Method for identifying target and use thereof

Abstract

Provided is a method for identifying a target, which method specifically comprising adjusting the expression or activity of a candidate target in a cell to be detected, bringing the cell to be detected into contact with an identification model for two periods of time, and determining the existence and / or number of a target adjusting system within a target tissue in the identification model during the first period of time and the second period of time. Further provided 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] The present invention discovered that current immune cell target screening systems are relatively simple and fail to accurately simulate the challenges faced by cell therapy products. Furthermore, systemic imperfections in the screening process can lead to a high rate of false-positive targets or miss important targets. The present invention also discovered that insufficient T cell tumor infiltration and an immunosuppressive tumor microenvironment, which allow tumor cells to escape the immune system and lead to tumor progression, are the main bottlenecks for cell therapy products in solid tumors.

[0004] Therefore, the present invention provides a novel target identification method that improves screening accuracy in a system for screening key targets and / or novel targets that regulate T cell function. The present invention also provides a method for screening and identifying key targets and / or novel targets that regulate T cell function in cancer (e.g., a specific cancer).

[0005] 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 by a target regulatory system; (2) contacting the test cell with an identification model for a first time period, and then determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; (3) contacting the test cell with the identification model for a second time period, and then determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; and (4) determining the effect of regulating the candidate target on the cell by reading the result obtained by the determination, thereby identifying the target.

[0006] On the other hand, the present invention also provides a system for identifying targets, which comprises: a measurement module, which allows the cells to be tested to contact the identification model for two time periods, and measures the presence and / or quantity of the target regulatory system in the target tissue in the identification model in the first time period and the second time period, and regulates the expression or activity of the candidate target in the cells to be tested before and / or simultaneously with the measurement.

[0007] In order to solve the existing technical problems, the present invention has designed a target gene screening process, which adopts a two-stage screening analysis to solve the problems of cell therapy products such as short in vivo persistence, limited infiltration, easy exhaustion and susceptibility to inhibition by the tumor microenvironment. The key targets and / or new targets found by the present invention can improve the survival and killing function of T cell products in the body.

[0008] Therefore, the present invention provides a method for identifying a target, which comprises the following steps: providing a cell to be tested, regulating the expression or activity of a candidate target in the cell to be tested, and contacting the cell to be tested with an identification model for one or two time periods, and determining the presence and / or amount of the target regulatory system in the target tissue in the identification model during the first time period and the optional second time period, thereby identifying the target.

[0009] In some embodiments, the method comprises the following steps: (1) providing a cell to be tested; (2) regulating the expression or activity of a candidate target in the cell to be tested by a target regulatory system; (3) after the cell to be tested is contacted with an identification model for a first time period, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; (4) optionally, after the cell to be tested is contacted with the identification model for a second time period, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; and (5) determining the effect of regulating the candidate target on the cell by reading the results obtained by the determination, thereby identifying the candidate target.

[0010] In some embodiments, the cells to be tested are immune cells.

[0011] In some embodiments, the cell to be tested is a T cell of a human or animal.

[0012] In some embodiments, the identification model comprises a non-human mammal.

[0013] In some embodiments, the target tissue comprises the spleen and / or a tumor.

[0014] In some embodiments, contacting the test cell with the identification model comprises administering the test cell to the non-human mammal by intravenous infusion.

[0015] In some embodiments, the identification model and the cells to be tested comprise a pair of molecules that can specifically bind to each other.

[0016] In some embodiments, the identification model comprises cells expressing tumor-specific antigen molecules, and the cells to be tested express receptors that can specifically bind to the tumor-specific antigen molecules.

[0017] In some embodiments, the method comprises contacting the test cell with the identification model for about 12 hours to 7 days, resulting in the target tissue being contacted with the identification model for a first period of time.

[0018] In some embodiments, the method comprises contacting the test cell with the identification model for about 7 to 14 days, resulting in the target tissue being contacted with the identification model for a second period of time.

[0019] In some embodiments, the target regulatory system comprises a target regulatory molecule that specifically recognizes the target sequence.

[0020] In some embodiments, the modulation step comprises separately introducing components of a target modulation system into the cells to be tested, wherein the target modulation system increases or decreases the expression or activity of the candidate target.

[0021] In some embodiments, the modulating step comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system, and / or an inhibitory RNA.

[0022] In some embodiments, the modulation step comprises targeting a specific nucleic acid sequence of the candidate target, introducing single-strand breaks, double-strand breaks, mutations and / or expression regulation upstream, upstream and / or downstream of the specific nucleic acid sequence.

[0023] In some embodiments, the modulating step comprises increasing or decreasing the expression or activity of the candidate target at the genomic level.

[0024] In some embodiments, the regulating step comprises introducing a target regulatory system comprising a guide nucleic acid molecule and a nuclease into the test cell.

[0025] In some embodiments, the guide nucleic acid molecule comprises a guide RNA (gRNA) that targets the target.

[0026] In some embodiments, the nuclease comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

[0027] In some embodiments, the nuclease comprises Cas 9 and / or Cas 12.

[0028] In some embodiments, in the test cell population obtained by the adjusting step, the proportion of cells expressing the candidate target is reduced to less than about 95%.

[0029] In some embodiments, the determining step comprises isolating immune cells from the target tissue and then determining the presence and / or amount of gRNA in the immune cells.

[0030] In some embodiments, the determining step comprises isolating immune cells from the spleen and / or the tumor, and then determining the presence and / or amount of gRNA in the immune cells.

[0031] In some embodiments, for a gRNA that is enriched in the first time period assay or in both the first time period assay and the second time period assay, the target gene corresponding to the gRNA is identified as a target with regulatory function.

[0032] In some embodiments, the determining step comprises determining the presence and / or amount of the target regulatory system in the target tissue in the identification model by gene sequencing.

[0033] In some embodiments, the presence and / or amount of the target regulatory system is determined by measuring the presence and / or amount of the target regulatory system in cells tested within a tissue of interest in an identification model.

[0034] In some embodiments, the cells to be tested comprise immune cells, such as phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0035] In some embodiments, the immune cells comprise monocytes, macrophages, and / or dendritic cells.

[0036] In some embodiments, the immune cells comprise B cells, T cells, natural killer cells, regulatory T cells, and / or natural killer-like T cells (NKT).

[0037] In some embodiments, the immune cells comprise αβ T cells and / or γδ T cells.

[0038] In some embodiments, the immune cells comprise tumor infiltrating lymphocytes (TILs).

[0039] In some embodiments, the TILs are TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and / or ascites, and / or TILs revived after cryopreservation.

[0040] The present invention also provides a method for identifying tumor-related targets, which comprises: (1) providing a test cell for targeted contact with a tumor identification model; (2) regulating the expression or activity of a candidate target in the test cell by a target regulation system; (3) after the test cell is contacted with the tumor identification model for a first time period, determining the presence and / or amount of the target regulation system in the target tissue in the identification model; (4) optionally, after the test cell is contacted with the tumor identification model for a second time period, determining the presence and / or amount of the target regulation system in the target tissue in the tumor identification model; and (5) identifying the candidate target by reading the results obtained by the determination.

[0041] In some embodiments, the tumor identification model is a tumor-bearing mouse or its tissue cells.

[0042] The present invention also provides a system for identifying targets, which comprises: a measurement module, which allows the cells to be tested to contact the identification model for two time periods, and measures the presence and / or quantity of the target regulatory system in the target tissue in the identification model in the first time period and the second time period, and regulates the expression or activity of the candidate target in the cells to be tested before and / or simultaneously with the measurement.

[0043] The present invention also provides a system for identifying a target, the system comprising: a module (1) for simultaneously or sequentially introducing components of a target regulation system selected from a CRISPR system, a zinc finger nuclease system, a TALEN system, a large-range nuclease 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 a candidate target; a module (2) for contacting the cell to be tested (a cell that has been or is about to be regulated with a candidate target) with an identification model for a first time period, and determining the presence and / or quantity of the target regulation system in the target tissue in the identification model; a module (3) for contacting the cell to be tested (a cell that has been or is about to be regulated with a candidate target) with the identification model for a second time period, and determining the presence and / or quantity of the target regulation system in the target tissue in the identification model; and a module (4) for determining the effect of regulating the candidate target on the cell through a reading result obtained by the determination; wherein there is no order restriction between modules (2) and (3), and modules (2) and (3) are optionally arranged in parallel. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The features and advantages of the present invention can be better understood by referring to the accompanying drawings, which are briefly described as follows:

[0045] FIG1 shows an exemplary screening flow chart of the present invention;

[0046] FIG2 shows the results of the multi-time point analysis panel screening of various gene targets for anti-depletion ability and pro-invasion ability;

[0047] Figure 3 shows the screening results of various gene targets in the pro-invasion analysis panel;

[0048] Figure 4 shows the screening results of each gene target in the anti-depletion analysis panel; and

[0049] FIG5 shows the screening results of various gene targets of the multi-time point analysis panel of the present invention. DETAILED DESCRIPTION

[0050] The following describes the embodiments of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0051] Definition of terms

[0052] In the present invention, the term "target" generally refers to a target (e.g., a target gene, target gene or target gene). For example, a target gene is obtained from a candidate gene through screening or identification. Depending on the results of the screening or identification, the protein encoded by the "candidate gene" or "target 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 is transcribed into RNA or translated into a polypeptide in vitro or in vivo when placed under the control of a suitable regulatory or control sequence. In some embodiments, the target is a target gene. In some embodiments, the target is a key target and / or a new target with a T cell function regulatory effect. In some embodiments, the target is a key target and / or a new target with a function of promoting T cell tumor infiltration. In some embodiments, the target is a key target and / or a new target with a function of promoting T cell anti-exhaustion.

[0053] In the present invention, the term "identification model" generally refers to a system for identifying a target (referred to as "identification system", which is synonymous with "identification model" and is used interchangeably). For example, the identification model of the present invention can be in vitro, ex vivo or in vivo. For example, the identification model of the present invention can include all or part of the functions of the circulatory system. For example, the identification model of the present invention can simulate the process of the cells to be tested approaching the target tissue. For example, the identification model of the present invention can be a non-human animal, for example, a non-human primate, such as an ape or monkey, and livestock, such as a dog, rabbit, cow, pig, sheep, goat, horse or donkey. For example, the identification model of the present invention can be a cow, a bull, a bison, a buffalo, a pig, a bighorn sheep, a horse, a mule, a deer, an elk, a llama, or an alpaca. For example, the identification model of the present invention can be a rodent, such as a mouse or a rat.

[0054] In the present invention, the term "target tissue" generally refers to a target, site, or biological tissue that is desired to be targeted by immune cells. For example, the target tissue of the present invention can be a target or site to be treated with cell therapy. For example, the tissue of the present invention can include biological tissue in vitro, ex vivo, or in vivo. For example, the tissue of the present invention can include biological tissue artificially induced.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0063] 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.

[0064] Detailed Description of the Invention

[0065] The present invention designs a target gene screening process to improve the screening accuracy in the system of screening key targets and / or new targets with T cell function regulation. The method of the present invention is proposed based on the present invention's discovery that in the screening process, the imperfection of the system may lead to a high proportion of false positive targets or miss important targets. The present invention also found that insufficient T cell tumor infiltration and the immunosuppressive tumor microenvironment enable tumor cells to achieve immune escape, leading to tumor progression, which is the main bottleneck encountered by cell therapy products in solid tumors.

[0066] Therefore, in one aspect, the present invention provides a method for identifying a target, the method comprising the steps of regulating the expression or activity of a candidate target in a cell to be tested, and contacting the cell to be tested with an identification model for one or two time periods, and determining the presence and / or amount of a target regulatory system in a target tissue in the identification model during a first time period and an optional second time period. For example, the present invention determines the presence and / or amount of a target regulatory system in a target tissue in the identification model during a first time period. For example, the present invention determines the presence and / or amount of a target regulatory system in a target tissue in the identification model during a first time period and an optional second time period.

[0067] In another aspect, the present invention provides a method for identifying a target, comprising the steps of: (1) regulating the expression or activity of a candidate target in a test cell by a target regulatory system; (2) contacting the test cell with an identification model for a first period of time, and then determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; (3) optionally, contacting the test cell with the identification model for a second period of time, and then determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; and (4) determining the effect of regulating the candidate target on the cell by reading the result obtained by the determination. In some embodiments, the test cell is a cell in which the expression or activity of the candidate target is regulated by the target regulatory system.

[0068] Figure 1 shows an exemplary screening method according to the present invention. In this screening process, mice expressing specific TCRs with transgenic genes (e.g., mice expressing specific OT-1 receptors with Cas9-positive transgenic genes) are used as donors to isolate the T cells to be tested. The gRNA screening library designed and constructed is then transferred to the target regulation system to construct a screening library knockout cell library, thereby regulating the expression or activity of the candidate targets in the cells to be tested. In the process of in vivo target screening, Cas9+ transgenic mice are used as recipient mice to construct specific tumor-bearing mice (e.g., inoculated with OVA tumor cells that can be specifically recognized by OT-1) as an identification model. The cells to be tested in the knockout cell library of the above-mentioned screening library are re-infused. After the first and / or second periods of contact, the presence and / or quantity of the target regulatory system in the target tissue in the identification model is determined (for example, the spleen and tumor of the recipient mouse are harvested, and the TIL cells in the tumor and T cells in the spleen of the mouse are separated, their genomes are extracted and separated, and the enrichment of gRNA sequences is captured and compared by high-throughput sequencing and bioinformatics analysis), thereby discovering targets (target genes) that are potentially related to the anti-tumor function of T cells (for example, enhancing T cell tumor infiltration and anti-exhaustion).

[0069] Another exemplary screening method utilizes a screening platform based on human T cells. In this screening platform, human T cells (e.g., human PBMC T cells) are used as test cells. A designed and constructed gRNA screening library is introduced to construct a knockout cell library, thereby modulating the expression or activity of candidate targets in the test cells. During in vivo target screening, immunodeficient mice (e.g., NOG tumor-bearing mice) are used to construct specific tumor-bearing mice (e.g., inoculated with cells carrying NYESO1 antigens specifically recognized by NYESO1-TCRs) as an identification model. Test cells from the knockout cell library are then infused back into the test cells. After the first and / or second periods of contact, the presence and / or quantity of the target regulatory system in the target tissue in the identification model is determined (for example, the spleen and tumor of the recipient mouse are harvested, and the TIL cells in the tumor and T cells in the spleen of the mouse are separated, their genomes are extracted and separated, and the enrichment of gRNA sequences is captured and compared by high-throughput sequencing and bioinformatics analysis), thereby discovering targets (target genes) that are potentially related to the anti-tumor function of T cells (for example, enhancing T cell tumor infiltration and anti-exhaustion).

[0070] In some embodiments, the identification model and the cells to be tested comprise a pair of molecules that can specifically bind. For example, the identification model comprises cells expressing tumor-specific antigen molecules, and the cells to be tested express receptors that can specifically bind to tumor-specific antigen molecules. In some embodiments, the identification model comprises cells expressing tumor-specific antigen molecules, and the cells to be tested express T cell receptors (TCRs) that can specifically bind to tumor-specific antigen molecules. In some embodiments, contact of the cells to be tested with the identification model comprises reinfusion of the cells to be tested in the screening library knockout cell bank to tumor-bearing mice (or contact with tissues or cells of tumor-bearing mice). In some embodiments, after the cells to be tested (e.g., T cells) are reinfused into tumor-bearing mice, the TCR specifically expressed by the T cells specifically recognizes the surface antigens of tumor cells in the tumor-bearing mice, thereby targetedly entering the tumor area. In some embodiments, through the design of the specific binding molecule pairs (specific recognition of TCR and tumor antigens), the methods described herein can be applied to identify targets (target genes) associated with T cell anti-tumor function (e.g., enhancing T cell tumor infiltration and anti-exhaustion) in specific cancers.

[0071] For example, in the identification method of the present invention, the presence and / or amount of the target regulatory system in the target tissue of the identification model is determined after the test cells are contacted with the identification model for about 12 hours to 7 days.

[0072] For example, the identification model described in the present invention comprises a non-human mammal. For example, the identification model of the present invention may comprise an in vitro, ex vivo, and / or in vivo model. For example, the identification model of the present invention may comprise all or part of the functions of the circulatory system. For example, the identification model of the present invention may simulate the process of the cells to be tested approaching and / or entering the target tissue. For example, the target tissue of the present invention comprises the spleen and / or tumor. For example, the target tissue of the present invention comprises tissue that can be targeted by immune cells. For example, the target tissue of the present invention comprises tumor tissue. For example, contacting the cells to be tested with the identification model of the present invention comprises administering the cells to be tested to the non-human mammal by intravenous reinfusion. In some embodiments, the identification model is a transgenic mouse. In some embodiments, the identification model is the spleen and / or tumor tissue of the animal model.

[0073] For example, in the present invention, the cells to be tested are contacted with the identification model for about 12 hours to 7 days to obtain the target tissue of the identification model in contact with the first time period. For example, in the present invention, the cells to be tested are contacted with the identification model for about 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, or 7 days to obtain the target tissue of the identification model in contact with the first time period. In some embodiments, after the cells to be tested are contacted with the identification model for about 12 hours to 7 days (e.g., 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days), the presence and / or quantity of the target regulatory system in the target tissue in the identification model is determined to thereby discover potential targets (target genes) associated with enhancing T cell tumor infiltration. In some embodiments, the cells to be tested are contacted with the identification model for about 2 days to thereby discover potential targets (target genes) associated with enhancing T cell tumor infiltration.

[0074] For example, in the present invention, the cells to be tested are contacted with the identification model for about 7 to 14 days to obtain the target tissue of the identification model in contact with the second time period. For example, in the present invention, the cells to be tested are contacted with the identification model for about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days to obtain the target tissue of the identification model in contact with the second time period. In some embodiments, after the cells to be tested are contacted with the identification model for about 7 to 14 days (for example, about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days), the presence and / or quantity of the target regulatory system in the target tissue in the identification model is determined to thereby discover potential targets (target genes) associated with enhancing T cell anti-exhaustion. In some embodiments, the cells to be tested are contacted with the identification model for about 7 days to thereby discover potential targets (target genes) associated with enhancing T cell anti-exhaustion.

[0075] For example, in the identification method of the present invention, the cells to be tested are contacted with the identification model for two or more time periods. For example, the identification model in the present invention is a mammalian model other than humans. For example, in the identification method of the present invention, the cells to be tested are contacted with mice for two or more time periods, and the presence and / or quantity of the target regulatory system in the target tissue in the identification model is determined. For example, in the identification method of the present invention, the mice in a test group are divided into two or more batches, wherein the cells to be tested are contacted with the first batch of mice for about 12 hours to 7 days before being measured, and the cells to be tested are contacted with the second batch of mice for about 7 days or more before being measured. For example, in the identification method of the present invention, the same mouse has two or more target tissues, wherein the cells to be tested are contacted with mice for about 12 hours to 7 days for the first time to measure one target tissue, and the cells to be tested are contacted with the second batch of mice for about 7 days or more for the second time to measure another target tissue. For example, in the identification method of the present invention, the same mouse has tumor tissue and / or spleen, wherein the test cells are placed in contact with the mouse for about 12 hours to 7 days to perform a first portion of tumor tissue and / or spleen measurement, and the test cells are placed in contact with a second batch of mice for about 7 days or more to perform a second portion of tumor tissue and / or spleen measurement.

[0076] For example, the target regulatory system of the present invention comprises a target regulatory molecule that specifically recognizes the target sequence. For example, the target regulatory system of the present invention comprises a gRNA in a CRISPR system. For example, determining the presence and / or amount of the target regulatory system may comprise determining the presence and / or amount of the gRNA in tumor tissue and / or spleen.

[0077] For example, the cells to be tested express a receptor that recognizes the target tissue in the identification model. For example, the cells to be tested express a receptor that recognizes the tumor antigen, and the tumor antigen is expressed in the tumor tissue of the identification model. For example, before or simultaneously with the determination, the cells are caused to express a cell receptor or a functional fragment thereof. For example, the cells to be tested in the present invention are derived from an animal model, and the immune cells of the animal model can express a receptor that can recognize the target tissue in the identification model. For example, the cell receptor in the present invention comprises a T cell receptor or an antigen-binding fragment thereof.

[0078] For example, the regulating step described in the present invention comprises introducing the components of the target regulatory system into the cells to be tested, respectively, and the target regulatory system increases or decreases the expression or activity of the candidate target. For example, the regulating step described in the present invention comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a large range of nuclease systems and / or inhibitory RNA. For example, the regulating step described in the present invention comprises targeting a specific nucleic acid sequence of the candidate target, and introducing single-strand breaks, double-strand breaks, mutations and / or expression regulation in the middle, upstream and / or downstream of the specific nucleic acid sequence. For example, the target regulatory system may include elements that increase gene expression, such as a CRISPRa system. For example, the target regulatory system may include elements that reduce gene expression, such as a CRISPRi system.

[0079] For example, the regulating step in the present invention comprises increasing or decreasing the expression or activity of the candidate target at the genomic level. For example, the regulating step in the present invention comprises introducing a target regulation system comprising a guide nucleic acid molecule and a nuclease into the test cell. For example, the guide nucleic acid molecule in the present invention comprises a guide RNA (gRNA) targeting the target. For example, the nuclease in the present invention comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the nuclease in the present invention comprises Cas 9 and / or Cas 12.

[0080] For example, the regulating step described in the present invention comprises 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 regulating step described 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 repeats (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.

[0081] For example, the regulating step 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 is 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 regulating step 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.

[0082] 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.

[0083] For example, the regulating step of the present invention comprises targeting a specific nucleic acid sequence of the target, introducing single-strand breaks, double-strand breaks and / or mutations midstream, upstream and / or downstream of the nucleic acid sequence. For example, the regulating step 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] For example, the regulation step of the present invention 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 vector 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 vector 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 the 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 of the present invention comprises a guide RNA (gRNA). For example, the nuclease of the present invention comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof. For example, the nuclease of the present invention 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] For example, wherein the regulating step comprises targeting a specific nucleic acid sequence of the target, introducing single-strand breaks, double-strand breaks and / or mutations in the middle, upstream and / or downstream of the specific nucleic acid sequence. For example, wherein the regulating step comprises increasing or decreasing the expression or activity of the candidate target at the genomic level. For example, wherein the regulating step 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.

[0101] For example, in the cell population obtained in the regulating step of the present invention, the proportion of cells expressing the candidate target is reduced to less than about 95%. For example, it can be reduced to about 95-1%, such as about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, or about 1%.

[0102] 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.

[0103] For example, in the present invention, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model comprises isolating the immune cells in the target tissue and then determining the presence and / or amount of the gRNA in the immune cells. For example, in the present invention, determining the presence and / or amount of the target regulatory system in the spleen and / or tumor in the identification model comprises isolating the immune cells in the spleen and / or the tumor and then determining the presence and / or amount of the gRNA in the immune cells.

[0104] For example, the presence and / or amount of the gRNA in the screening panel in which the candidate target is edited is increased or decreased by about 0.001% or more, such as about 0.001% to about 10,000-fold, for example, about 0.001%, about 0.005%, about 0.01%, about 0.05%, 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%, or more, compared to a control group in which the candidate target is not edited. , about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, 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 the corresponding candidate target as a target that can affect immune cells. For example, compared to a control group in which the candidate target is not edited, a candidate target corresponding to the absence of a gRNA in the screening group in which the candidate target is edited (i.e., the number of gRNAs is reduced to about 0) in the tumor can be used as a target that can affect immune cells.

[0105] For example, optionally, the presence and / or amount of the gRNA in the screening panel for editing the candidate target in the tumor is increased by about 0.001% or more, e.g., about 0.001% to about 10,000-fold, e.g., about 0.001%, about 0.005%, about 0.01%, about 0.05%, 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 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 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78 %, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, 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 the corresponding candidate target as a target that can significantly affect immune cells in tumor tissue.

[0106] For example, the enriched guide RNA corresponding to the enriched cells after screening can be statistically processed. 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.

[0107] For example, for a gRNA that is enriched in the first time period or in both the first time period and the second time period in the present invention, the target gene corresponding to the gRNA is identified as a target with regulatory function. For example, the determination in the present invention includes determining the presence and / or amount of the target regulatory system in the target tissue in the identification model by gene sequencing.

[0108] For example, in the present invention, the cells to be tested are contacted with mice for about 12 hours to 7 days to obtain tumors in the mice contacted for the first time period; the candidate target corresponding to the presence and / or quantity of gRNA in the screening group in which the candidate target is edited in the tumor is increased by 0.3 times compared to the control group in which the candidate target is not edited is used as a target that can affect immune cells.

[0109] For example, in the present invention, the cells to be tested are contacted with mice for about 7 to 14 days to obtain tumors in the mice contacted for a second time period; the candidate target corresponding to the presence and / or quantity of gRNA in the screening group in which the candidate target is edited in the tumor is increased by 0.5 times compared to the control group in which the candidate target is not edited is used as a target that can affect immune cells.

[0110] For example, the presence and / or quantity of gRNA in the screening group edited by the candidate target in the mouse tumor of the first time period is increased by 0.3 times the corresponding candidate target, as a target that can affect immune cells.For example, the presence and / or quantity of gRNA in the screening group edited by the candidate target in the mouse tumor of the first time period is increased by 0.3 times and the presence and / or quantity of gRNA in the screening group edited by the candidate target in the mouse tumor of the second time period is increased by 0.5 times, as a target that can affect immune cells.In some embodiments, the presence and / or quantity of gRNA in the screening group edited by the candidate target in the identification model (e.g., mouse) tumor in a specific time period (e.g., the first time period or the second time period) is increased to a greater extent than the presence and / or quantity of gRNA in the screening group edited by the candidate target in the spleen of the identification model (e.g., mouse) in the same time period.

[0111] In some embodiments, the presence and / or amount of the target regulatory system is determined by the presence and / or amount of the target regulatory system in the test cells within the target tissue in the identification model. For example, the test cells 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 (iPSCs), 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 (TILs). 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.

[0112] Those skilled in the art are familiar with the definition of "tumor". For example, tumors or cancers (used interchangeably herein) can include breast cancer, ovarian cancer, uterine cancer, cervical cancer, brain tumor, thyroid cancer, esophageal cancer, lung cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, colorectal cancer, bladder cancer, leukemia, lymphoma, melanoma, prostate cancer, testicular cancer and penile cancer, etc.

[0113] Also provided herein are methods for diagnosing and treating cancer. For example, targets identified by the methods described herein can be used as cancer-specific biomarkers for diagnosing and treating cancer. In some embodiments, the present invention provides a method for treating cancer, comprising detecting a cancer-specific target in a subject and administering an inhibitor of the specific target to the subject. In some embodiments, the treatment methods described herein can be combined with other treatment methods known in the art.

[0114] On the other hand, the present invention provides a system for identifying targets, which comprises: a measurement module, which contacts the cells to be tested with the identification model for two time periods, measures the presence and / or amount of the target regulatory system in the target tissue in the identification model in the first time period and the second time period, and regulates the expression or activity of the candidate target in the cells to be tested before and / or simultaneously with the measurement.

[0115] In another aspect, the present invention provides a method for identifying a target, the method comprising:

[0116] 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;

[0117] Step (2) contacting the test cells (cells that have been or are about to be regulated by the candidate target) with the identification model for a first period of time, and determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; for example, in the present invention, the test cells are contacted with the identification model for about 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, or 7 days to obtain the target tissue of the identification model that has been contacted for the first period of time;

[0118] Optional step (3) is to contact the test cells (cells that have been or are about to be regulated by the candidate target) with the identification model for a second time period, and determine the presence and / or amount of the target regulatory system in the target tissue of the identification model; for example, in the present invention, the test cells are contacted with the identification model for about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days to obtain the target tissue of the identification model that has been contacted for the second time period;

[0119] Step (4), determining the effect of regulating the candidate target on the cell by reading the results obtained by the assay;

[0120] There is no restriction on the order of step (2) and step (3), and step (2) and step (3) can be optionally performed simultaneously.

[0121] In another aspect, the present invention provides a system for identifying a target, the system comprising:

[0122] 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 a candidate target;

[0123] Module (2) is used to contact the test cells (cells that have been or are about to be regulated by the candidate target) with the identification model for a first time period, and determine the presence and / or amount of the target regulatory system in the target tissue in the identification model; for example, in the present invention, the test cells are contacted with the identification model for about 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, or 7 days to obtain the target tissue of the identification model that has been contacted for the first time period;

[0124] Optional module (3) for contacting the test cells (cells that have been or are about to be regulated by the candidate target) with the identification model for a second time period to determine the presence and / or amount of the target regulatory system in the target tissue in the identification model; for example, in the present invention, the test cells are contacted with the identification model for about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days to obtain the target tissue of the identification model contacted for the second time period;

[0125] Module (4) is used to determine the effect of regulating the candidate target on the cell by reading the results obtained by the assay;

[0126] There is no restriction on the order of modules (2) and modules (3), and modules (2) and modules (3) can be optionally arranged in parallel.

[0127] 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.

[0128] 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.

[0129] Example

[0130] Example 1 Screening Platform Using Transgenic Mice as Donors Screening Process

[0131] Figure 1 shows an example screening flow chart of the present invention. In this screening process, mice expressing specific TCRs with transgenic genes (e.g., mice expressing specific OT-1 receptors with Cas9-positive transgenic genes) are used as donors to isolate their T cells, which are then transferred to a designed and constructed gRNA screening library to construct a screening library knockout cell library. In vivo screening uses Cas9+ transgenic mice as recipient mice to construct specific tumor-bearing mice (e.g., inoculated with OVA tumor cells that can be specifically recognized by OT-1). The cells in the above-mentioned screening library knockout cell library are re-infused. After a period of time, the spleens and tumors of the recipient mice are harvested, and the TIL cells in the tumors and the T cells in the spleens of the mice are isolated, their genomes are extracted and separated, and the enrichment of gRNA sequences is captured and compared by high-throughput sequencing and bioinformatics analysis, thereby discovering target genes with potential T cell anti-tumor functions (e.g., enhancing T cell tumor infiltration and anti-exhaustion).

[0132] In vivo screening library plasmid construction

[0133] The size of the screening library designed by the present invention is a few tenths to a few hundredths of the number of T cells carrying gRNA (guide RNA) that can be harvested in the mouse model, so as to avoid the loss of some gRNA due to random reasons. The screening library of the present invention can adopt a target gene library, for example, a library designed by big data and / or literature analysis and prediction of possible immune regulatory genes, and add a non-targeted or non-functional region-targeted control gRNA as a quality control parameter. High-throughput sequencing detected that its gRNA coverage was 100%. Knockout experiments showed that the transduction efficiency of gRNA virus in mouse spleen T cells can reach more than 60%, and the knockout efficiency of transduction-positive Cas9+T cells for target proteins can reach more than 90%.

[0134] Then, 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 a designed retroviral vector using the Gibson kit. The vector also carries a sequence encoding a fluorescent protein, such as VEX fluorescent protein, which can be used to indicate the transduction and expression of the subsequent library in T cells and can be used to enrich transduced cells. The assembled library plasmid is electroporated into Endura competent cells, cultured overnight to amplify the library plasmid, and then the plasmid is extracted to prepare the library plasmid. High-throughput NGS sequencing is used to control the quality of the prepared library plasmid and detect the coverage and distribution of gRNA.

[0135] Screening library knockout cell library construction

[0136] The library virus is packaged in HEK293T cells using retrovirus from the in vivo screening library. This library virus is then transduced into splenic T cells of transgenic mice carrying Cas9, achieving a transduction efficiency greater than 30%, for example, controlled at 50-60%. After transduction, enrichment is no longer necessary before reinfusion into the mice. Downstream bioinformatics analysis confirms that the increased transduction efficiency, resulting in certain double or multiple knockouts, does not affect analytical results, allowing key functional genes to be identified.

[0137] In vivo screening model construction and screening process

[0138] In vivo screening uses Cas9+ transgenic mice as recipients to prevent immune rejection after infusion of Cas9+ T cells. Cas9+OT-1+ bi-positive mice (Cas9-expressing screening group) and Cas9-OT-1+ mice (Cas9-non-expressing control group) serve as donors. Spleens are harvested to purify CD8+ T cells and transduce them with the library-carrying virus. In the screening group donor mice, endogenous Cas9 enables editing of the target gene after transduction with the library virus, whereas the control group donor mice lack Cas9, eliminating the possibility of gRNA accumulation or loss due to accidental factors.

[0139] Cas9+ recipient mice were inoculated with tumor cells carrying the antigen OVA (specifically recognized by the TCR of OT-1 cells). After the tumors grew stably to a certain size, the edited CD8+ T cells from the two groups of donor mice (screening group and control group) were reinfused through the tail vein.

[0140] From 12 hours to 7 days after the reinfusion of the above-mentioned edited T cells, for example, on the 2nd day, the spleen and tumor of the recipient mouse are harvested, and the TIL cells and T cells in the spleen of the mouse are separated, and their genomes are extracted and isolated. The gRNA sequences enriched in the screening group expressing Cas9 in the tumor and the control group not expressing Cas9 are captured and compared through high-throughput sequencing and bioinformatics analysis, thereby discovering target genes that potentially enhance T cell tumor infiltration.

[0141] From the 7th to the 14th day after the reinfusion of the above-mentioned edited T cells, for example, on the 7th day, the spleen and tumor of the recipient mouse are harvested, and the TIL cells and T cells in the spleen of the mouse are separated, and their genomes are extracted and isolated. The gRNA sequences enriched in the screening group expressing Cas9 in the tumor and the control group not expressing Cas9 are captured and compared by high-throughput sequencing and bioinformatics analysis, thereby discovering target genes that have the potential to enhance the tumor-killing function of T cells in vivo and / or have resistance to exhaustion.

[0142] Example 2 Analysis of in vivo screening accuracy results

[0143] Combined with the multi-time-point sample collection and sequencing scheme proposed and designed by the present invention, the present invention can solve the problem of a high proportion of false positive targets in existing screening systems.

[0144] Analysis of screening accuracy of sequencing collected at different time points

[0145] Pro-infiltration analysis group: From 12 hours to 7 days after the reinfusion of the above-mentioned edited T cells (day 2 in this experiment), the spleen and tumor of the recipient mice were harvested, and the TIL cells and T cells in the spleen of the mice were isolated, and the enriched gRNA sequences were sequenced.

[0146] Anti-exhaustion analysis group: From the 7th to the 14th day after the reinfusion of the above-mentioned edited T cells (day 7 in this experiment), the spleen and tumor of the recipient mice were harvested, and the TIL cells and T cells in the spleen of the mice were isolated, and the enriched gRNA sequences were sequenced.

[0147] Multi-time point analysis group of the present invention: from 12 hours to 7 days (day 2 in this experiment) after the reinfusion of the above-mentioned edited T cells, and from 7 days to 14 days (day 7 in this experiment) after the reinfusion of the above-mentioned edited T cells, the spleens and tumors of the recipient mice were harvested, and the TIL cells and T cells in the spleen of the mice were isolated, and the enriched gRNA sequences were sequenced.

[0148] Figure 2 shows the results of the multi-time point analysis panel screening of various gene targets for anti-exhaustion and pro-invasive abilities. Each point in the figure represents the analyzed target gene; the horizontal axis represents the fold increase in gRNA analyzed on day 2 in the Cas9-expressing screening group with target gene knockout compared to the Cas9-non-expressing control group; the vertical axis represents the fold increase in gRNA analyzed on day 7 in the Cas9-expressing screening group with target gene knockout compared to the Cas9-non-expressing control group.

[0149] Figure 3 shows the screening results of each gene target in the pro-infiltration analysis group. Among them, each point in the figure represents the target gene analyzed. Among them, the data on the second day were selected as the basis for analysis reference, and the genes with a pro-infiltration increase of more than 0.3 times were used as the target genes obtained by screening. Among them, among the 49 genes identified in this experiment, 18 genes (darkened points) were verified to have enhanced in vivo anti-tumor ability of immune cells after knockout. The above results show that the target identification method of the present invention has an identification accuracy of at least 36.7% in the pro-infiltration analysis group.

[0150] Figure 4 shows the screening results of each gene target in the anti-depletion analysis group. Among them, each point in the figure represents the target gene analyzed. Among them, the data on the 7th day was selected as the basis for analysis reference, and the genes with an anti-depletion increase multiple higher than 0.5 times were used as the screened genes. Among them, among the 34 genes identified in this experiment, 19 genes (darkened points) were verified to have enhanced in vivo anti-tumor ability of immune cells after knockout. The above results prove that the target identification method of the present invention has an identification accuracy of at least 55.9% in the anti-depletion analysis group.

[0151] Figure 5 shows the screening results of each gene target of the multi-time point analysis group of the present invention. Among them, each point in the figure represents the target gene analyzed. Among them, the data on the 2nd day and the 7th day were selected as the basis for analysis reference, and the genes with a pro-infiltration increase multiple higher than 0.3 times and an anti-depletion increase multiple higher than 0.5 times were used as the screened genes. Among them, all 6 genes identified in this experiment have been verified to have enhanced in vivo anti-tumor ability of immune cells after knockout. The above results prove that the target identification method of the present invention has an identification accuracy of 100% in the pro-infiltration and anti-depletion analysis groups.

[0152] For example, the genes screened through multi-time point analysis of the present invention, after being knocked out in immune cells, have improved anti-tumor ability in vivo compared to unedited immune cells.

[0153] The above results show that the infiltration-promoting analysis group, the anti-exhaustion analysis group and the multi-time point analysis group of the present invention all effectively identify target genes related to anti-tumor function in immune cells.

[0154] Example 3 Screening platform based on human T cells

[0155] This example describes a screening platform based on human T cells. In this screening platform, a designed gRNA screening library is introduced into human T cells (e.g., human PBMC T cells) to construct a knockout cell library. In vivo screening is performed using immunodeficient mice (e.g., NOG tumor-bearing mice) to create specific tumor-bearing mice (e.g., by inoculating cells carrying the NYESO1 antigen specifically recognized by the NYESO1-TCR). Cells from the knockout cell library are then infused back into the recipient mice. After a period of time, the spleens and tumors of the recipient mice are harvested, and TILs from the tumors and T cells from the spleens are isolated. Their genomes are then extracted and separated. High-throughput sequencing and bioinformatics analysis are used to capture and compare the enrichment of gRNA sequences, thereby identifying target genes potentially associated with T cell anti-tumor functions (e.g., enhancing T cell tumor infiltration and resistance to exhaustion).

[0156] Screening library plasmid construction

[0157] The method for constructing the screening library plasmid in this experiment is similar to that in Example 1. The screening library in this experiment can use a target gene library, for example, a library designed by using big data and / or literature analysis and prediction of possible immune regulatory genes, and add a non-targeted or non-functional region-targeted control gRNA as a quality control parameter. High-throughput sequencing detected that its gRNA coverage was 100%. Knockout experiments showed that the transduction efficiency of gRNA virus in human T cells can reach more than 80% after enrichment, and the knockout efficiency of the target protein by electroporating Cas9 protein in transduced positive cells can reach more than 90%.

[0158] Then, 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 vector using the Gibson kit. The vector also carries a sequence encoding a fluorescent protein, such as VEX fluorescent protein, or a tag protein sequence, such as CD90.1 protein, which can be used to indicate the transduction and expression of the subsequent library in T cells and can be used to enrich transduced cells. The assembled library plasmid is electroporated into Endura competent cells, cultured overnight to amplify the library plasmid, and then the plasmid is extracted to prepare the library plasmid. High-throughput NGS sequencing is used to control the quality of the prepared library plasmid and detect the coverage and distribution of gRNA.

[0159] Screening library knockout cell library construction

[0160] The lentivirus from the screening library was packaged in HEK293T cells to obtain the library virus, which was then 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. The lentivirus carrying the screening library and a retrovirus expressing the NYESO1-TCR were co-transduced into activated human PBMC T cells according to the exemplary methods 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.

[0161] An exemplary pre-transduction treatment method involves coating a 24- or 6-well suspension culture plate with recombinant human fibrin fragment (Retronectin, Takara) at a final concentration of 15 μg / mL one day before viral transduction. 250 μL per well of a 24-well plate and 1000 μL per well of a 6-well plate were added. Protect from light and incubate at 4°C overnight until ready for use. Remove the coated plate, discard the coating solution, and block with 2% BSA blocking solution at room temperature for 30 minutes. Discard the blocking solution and wash the plate twice with a plate washer containing 2.5% HEPES, which was then discarded.

[0162] An exemplary method for transducing a specific receptor is as follows: the test group is subjected to viral transduction using an appropriate amount of viral dilution, and the retroviral vector contains a nucleic acid fragment encoding NYESO1-TCR (an exemplary α chain variable region is shown in SEQ ID NO: 1, and an exemplary β chain variable region is shown in SEQ ID NO: 2). The viral vector may contain a guide RNA for 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.

[0163] The variable region of the TCR α chain is shown in SEQ ID NO: 1:

[0164] The variable region of the TCRβ chain is shown in SEQ ID NO: 2:

[0165] The marker protein of the screening library and the magnetic beads carrying the corresponding antibodies were used to enrich the T cells that were positive for viral transduction in the screening library for the method of introducing the CRISPR Cas system in the embodiment of the present invention. Cas9 (Kactus Biosystem: Cas9Nuclease) was electroporated with the cells using a Lonza electroporator to obtain a knockout cell library of the screening library. After the Cas9 protein electroporation was completed, according to the cell count density and viability, an appropriate amount of T cell culture medium was added 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.

[0166] In vivo screening model construction and screening process

[0167] In vivo screening involved subcutaneous implantation of human malignant melanoma A375 cells (expressing the NYESO1 antigen, with an exemplary amino acid sequence: SLLMWITQC (SEQ ID NO: 3), which is specifically recognized by the TCR of the library cells) in NOG tumor-bearing mice. After the tumors had grown to a stable volume, cells from the edited human TCR-T cell library were reinfused via the tail vein.

[0168] From 12 hours to 7 days after the infusion of the edited T cells (day 2 in this experiment), the spleen and tumors of the mice were harvested, and the T cells in the tumor and spleen were separated. Their genomes were extracted and isolated, and the gRNA sequences enriched in the screening group electroporated with Cas9 and the control group without electroporated with Cas9 in the tumor were captured and compared through high-throughput sequencing and bioinformatics analysis, thereby discovering target genes that potentially enhance T cell tumor infiltration.

[0169] From the 7th to the 14th day after the infusion of the above-mentioned edited T cells (the 7th day in this experiment), the spleen and tumor of the mice were harvested, and the T cells in the tumor and spleen of the mice were separated. Their genomes were extracted and isolated, and their gRNA sequences enriched in the screening group electroporated with Cas9 and the control group without electroporated with Cas9 in the tumor were captured and compared through high-throughput sequencing and bioinformatics analysis. This can help to discover target genes that have the potential to enhance the tumor-killing function of T cells in vivo and / or have resistance to exhaustion.

[0170] 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.

[0171] Exemplary embodiments

[0172] 1. A method for identifying a target, the method comprising the steps of regulating the expression or activity of a candidate target in a cell to be tested, contacting the cell to be tested with an identification model for one or two time periods, and determining the presence and / or amount of the target regulatory system in the target tissue in the identification model during the first time period and the optional second time period.

[0173] 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 by a target regulatory system; (2) after the test cell is contacted with an identification model for a first time period, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; (3) optionally, after the test cell is contacted with the identification model for a second time period, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; and (4) determining the effect of regulating the candidate target on the cell by reading the results obtained by the determination.

[0174] 3. The method of any one of embodiments 1-2, wherein the identification model comprises a non-human mammal.

[0175] 4. The method of any one of embodiments 1-3, wherein the target tissue comprises a spleen and / or a tumor.

[0176] 5. The method of any one of embodiments 3-4, wherein contacting the cells to be tested with the identification model comprises administering the cells to be tested to the non-human mammal by intravenous infusion.

[0177] 6. The method of any one of embodiments 1-5, wherein the cells to be tested are contacted with the identification model for about 12 hours to 7 days to obtain the target tissue contacted with the identification model for a first period of time.

[0178] 7. The method of any one of embodiments 1-6, wherein the cells to be tested are contacted with the identification model for about 7 to 14 days to obtain the target tissue contacted with the identification model for a second period of time.

[0179] 8. The method of any one of embodiments 1-7, wherein the target regulatory system comprises a target regulatory molecule that specifically recognizes the target sequence.

[0180] 9. A method as described in any one of embodiments 1-8, wherein the regulation step comprises introducing components of a target regulation system into the cells to be tested, respectively, and the target regulation system increases or decreases the expression or activity of the candidate target.

[0181] 10. The method of any one of embodiments 1-9, wherein the regulating step comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system, and / or an inhibitory RNA.

[0182] 11. The method of any one of embodiments 1-10, wherein the regulating step comprises targeting a specific nucleic acid sequence of the candidate target, introducing single-strand breaks, double-strand breaks, mutations and / or expression regulation upstream, upstream and / or downstream of the specific nucleic acid sequence.

[0183] 12. The method of any one of embodiments 1-11, wherein the modulating step comprises increasing or decreasing the expression or activity of the candidate target at the genomic level.

[0184] 13. The method of any one of embodiments 1-12, wherein the regulating step comprises introducing a target regulation system comprising a guide nucleic acid molecule and a nuclease into the test cell.

[0185] 14. The method of embodiment 13, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA) that targets the target.

[0186] 15. The method of any one of embodiments 13-14, wherein the nuclease comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

[0187] 16. The method of any one of embodiments 13-15, wherein the nuclease comprises Cas 9 and / or Cas 12.

[0188] 17. The method of any one of embodiments 1-16, wherein in the cell population to be tested obtained in the adjustment step, the proportion of cells expressing the candidate target is reduced to less than about 95%.

[0189] 18. The method of any one of embodiments 1-17, wherein the determining step comprises isolating the immune cells in the target tissue and then determining the presence and / or quantity of the gRNA in the immune cells.

[0190] 19. The method of any one of embodiments 1-18, wherein the determining step comprises isolating the immune cells in the spleen and / or the tumor and then determining the presence and / or quantity of the gRNA in the immune cells.

[0191] 20. The method of any one of embodiments 1-19, wherein for a gRNA that is enriched in the first time period determination or in both the first time period determination and the second time period determination, the target gene corresponding to the gRNA is identified as a target with regulatory function.

[0192] 21. The method of any one of embodiments 1-20, wherein the determining step comprises determining the presence and / or amount of the target regulatory system in the target tissue in the identification model by gene sequencing.

[0193] 22. The method of any one of embodiments 1-21, wherein the cells to be tested comprise immune cells.

[0194] 23. The method of embodiment 22, wherein the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0195] 24. The method of any one of embodiments 22-23, wherein the immune cells comprise monocytes, macrophages, and / or dendritic cells.

[0196] 25. The method of any one of embodiments 22-24, wherein the immune cells comprise B cells, T cells, natural killer cells, regulatory T cells, and / or natural killer-like T cells (NKT).

[0197] 26. The method of any one of embodiments 22-25, wherein the immune cells comprise αβ T cells and / or γδ T cells.

[0198] 27. The method of any one of embodiments 22-26, wherein the immune cells comprise tumor infiltrating lymphocytes (TILs).

[0199] 28. A method as described in embodiment 27, wherein the TILs are TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of adjacent cancerous tissue, pleural effusion and / or ascites and / or TILs revived after cryopreservation.

[0200] 29. A system for identifying targets, the system comprising: a measurement module, which allows the cells to be tested to contact the identification model for two time periods, determines the presence and / or amount of the target regulatory system in the target tissue in the identification model in the first time period and the second time period, and regulates the expression or activity of the candidate target in the cells to be tested before and / or simultaneously with the measurement.

[0201] 30. A system for identifying a target, the system comprising:

[0202] 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 a candidate target;

[0203] Module (2) is used to contact the cells to be tested (cells that have been or are about to be regulated by the candidate target) with the identification model for a first period of time, and to determine the presence and / or amount of the target regulatory system in the target tissue in the identification model;

[0204] Module (3) is used to contact the cells to be tested (cells that have been or are about to be regulated by the candidate target) with the identification model for a second period of time, and to determine the presence and / or amount of the target regulatory system in the target tissue in the identification model;

[0205] Module (4) is used to determine the effect of regulating the candidate target on the cell by reading the results obtained by the assay;

[0206] There is no restriction on the order of modules (2) and modules (3), and modules (2) and modules (3) can be optionally arranged in parallel.

Claims

1. A method for identifying a target, the method comprising the following steps: providing a cell to be tested, regulating the expression or activity of a candidate target in the cell to be tested, and contacting the cell to be tested with an identification model for one or two time periods, and determining the presence and / or amount of the target regulatory system in the target tissue in the identification model during the first time period and the optional second time period, thereby identifying the target.

2. A method for identifying a target, comprising the steps of: (1) providing a cell to be tested; (2) regulating the expression or activity of the candidate target in the test cells through the target regulation system; (3) after contacting the test cells with the identification model for a first period of time, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; (4) optionally, after contacting the test cells with the identification model for a second period of time, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; as well as (5) Determining the effect of modulating the candidate target on the cell by reading the results obtained by the assay, thereby identifying the candidate target. The method according to claim 1 or 2, wherein the cells to be tested are immune cells. The method according to claim 3 , wherein the cells to be tested are human or animal T cells.

5. The method of any one of claims 1-4, wherein the identification model comprises a non-human mammal.

6. The method of any one of claims 1-5, wherein the target tissue comprises a spleen and / or a tumor.

7. The method of claim 5 or 6, wherein contacting the test cells with the identification model comprises administering the test cells to the non-human mammal by intravenous infusion.

8. The method according to any one of claims 1 to 7, wherein the identification model and the cells to be tested comprise a pair of molecules capable of specific binding.

9. The method of claim 8, wherein the identification model comprises cells expressing tumor-specific antigen molecules, and the cells to be tested express receptors that can specifically bind to the tumor-specific antigen molecules.

10. The method of any one of claims 1 to 9, wherein the cells to be tested are contacted with the identification model for about 12 hours to 7 days, resulting in the target tissue being contacted with the identification model for a first period of time.

11. The method of any one of claims 1 to 10, wherein the test cells are contacted with the identification model for about 7 to 14 days to obtain the target tissue contacted with the identification model for a second period of time.

12. The method of any one of claims 1 to 11, wherein the target regulatory system comprises a target regulatory molecule that specifically recognizes the target sequence.

13. The method of any one of claims 1 to 12, wherein the regulating step comprises separately introducing components of a target regulatory system into the cells to be tested, wherein the target regulatory system increases or decreases the expression or activity of the candidate target.

14. The method of any one of claims 1 to 13, wherein the modulating step comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system, and / or an inhibitory RNA.

15. The method of any one of claims 1 to 14, wherein the regulating step comprises targeting a specific nucleic acid sequence of the candidate target, introducing single-strand breaks, double-strand breaks, mutations and / or expression regulation in the middle, upstream and / or downstream of the specific nucleic acid sequence.

16. The method of any one of claims 1 to 15, wherein the modulating step comprises increasing or decreasing the expression or activity of the candidate target at the genomic level.

17. The method of any one of claims 1-16, wherein the regulating step comprises introducing a target regulation system comprising a guide nucleic acid molecule and a nuclease into the test cell.

18. The method of claim 17, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA) that targets the target.

19. The method of claim 17 or 18, wherein the nuclease comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

20. The method of any one of claims 17-19, wherein the nuclease comprises Cas 9 and / or Cas 12.

21. The method according to any one of claims 1 to 20, wherein in the test cell population obtained in the adjusting step, the proportion of cells expressing the candidate target is reduced to less than about 95%.

22. The method of any one of claims 1-21, wherein the determining step comprises isolating immune cells from the target tissue and then determining the presence and / or quantity of gRNA in the immune cells.

23. The method of any one of claims 1-22, wherein the determining step comprises isolating immune cells in the spleen and / or the tumor, and then determining the presence and / or quantity of gRNA in the immune cells.

24. The method of any one of claims 1 to 23, wherein for a gRNA that is enriched in the first time period assay or in both the first time period assay and the second time period assay, the target gene corresponding to the gRNA is identified as a target with regulatory function.

25. The method of any one of claims 1 to 24, wherein the determining step comprises determining the presence and / or amount of the target regulatory system in the target tissue in the identification model by gene sequencing.

26. The method of any one of claims 1-25, wherein the presence and / or amount of the target regulatory system is determined by determining the presence and / or amount of the target regulatory system in cells to be tested within a target tissue in an identification model.

27. The method of claim 26, wherein the cells to be tested comprise immune cells, such as phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

28. The method of claim 26 or 27, wherein the immune cells comprise monocytes, macrophages and / or dendritic cells.

29. The method of any one of claims 26-28, wherein the immune cells comprise B cells, T cells, natural killer cells, regulatory T cells, and / or natural killer-like T cells (NKT).

30. The method of any one of claims 26-29, wherein the immune cells comprise αβ T cells and / or γδ T cells.

31. The method of any one of claims 26-30, wherein the immune cells comprise tumor infiltrating lymphocytes (TILs).

32. The method of claim 31, wherein the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and / or ascites and / or is derived from TIL revived after cryopreservation.

33. A method for identifying a tumor-associated target, comprising: (1) providing a test cell that is in targeted contact with a tumor identification model; (2) regulating the expression or activity of the candidate target in the test cells through the target regulation system; (3) after contacting the test cells with the tumor identification model for a first period of time, determining the presence and / or amount of the target regulatory system in the target tissue in the identification model; (4) optionally contacting the test cells with the tumor identification model for a second period of time, and determining the presence and / or amount of the target regulatory system in the target tissue in the tumor identification model; as well as (5) Identifying candidate targets by reading the results obtained by the assay.

34. The method of claim 33, wherein the tumor identification model is a tumor-bearing mouse or its tissue cells.

35. A system for identifying targets, the system comprising: a measurement module, which contacts the cells to be tested with an identification model for two time periods, measures the presence and / or quantity of the target regulatory system in the target tissue in the identification model in the first time period and the second time period, and regulates the expression or activity of the candidate target in the cells to be tested before and / or simultaneously with the measurement.

36. A system for identifying a target, the system comprising: 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 a candidate target; Module (2) is used to contact the cells to be tested (cells that have been or are about to be regulated by the candidate target) with the identification model for a first period of time, and to determine the presence and / or amount of the target regulatory system in the target tissue in the identification model; Module (3) is used to contact the cells to be tested (cells that have been or are about to be regulated by the candidate target) with the identification model for a second period of time, and determine the presence and / or amount of the target regulatory system in the target tissue in the identification model; Module (4) is used to determine the effect of regulating the candidate target on the cell by reading the results obtained by the assay; There is no restriction on the order of modules (2) and modules (3), and modules (2) and modules (3) can be optionally arranged in parallel.