stage hypofunctional state culture was used, and the ratio of effector cells (immune cells to be tested, E): target cells (A375 cells, T) in the second stage was preliminarily verified.

[0151] Effector cells were generated by expressing a TCR targeting NYESO1 in PBMC T cells from two different donors; A375 cells expressing the NYESO1 antigen served as target cells. The first phase (Day 0) began with co-culture of edited T cells with A375 cells at a ratio of 1:3 to 1:30 for effector cells (immune cells to be tested, E): target cells (A375 cells, T). For example, at an E:T ratio of 1:4.23 (donor one and donor two), co-culture was performed for approximately four days.

[0152] At the start of the second phase (Day 4), A375 cells were added to the co-culture system to achieve an effector cell (test immune cell, E) to target cell (A375 cell, T) ratio of 1:3 to 1:30, depending on the number of T cells in the co-culture system. For example, based on the number of viable T cells counted by flow cytometry, A375 cells were added to achieve an E:T ratio of 1:8.84 (donor one) or 1:6.67 (donor two), and the co-culture was continued for approximately 3 days.

[0153] FIG2 shows the number of tumor cells after 4 and 7 days of co-culture at two stages so that the ratio of effector cells (immune cells to be tested, E): target cells (A375 cells, T) was 1:3 to 1:30.

[0154] The results showed that when co-cultured with tumor cells in both stages, the tested immune cells could kill tumor cells on Day 4 after 4 days, and on Day 7 after 7 days, the tested immune cells basically reached a functionally impaired state.

[0155] Verification of the ratio of effector cells (immune cells to be tested, E): target cells (A375 cells, T) in the first and second stages

[0156] Effector cells were generated by expressing a TCR targeting NYESO1 in PBMC T cells from two different donors; A375 cells expressing the NYESO1 antigen served as target cells. At the beginning of the first phase (designated Day 0), the edited T cells were co-cultured with A375 cells at a ratio of 1:3 to 1:30 for effector cells (immune cells to be tested): target cells (A375 cells, T). For example, co-culture was performed for approximately four days using E:T ratios of 1:30, 1:10, 1:3, or 1:1 for each experimental group.

[0157] At the beginning of the second phase (recorded as Day 4), A375 cells are continuously added to the above co-culture system; depending on the number of T cells contained in the co-culture system, A375 cells are added to achieve a cell ratio of effector cells (immune cells to be tested, E): target cells (A375 cells, T) of 1:3 to 1:30. For example, based on the number of viable T cells counted by flow cytometry, A375 cells are added to achieve an E:T ratio of 1:30, 1:10, 1:3 or 1:1, and the co-culture is carried out for about 3 days. Among them, in this test experiment:

[0158] The 1:30 group had an E:T ratio of 1:30 in the first and second phases;

[0159] The 1:10 group had an E:T ratio of 1:10 in the first and second phases;

[0160] The 1:3 group had an E:T ratio of 1:3 in the first and second phases;

[0161] The E:T ratio of the 1:1 group was 1:1 in the first and second phases (as shown in the figure legends, respectively).

[0162] The supernatant of each experimental group was taken and the cytokine secretion was analyzed using a CBA kit.

[0163] FIG3 shows the release capacity of IFN-γ and TNF by the immune cells to be tested after two-stage culture.

[0164] The results showed that after two stages of culture, the immune cells in the 1:30, 1:10, and 1:3 groups basically stopped releasing cytokines, essentially reaching a state of hypofunction. However, in the 1:1 group, the immune cells still released cytokines after two stages of culture, but did not reach a state of hypofunction.

[0165] FIG4 shows the number of tumor cells after two-stage culture.

[0166] The results showed that after two-stage culture, the immune cells in the 1:30, 1:10, and 1:3 groups essentially reached a state of functional impairment on Day 7. In the 1:1 group, the immune cells still killed tumor cells after two-stage culture and did not reach a state of functional impairment.

[0167] FIG5 shows the difference in expression ratio of apoptotic cells in the tested immune cells on day 7 relative to day 3 after two-stage culture.

[0168] The results showed that after two-stage culture, the apoptosis level of dysfunctional cells in the tested immune cells in the 1:30, 1:10, and 1:3 groups was higher on Day 7 than on Day 3 compared with the 1:1 group.

[0169] FIG6 shows the difference in expression ratio of exhaustion markers in the tested immune cells on day 7 relative to day 3 after two-stage culture.

[0170] The results showed that after two-stage culture, the expression levels of exhaustion markers in the tested immune cells in the 1:30, 1:10, and 1:3 groups were significantly higher on Day 7 than on Day 3, compared with the 1:1 group.

[0171] Example 3 Functional verification of the screened targets

[0172] The optimized screening platform of the present invention was used for genome-wide functional screening. Using an optimized method for culturing cells in a hypofunctional state, hypofunctional immune cells were obtained for target screening. This subpopulation was collected using flow cytometry and subjected to NGS sequencing analysis. The results showed that the distribution of Safe Harbor guides did not change significantly in the harvested samples. This result demonstrates that the screening platform of the present invention can effectively exclude genes unrelated to cell function.

[0173] Figure 7 shows the gRNA distribution results analyzed by this screening platform.

[0174] In the gRNA distribution results, a large number of gRNAs, such as POLR2L, HMGCS1, and UBA1, showed a decrease in expression. These targets are also known in the art as essential genes related to T cells (such as those documented by the Broad Institute's Achilles Project), and knocking out these target genes inhibits T cell proliferation. For example, the targets in the upper left corner help maintain cell function, and knocking out these targets will result in a decrease in the number of cells in the knockout analysis group.

[0175] In addition, a small number of gRNAs for targets such as RASA2 and BCL2L11 in the upper right corner were enriched. These targets are also T cell inhibition-related targets known in the art, for example, they can potentially inhibit cell function. Knocking out these targets will lead to an increase in the number of cells in the knockout analysis group.

[0176] The results showed that the screening platform of the present invention can detect candidate genes related to T cell effector function with high sensitivity.

[0177] Example 4: Individual verification of the selected targets

[0178] The present invention also designs targeted knockout tools for the screened targets such as RASA2 and BCL2L11, and independently verifies them.

[0179] Culture of tumor-infiltrating lymphocytes

[0180] 1.1 Tumor tissue receipt and processing

[0181] 1.1.1 Organization reception

[0182] Receive tumor tissue and blood samples from donors, verify and record sample information, and print corresponding sample labels.

[0183] 1.1.2 Tissue processing and culture

[0184] Use 75% alcohol to disinfect the sample tube and blood collection tube and transfer them to a biosafety cabinet. Isolate PBMC cells from the blood sample and freeze them according to the above-mentioned PBMC manual isolation and freezing procedures. Take a culture flask or culture bag with a breathable surface, such as a culture bag (Origen), and add 300 mL of thawed complete culture medium. The complete culture medium can be arbitrarily selected from X-vivo15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and can be supplemented with essential amino acids and antibiotics, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL). Take several 10 cm culture dishes, add an appropriate amount of culture medium, use sterile ophthalmic forceps to remove the tumor tissue from the sample tube into a 10 cm culture dish, wash the tissue and change the culture dish. Use ophthalmic scissors and forceps to perform initial shearing, removing adipose and necrotic tissue. Each tissue block is then minced to approximately 27 cubic millimeters. A non-suspended tumor tissue block is obtained. A 20 mL syringe is used to remove the internal stopcock and connect to a culture bag. Using a pipette, approximately 1 g of tissue is transferred from the syringe into the culture bag. The culture bag is placed in a CO2 incubator for incubation. The scissors and forceps are cleaned and initially disinfected with 75% alcohol. After ultrasonic cleaning, they are sterilized to obtain the first TIL population.

[0185] 1.2 Step (A) In vitro expansion and harvesting

[0186] 1.2.1 Step (A) In vitro amplification

[0187] Depending on the cell growth status, the medium should be replenished or half-replaced every 3-7 days to ensure cell nutrition. Complete culture medium can be arbitrarily selected from X-vivo 15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 (double heron and / or tetracycline) at a concentration of 300-9000 IU / mL (e.g. 1000-9000 IU / mL, such as 6000 IU / mL) can be added. 3-14 days in step (A), for example, samples can be taken and counted on the 13th or 14th day. If the cell number is between 5×10 5 to 5×10 8 During this time, the harvesting step (A) is entered.

[0188] 1.2.2 Results of Step (A)

[0189] Collect the cells after in vitro expansion in step (A), centrifuge, discard the culture medium, wash the cells once with PBS or normal saline, obtain the TILs (second TIL population) expanded in vitro in step (A), and take samples for counting and retain about 5×10 5 to 2×10 8 cells into the subsequent in vitro expansion step; about 5×10 5 The remaining cells can be added to the cryopreservation medium and cryopreserved as cryopreserved preREP TIL in vitro cells.

[0190] 1.3 Step (B) TIL activation

[0191] Continue to culture the TILs (second TIL population) expanded in vitro in step (A), or recover the frozen preREP TIL cells in vitro and perform TIL activation in step (B).

[0192] Complete culture medium can be selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi Biotech, etc. Essential amino acids and antibiotics can be added to adjust the cell density to 5×10 5 to 2×10 6 Cells are suspended in a 24-well culture plate at a concentration of 1 mL / well and IL-2 is added at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, for example, 6000 IU / mL). T cell activators, such as CD3 agonists and / or CD28 agonists, can be added to the culture medium of each TIL population, for example, approximately 30 ng / mL of CD3 antibody (Miltenyi Biotech, OKT3), approximately 30 ng / mL of CD28 antibody (Merck, 15E8), magnetic beads (Dynabeads, approximately 1 to 10 μm diameter, Thermo Fisher) at a ratio of approximately 1:2-2:1 beads to TILs, and / or transACT (Miltenyi, approximately 100 to 500 nm diameter, TILs) at a ratio of approximately 1:100-1:2000. Culture is continued for approximately 0-4 days to obtain a third TIL population.

[0193] 1.4 Step (C) TIL cell gene editing

[0194] Based on the target gene screened by the present invention, a guide sequence was designed, thawed and added with nuclease-free water to a concentration of about 100 μM. About 2 μL of gRNA (50 μM) was incubated at 95°C for 2 minutes to anneal and then added to P3 buffer, and 0.3-1 μL of Cas9 (such as Kaixia, Ke Rui, Acro, 10 mg / mL) was added and incubated at 25°C for 10 minutes to form a ribonucleoprotein complex (RNP). In P3 buffer (Lonza), the above RNP was mixed with about 1×10 TILs of the third group using a Lonza electroporator. 6 The cells are electroporated. For example, the electroporation procedure can be human T cell stim (EO115). The electroporated cells are cultured for about 0-4 days after gene editing to obtain a fourth TIL population.

[0195] 1.5 Step (D) TIL cell culture after gene editing

[0196] Feeder cells (irradiated healthy donor PBMC T cells) are added to the fourth TIL cell population for culture. The time for contacting TIL with feeder cells needs to be several times T after the TIL is contacted with IL-2 and T cell activator (such as CD3 antibody or a nanomatrix containing CD3 antibody and CD28 antibody, such as transACT) in step (B). n Afterwards (T n The time period can be from 0 hours to 12 days, for example, 24 hours or 48 hours. First, resuscitate the mixed feeder cells from 1-5 donors; mix the activated TIL cells and feeder cells at a ratio of approximately 1:200, transfer them to a G-Rex100 culture flask or breathable bag, and supplement with complete culture medium. Samples are taken and counted every 1-3 days, and the medium is replenished or half-filled depending on the cell status until the total number of cells is greater than 1×10 9 Alternatively, the in vitro expansion culture of step (D) is carried out for about 5 days to about 14 days, and the in vitro expansion culture of step (D) is terminated.

[0197] 1.6 Harvesting of Tumor-Infiltrating Lymphocytes

[0198] Take the cells amplified in step (D), centrifuge and discard the culture supernatant, and wash three times with PBS or saline or compound electrolyte solution to obtain TILs amplified in step (D) (fifth TIL population). Samples are counted during the third wash. According to the counting results, after the final centrifugation, discard the supernatant and take 3×10 6 The cells were sent for quality control testing; all the remaining cells were added to the freezing solution and the cell density was adjusted to 1-3×10 8 cells / mL for cryopreservation.

[0199] RASA2 Authentication

[0200] Gene knockout detection

[0201] Reagents and materials: DNA extraction solution (QuickExtract DNA extraction solution, Lucigen, QE09050), RNase / DNase free water (Tiangen), EDTA (Shenggong, 0.5 M), Recombinant DNase I (RNase-free, TAKARA).

[0202] Extract genomic DNA: About 2-7 days after tumor-infiltrating lymphocyte T cell knockout, take about 1×10 5 to about 2×10 5 Wash the cells once with PBS, then resuspend the gene-edited cells in 44 μL PBS. Add 6 μL of the prepared nuclease mix (containing 1 μL DNase I and 5 μL 10× DNase I Buffer) and incubate at 37°C for 5 minutes. Add 2.5 μL of 0.5 M EDTA to the sample and incubate at 80°C for 10 minutes. After centrifugation and discarding the supernatant, add 50 μL of DNA extraction solution to the cell pellet. After brief centrifugation, run the following program: 75°C for 10 minutes; 95°C for 5 minutes; 4°C for hold. DNA sample concentration can be measured using a spectrophotometer (NanoDrop™).

[0203] Sequencing: PCR primers can be designed in the region approximately 100 to 200 nucleotides upstream and downstream of the PAM site. Design the PCR reaction system as follows:

[0204] And amplify according to the following PCR program:

[0205] The PCR products were analyzed by Sanger sequencing.

[0206] Analyzing Crispr Cas9 knockout efficiency

[0207] Crispr Cas9 knockout efficiency was analyzed using the Tracking of Indels by DEcomposition (Tide) method based on Sager sequencing data. For specific methods, see (Brinkman et al, Nucl. Acids Res. (2014) or shinyapps.datacurators.nl / tide / ). Knockout efficiency analysis was performed by inputting the corresponding sgRNA sequence of the present invention, the pre-knockout control sequence, and the test sequence after Crispr Cas9 knockout, with a P-value threshold of 0.001.

[0208] Donor 812 was a patient with oral mucosal melanoma, and donor 904 was a patient with lung cancer. The sgRNA targeting RASA2, in cells derived from donor 812, the knockout efficiency of the CRISPR tool composed of two different sequences of guides targeting RASA2 was 35%, and in cells derived from donor 904, the knockout efficiency was 93.3%. The results show that the various gene editing methods of the present invention can achieve a certain proportion of knockout efficiency. Among them, in Figures 8A to 8F, the RA1 group, the RA2 group, and the RA4 group represent the experimental groups edited with gRNA targeting RASA2 coded as RA1 (SEQ ID NO: 3), RA2 (SEQ ID NO: 4), and RA4 (SEQ ID NO: 5), respectively.

[0209] Detection of cell proliferation

[0210] Experimental preparation

[0211] For the CD3 antibody group, 30 ng / ml CD3 antibody (Miltenyi Biotech, OKT3) was used to coat a flat-bottom 96-well plate one day in advance at 4°C overnight.

[0212] Starting from the 7th day after gene editing, tumor-infiltrating lymphocytes in each group were re-plated with the same total number of cells. The unstimulated group did not require CD3 antibody stimulation in the expansion efficiency test. The stimulated group was stimulated with 30 ng / mL CD3 antibody (Miltenyi Biotech, OKT3), and the fluorescence of T cells at the time of plating was analyzed using the CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega). The fluorescence of T cells was analyzed using the CTG kit 3 days later. The expansion efficiency of T cells was characterized by the fluorescence on the third day / the fluorescence at the time of plating.

[0213] Figure 8A shows that gene-edited T cells in the no-stimulation group can have significant expansion capacity.

[0214] Figure 8B shows that the gene-edited T cells in the CD3 antibody stimulation group can have a significant expansion capacity. The results show that compared with the control group (NT), the gene-edited T cells of the present invention can have a significant expansion capacity.

[0215] Cell killing ability assay

[0216] Starting six days after gene editing, tumor target cells were plated in 96-well flat-bottom plates. The following day, tumor-infiltrating lymphocytes (TILs) from each group were co-cultured with target cells at varying effector-to-target ratios (E:T). 100 μL of target cells and 100 μL of T cells were added, with triplicate wells set up for each group. A control group containing only target cells was also established.

[0217] According to the instructions for the apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL of the apoptosis detection reagent was added per well, and 25 μL of culture medium diluted with Caspase 3 / 7 Green Dye was added per well. Caspase 3 / 7 activity was recorded using an Incucyte recorder (Sartorius) to analyze T cell cytotoxicity, with recordings every 3 hours for approximately 2 days.

[0218] Figure 8C shows the target cell killing ability of gene-edited T cells. The results show that compared with the control group (NT), gene-edited T cells can have more significant target cell killing ability.

[0219] Cell flow cytometry

[0220] The tumor-infiltrating lymphocyte T cell population obtained on the 8th day after gene editing was used to detect cell expression by flow cytometry.

[0221] Sources of experimental materials for T cell flow cytometry

[0222] V-bottom 96-well plate, manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.

[0223] The flow cytometry antibodies in this example were purchased from BD or Biolegend. 5 to 5×10 5 Add a cell sample to a flow tube or a V-bottom 96-well plate. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, add 1mL / tube to the flow tube and 200μL / well to the 96-well plate, and discard the supernatant. Add the prepared antibody working solution for cell surface staining. The antibody (BD or Biolegend) concentration is 1:100 to 1:200, containing activity detection dye at 1:10000. Stain 100μL / tube of the flow tube and 50μL / well of the 96-well plate, and incubate at 2-8℃ in the dark for 30 minutes. After surface staining, wash the cells once with PBS (200μL / time for 96-well plate and 1mL / time for flow tube), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant after centrifugation. Resuspend the cells in 100-500μL PBS and perform flow cytometry detection.

[0224] Figure 8D shows that gene-edited T cells have a lower proportion of T cells expressing exhaustion markers. For example, T cells expressing exhaustion markers can be CD38-positive and / or CD101-positive cells.

[0225] Cytokine expression flow cytometry

[0226] The cytokine expression of tumor-infiltrating lymphocyte T cell populations obtained on the 7th or 8th day after gene editing was detected by flow cytometry.

[0227] Experimental preparation

[0228] For the CD3 antibody group, 30 ng / ml CD3 antibody (Miltenyi Biotech, OKT3) was used to coat a flat-bottom 96-well plate one day in advance at 4°C overnight.

[0229] Prepare the culture medium required for intracellular factor expression analysis: Take T cell culture medium and add the following volume ratios: Golgistop 0.7:1000, Golgiplug 1:1000, and CD107a antibody 1:500, for a total of 2 μL / mL. Do not add interleukins.

[0230] Detection steps

[0231] After centrifugation, the tumor-infiltrating lymphocytes of each experimental group were resuspended in 600 μL of the culture medium required for the above-mentioned intracellular factor expression detection to a concentration of 1×10 6 cells / mL, added into a 96-well plate, 200 μL / well, and incubated in a 37°C incubator overnight.

[0232] After incubation, wash once with 200 μL / well PBS, centrifuge at 600 g for 3 minutes, and discard the supernatant. Prepare a mixed antibody working solution for cell surface staining of CD3 / CD4 / CD8 (BD) at an antibody concentration of 1:100 and a cell viability assay dye concentration of 1:10,000. Stain 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube. Incubate at 2-8°C in the dark for 30 minutes. During the staining process, prepare the reagents required for transcription factor staining: dilute 4× Fixation / Permeabilization Buffer (BD) with Transcription Factor Buffer Set to 1× Working Solution A; dilute 5× Perm / Wash Buffer (BD) with double-distilled water to 1× Working Solution B. Pre-cool at 4°C until use. After staining, wash the cells twice with an appropriate amount of PBS (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes), centrifuge at 600 g for 3 minutes, and discard the supernatant. Cell fixation and membrane permeabilization: Resuspend the cells thoroughly and add an appropriate amount of 1× working solution A (100 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes) to fix and permeabilize the membrane. Incubate at 2-8°C in the dark for 40-50 minutes. After fixation and permeabilization, wash the cells with 1× working solution B (200 μL / well for 96-well plates, 2 mL / well for flow cytometry tubes), centrifuge at 2-8°C, centrifuge at 350 g for 6 minutes, and wash twice. Intracellular antibodies (CD107a, GZMB, TNF-α, and IFN-γ, BD / BioLegend) were prepared using 1× Working Solution B at a concentration of 1:100 to 1:200. 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube were added and stained for 30 minutes at 2-8°C in the dark. After staining, cells were washed with 1× Working Solution B (200 μL / well of a 96-well plate and 2 mL / well of a flow cytometry tube) and centrifuged twice at 350 g for 6 minutes at 2-8°C. Cells were resuspended in 100-500 μL of PBS and analyzed by flow cytometry.

[0233] Figure 8E shows that the gene-edited T cells in the unstimulated group had a higher cytokine expression ratio, for example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression.

[0234] Figure 8F shows that the gene-edited T cells in the stimulated group had a higher cytokine expression ratio, for example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression.

[0235] BCL2L11 validation

[0236] Using a similar experimental method as described above, BCL2L11 was separately verified in tumor-infiltrating lymphocytes. Donor 906 was a patient with cervical cancer, donor 107 was a patient with lung cancer, and donor 812 was a patient with melanoma. The knockout efficiencies of the CRISPR tool composed of sgRNA targeting BCL2L11 and two guides targeting BCL2L11 with different sequences were 92.4% and 80.3%, respectively. The results showed that various gene editing methods of the present invention can achieve a certain proportion of knockout efficiency. Among them, in Figures 9A to 9F, the BCL2L11-3 group and the BCL2L11-4 group represent the experimental groups edited with gRNAs code-named BCL2L11-3 (SEQ ID NO: 6) and BCL2L11-4 (SEQ ID NO: 7), respectively.

[0237] Figure 9A shows the expansion fold of TILs with BCL2L11 single-target gene editing in the non-stimulation culture medium group.

[0238] Figure 9B shows the expansion fold of TILs with BCL2L11 single-target gene editing in the TransACT stimulation group.

[0239] Figure 9C shows the central memory T cell ratio of TIL cells after BCL2L11 editing. For example, central memory T cells can be CD45RO-positive CD62L-positive cells.

[0240] Figure 9D shows the proportion of naive T cells in TIL cells after BCL2L11 editing. For example, naive T cells can be CD45RO-negative and CD62L-positive cells.

[0241] Figure 9E shows the proportion of cells expressing exhaustion markers of TIL cells after BCL2L11 editing. For example, T cells expressing exhaustion markers can be PD-1 positive, LAG-3 positive, TIM-3 positive, CD38 positive and / or CD101 positive cells.

[0242] Figure 9F shows the proportion of stem-like T cells in TIL cells after BCL2L11 editing. For example, stem-like T cells can have a CD39-negative and CD69-negative phenotype.

[0243] Figure 9G shows that BCL2L11-edited TIL cells in the non-stimulation medium group had a higher cytokine expression ratio. Figure 9H shows that BCL2L11-edited TIL cells in the non-stimulation medium group had a higher cytokine expression ratio. Figures 9I and 9J show that BCL2L11-edited TIL cells in the TransACT stimulation group had a higher cytokine expression ratio.

[0244] Figure 9K shows that BCL2L11-edited cells in TCR-T cells have higher cytokine release levels.

[0245] The above-mentioned independent verification results show that the candidate genes related to cell functions that can be screened out by the screening platform of the present invention have ideal application prospects, which proves the advantageous effect of the screening platform of the present invention.

[0246] Example 5 Further screening of the screening platform of the present invention

[0247] By using the optimized screening platform of the present invention, more donor-derived T cells are obtained as cell samples, and then the function-related candidate genes of the present invention are screened.

[0248] Figure 10 shows the gRNA distribution results analyzed after expanding the sample size of this screening platform.

[0249] On the one hand, the targets screened out, such as RPL8, CDCA8, PSMB4, RPS8, RPL6, IL2RG, RPL23, JAK3, GRB2, TOP2A, IKBKG, PDPK1, etc., with a decreased number of gRNAs, are also genes known in the art to be related to T cell proliferation and activation signal transduction. Knocking out these target genes leads to immunodeficiency and inhibition of T cell proliferation. On the other hand, the screening platform of the present invention also sensitively enriches targets with an increased number of gRNAs such as BCL2L11, ARIH2, RASA2, ZBTB7B, RARG, etc., which are also T cell inhibition or exhaustion signal-related targets known in the art, for example, potential inhibitory cell function, and knocking out this target will result in increased expression of the preferred screening function marker of the knockout analysis group.

[0250] The above results show that the screening platform of the present invention can detect candidate genes related to cell effector functions with high sensitivity and stability.

[0251] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

[0252] Sequence information of the present invention

Claims

1. A method for identifying a target, the method comprising, after substantially eliminating the killing ability of a test cell, measuring the effect of a candidate target on the activation level of the test cell, and before and / or simultaneously with the measurement, the expression or activity of the candidate target in the test cell is regulated.

2. A method for identifying a target, the method comprising the following steps: (1) regulating the expression or activity of a candidate target in a test cell; (2) substantially eliminating the killing ability of the test cell; (3) measuring the activation level of the regulated test cell; and (4) determining the effect of regulating the candidate target on the cell based on the reading result obtained from the measurement.

3. The method according to any one of claims 1-2, wherein substantially eliminating the killing ability of the test cell comprises co-culturing the test cell with a tumor antigen.

4. The method according to any one of claims 1-3, wherein the test cell expresses a receptor that recognizes the tumor antigen, the tumor antigen is expressed on a tumor cell, and substantially eliminating the killing ability of the test cell comprises co-culturing the test cell with the tumor cell.

5. The method according to claim 4, wherein the co-culture comprises a co-culture in at least two stages: in the first stage, co-culturing at a ratio of the test cell to the tumor cell of 1:3 to 1:30; in the second stage, co-culturing at a ratio of the test cell to the tumor cell of 1:3 to 1:

30.

6. The method according to claim 5, wherein the first stage lasts about 2 to 6 days.

7. The method according to any one of claims 5-6, wherein the second stage lasts about 2 to 6 days.

8. The method according to any one of claims 1-7, wherein after substantially eliminating the killing ability of the test cell, the expression of PD-1, CD38, and / or CD101 in the test cell is increased relative to the test cell that has not lost its killing ability.

9. The method according to any one of claims 1-8, wherein regulating the expression or activity of a candidate target in a test cell comprises separately introducing components of a target regulation system into the test cell, and the target regulation system increases or decreases the expression or activity of the candidate target.

10. The method according to any one of claims 1-9, wherein the regulation comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system, and / or inhibitory RNA.

11. The method according to any one of claims 1-10, wherein the regulation comprises targeting a specific nucleic acid sequence of the candidate target and introducing a single-strand break, a double-strand break, and / or a mutation therein, upstream, and / or downstream.

12. The method according to any one of claims 1-11, wherein the regulation comprises increasing or decreasing the expression or activity of the candidate target at the genomic level.

13. The method according to any one of claims 1-12, wherein the regulation comprises introducing a target regulation system containing a guide nucleic acid molecule and a nuclease into the test cell.

14. The method according to claim 13, wherein the guiding nucleic acid molecule comprises a guiding RNA (gRNA) targeting the target.

15. The method according to any one of claims 13-14, wherein the nuclease comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

16. The method according to any one of claims 13-15, wherein the nuclease comprises Cas9 and / or Cas12.

17. The method according to any one of claims 1-16, wherein in the adjusted cell population to be tested, the proportion of cells expressing the candidate target is reduced to less than about 95%.

18. The method according to any one of claims 1-17, wherein the activation level of the cell to be tested comprises the level of a substance selected from the following groups expressed by the cell to be tested: members of the interleukin family, members of the interferon family, members of the tumor necrosis factor family, and members of the lysosome-associated membrane protein family.

19. The method according to any one of claims 1-18, wherein the activation level of the cell to be tested comprises the level of CD107a, GZMB, IFN-γ, TNF-α, IL2, and / or IL6 expressed by the cell to be tested.

20. The method according to any one of claims 1-19, wherein the determination comprises measuring the level of cytokines selected from CD107a, GZMB, IFN-γ, TNF-α, IL2, and IL6 expressed by the adjusted cell to be tested by flow cytometry, Western blotting, or ELISA.

21. The method according to any one of claims 1-20, wherein the determination comprises measuring, by flow cytometry, the magnification and / or increase in the level of the cytokine expressed by the cell to be tested whose expression or activity of the candidate target is adjusted compared to the corresponding cell to be tested whose expression or activity of the candidate target is not adjusted.

22. The method according to any one of claims 1-21, wherein the cell to be tested comprises an immune cell.

23. The method according to claim 22, wherein the immune cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil, and / or a basophil.

24. The method according to any one of claims 22-23, wherein the immune cell comprises a monocyte, a macrophage, and / or a dendritic cell.

25. The method according to any one of claims 22-24, wherein the immune cell comprises a B cell, a T cell, a natural killer cell, a regulatory T cell, and / or a natural killer-like T cell (NKT).

26. The method according to any one of claims 22-25, wherein the immune cell comprises an αβ T cell and / or a γδ T cell.

27. The method according to any one of claims 22-26, wherein the immune cell comprises tumor-infiltrating lymphocytes (TIL).

28. The method according to claim 27, wherein the TIL is TIL derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis foci, fragments of tissue adjacent to cancer, pleural effusion and / or ascites, and / or TIL derived from TIL recovered after cryopreservation.

29. A system for identifying a target, the system comprising: a determination module for determining the effect of a candidate target on the activation level of a test cell after the test cell is substantially deprived of its killing ability, wherein the expression or activity of the candidate target in the test cell is regulated before and / or simultaneously with the determination.

30. A system for identifying a target, the system comprising: Module (1) for introducing components of a target regulation system selected from a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system, and / or an inhibitory RNA into a test cell simultaneously or sequentially, the target regulation system increasing or decreasing the expression or activity of a candidate target; Module (1-a) for causing the test cell to express a receptor that recognizes the tumor antigen; Module (2) for co-culturing a test cell (a cell that has been or is about to have its candidate target regulated) with a tumor antigen or a tumor cell expressing the tumor antigen, such that the test cell is substantially deprived of its killing ability; Module (3) for measuring the levels of GZMB, IFN-γ, TNF-α, and / or CD107a expressed by the test cell; Module (4) for determining the effect of regulating the candidate target on the cell based on the reading results obtained from the measurement; wherein there is no order restriction among Module (1), Module (1-a), and Module (2), and Module (1), Module (1-a), and Module (2) are optionally arranged in parallel. 3l. A system for identifying a target, the system comprising: Module (1) for introducing components of a target regulation system selected from a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system, and / or an inhibitory RNA into a test cell simultaneously or sequentially, the target regulation system increasing or decreasing the expression or activity of a candidate target; Module (1-a) for causing the test cell to express an exogenous cell receptor or a functional fragment thereof; Module (2) for co-culturing a test cell (a cell that has been or is about to have its candidate target regulated) with a specific antigen of the exogenous cell receptor or a functional fragment thereof or a cell expressing the specific antigen, such that the test cell is substantially deprived of its killing ability; Module (3) for measuring the levels of GZMB, IFN-γ, TNF-α, and / or CD107a expressed by the test cell; Module (4) for determining the effect of regulating the candidate target on the cell based on the reading results obtained from the measurement; wherein there is no order restriction among Module (1), Module (1-a), and Module (2), and Module (1), Module (1-a), and Module (2) are optionally arranged in parallel.

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