A method for identifying a target and use thereof

Abstract

A method of identifying a target is provided, which specifically comprises determining the effect of a candidate target on a cell by assaying the level of a cytokine expressed by the cell, the expression or activity of the candidate target in the cell being modulated prior to the assay. A system for identifying a target is also provided.

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 identifying gene targets that can regulate cellular function holds significant application prospects. Existing target screening methods and systems still have drawbacks, such as low sensitivity, whereby certain targets that can affect cells after expression manipulation are not detected by existing screening methods, resulting in missed screenings; or low specificity, whereby certain targets that cannot affect cells after expression manipulation are mistakenly detected by existing screening methods, resulting in misidentification. Therefore, a method for sensitive and specific gene target screening is urgently needed in this field.

[0003] Summary of the Invention

[0004] In order to solve the above-mentioned technical problems, the present invention designs specific screening readouts and screening parameters, and provides a sensitive and specific screening method.

[0005] On the one hand, the present invention provides a method for identifying a target, the method comprising determining the effect of a candidate target on the cell by measuring the level of a cytokine expressed by the cell. For example, the cytokine provided in the present invention for measuring comprises a member selected from the interferon family, a tumor necrosis factor family, and a lysosome-associated membrane protein family. For example, before and / or simultaneously with the determination, the expression or activity of the candidate target in the cell is regulated.

[0006] On the other hand, the present invention provides a method for identifying a target, comprising the following steps: (1) regulating the expression or activity of the candidate target in a cell; (2) measuring the level of a cytokine expressed by the test cell, wherein the cytokine is selected from the group consisting of an interferon family member, a tumor necrosis factor family member, and a lysosome-associated membrane protein family member; and (3) determining the effect of regulating the candidate target on the cell based on the reading result obtained by the determination.

[0007] On the other hand, the present invention also provides a system for identifying targets, which comprises: a measurement module for measuring the level of cytokines expressed by the cells to be tested and determining the effect of the candidate target on the cells; wherein the cytokines are selected from interferon (IFN) family members, tumor necrosis factor (TNF) family members and lysosome-associated membrane protein (LAMP) family members; and before and / or simultaneously with the measurement, the expression or activity of the candidate target in the cells to be tested is regulated.

[0008] Those skilled in the art will readily appreciate other aspects and advantages of the present invention from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present invention. As will be appreciated by those skilled in the art, the disclosure of the present invention enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the descriptions in the drawings and specification of the present invention are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The features and advantages of the present invention can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. The accompanying drawings are briefly described as follows:

[0010] FIG1 shows the process for constructing a whole genome knockout cell library of the present invention.

[0011] FIG2 shows an exemplary high-throughput genome-wide effector function gene screening process of the present invention.

[0012] The results in Figures 3A-3B show that the optimal screening function marker positive cell population (IFN-γ + TNF-α - IFN-γ - TNF-α + IFN-γ + TNF-α + The ratio of the total cell population in A375 cells was calculated between stimulated (Figure 3B) and unstimulated (Figure 3A).

[0013] The results in Figures 4A-4B show the ratio of the positive cell population using CD107a as a marker in A375 cells stimulated (Figure 4B) and unstimulated (Figure 4A).

[0014] The results in Figures 5A-5B show the ratio of the positive cell population using GZMB as a marker in A375 cells stimulated (Figure 5B) and unstimulated (Figure 5A).

[0015] Figure 6 shows that the optimal screening function marker positive cell population (IFN-γ + TNF-α - IFN-γ - TNF-α + IFN-γ + TNF-α + The ratio of the total cell population was determined when the target cells were incubated for 28 h and the cytokine secretion was inhibited for 4 h under stimulation conditions or without stimulation.

[0016] Figure 7 shows that the optimal screening function marker positive cell population (IFN-γ + TNF-α - IFN-γ - TNF-α + IFN-γ + TNF-α + The ratio of the total cell population was determined under the stimulation condition with a target cell incubation time of 16 h and a cytokine secretion inhibition time of 4 h.

[0017] 8A-8F show the system quality control results of virus packaging, cell editing and editing efficiency of the screening platform of the present invention.

[0018] Figure 9 shows the gRNA distribution results analyzed by this screening platform.

[0019] Figure 10A shows that gene-edited T cells in the no-stimulation group can have significant expansion capacity.

[0020] Figure 10B shows that the gene-edited T cells in the CD3 antibody stimulation group can have a significant expansion capacity. The results show that compared with the control group (NT), the gene-edited T cells of the present invention can have a significant expansion capacity.

[0021] FIG10C shows the target cell killing ability of gene-edited T cells.

[0022] Figure 10D shows that gene-edited T cells have a lower proportion of exhausted T cells. For example, exhausted T cells can be CD38-positive and / or CD101-positive cells.

[0023] Figure 10E shows that the gene-edited T cells in the unstimulated group had a higher cytokine expression ratio, for example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression.

[0024] Figure 10F shows that the gene-edited T cells in the stimulated group had higher cytokine expression rates. For example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression. In Figures 10A to 10F, the RA2 and RA4 groups represent experimental groups edited with gRNA targeting RASA2, codenamed RA2 and RA4, respectively.

[0025] Figure 11A shows that gene-edited T cells in the no-stimulation group can have significant expansion capacity.

[0026] Figure 11B shows that the gene-edited T cells in the CD3 antibody stimulation group can have a significant expansion capacity. The results show that compared with the control group (NT), the gene-edited T cells of the present invention can have a significant expansion capacity.

[0027] Figure 11C shows that gene-edited T cells have a higher proportion of central memory T cells. For example, central memory T cells can be CD45RO-positive and CD62L-positive cells.

[0028] Figure 11D shows that gene-edited T cells have a lower proportion of exhausted T cells. For example, exhausted T cells can be CD38-positive, CD101-positive, PD-1-positive and / or TIM-3-positive cells.

[0029] Figure 11E shows that the gene-edited T cells in the unstimulated group had a higher cytokine expression ratio, for example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression.

[0030] Figure 11F shows that the gene-edited T cells in the stimulation group have a higher cytokine expression ratio. For example, higher CD107a expression ability, higher IFN-γ expression ability, higher TNF-α expression ability or higher GZMB expression ability. The results show that the gene-edited T cells have a more advantageous cell subset composition and / or cytokine expression ability. Among them, in Figures 11A to 11F, the BD3 group and the BD4 group represent the experimental groups edited with gRNA targeting BRD4, code-named BD3 and BD4, respectively.

[0031] Figure 12 shows the gRNA distribution results of the screening platform after expanding the sample analysis. DETAILED DESCRIPTION

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

[0033] Definition of terms

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

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

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

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

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

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0046] 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.

[0047] Detailed Description of the Invention

[0048] The present invention designs specific screening readout (readout) and screening parameters, and provides a kind of can sensitive and specific screening method.On the one hand, the present invention provides a method for identifying a target, the method comprising determining the level of the cytokine expressed by the cell by measuring the cell, determining the influence of the candidate target on the cell.For example, the cytokine for measuring provided in the present invention comprises and is selected from interferon (interferon) family member, tumor necrosis factor (tumor necrosis factor) family member and lysosome-associated membrane protein (Lysosome-Associated Membrane Protein) family member.For example, before and / or simultaneously with the mensuration, the expression or activity of the candidate target in the cell is regulated.

[0049] For example, in the identification method of the present invention, in order to screen effective targets, the cells to be tested are regulated against the candidate target. For example, in the cells to be tested, the expression or activity of the candidate target is regulated. For example, in the cells to be tested, the expression or activity of the candidate target is regulated 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 ... 8.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17%, about 16.75%, about 16.50%, about 16.25%, about 16%, about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14%, about 13.75%, about 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25 %, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75%, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%, about 9%, about 8.75%, about 8.50%, about 8.25%, about 8%, about 7.75%, about 7.50%, about 7.25 %, about 7%, about 6.75%, about 6.50%, about 6.25%, about 6%, about 5.75%, about 5.50%, about 5.25%, about 5%, about 4.75%, about 4.50%, about 4.25%, about 4%, about 3.75%, about 3.50%, about 3.25%, about 3%, about 2.75%, about 2.50%, about 2.25%, about 2%, about 1.7 5%, 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 0.00 7%, 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.

[0050] For example, in the prior art, for the determination method of the cells to be tested after adjustment, the determination process is too complicated, difficult to operate, the determination efficiency is too low, or the result of the determination is poor in sensitivity and specificity, and the difference between the cells to be tested after adjustment cannot be accurately distinguished, resulting in high false positive and false negative of the target obtained by screening. The present invention designs a determination method in order to improve the sensitivity and specificity of identifying the target. For example, specifically, the present invention determines the influence of the candidate target to be tested on the cells to be tested by measuring the level of interferon (interferon) family members, tumor necrosis factor (tumor necrosis factor) family members and / or lysosome-associated membrane protein (Lysosome-Associated Membrane Protein) family members expressed by the cells to be tested. For example, the present invention determines the level of IFN-γ expressed by the cells to be tested after adjustment. For example, the present invention determines the level of TNF-α expressed by the cells to be tested after adjustment. For example, the present invention determines the level of CD107a expressed by the cells to be tested after adjustment.

[0051] For example, the present invention measures the level of IFN-γ, TNF-α, and / or CD107a expressed by the cells to be tested after adjustment. For example, the level of cytokine expression includes the expression amount of the cytokine, the secretion release amount, the level of the effect of the cytokine on the downstream signaling pathway, and / or the effect of the cytokine on cell function and / or cell characteristics. For example, the present invention measures the content of IFN-γ, TNF-α, and / or CD107a in the cells to be tested after adjustment. For example, the present invention measures the release amount of IFN-γ, TNF-α, and / or CD107a in the cells to be tested after adjustment. For example, the present invention measures the level of downstream pathway signals regulated by IFN-γ, TNF-α, and / or CD107a in the cells to be tested after adjustment. For example, the present invention measures the cell function and / or cell characteristics affected by IFN-γ, TNF-α, and / or CD107a regulation in the cells to be tested after adjustment. For example, the present invention can determine the expression level of the cytokine in the cells to be tested by flow cytometry, Western blotting or ELISA.

[0052] For example, based on the level of cytokines expressed by cells, targets that can affect cells can be determined. For example, the determined targets can affect cell proliferation ability, affect cell survival ability, affect cell subpopulation ratios, affect cytokine secretion ability, affect in vitro tumor cell killing ability and / or affect in vivo tumor killing ability. For example, the determined targets can improve cell proliferation ability, improve cell survival ability, improve cytokine secretion ability, improve in vitro tumor cell killing ability and / or improve in vivo tumor killing ability. For example, the determined targets can reduce cell proliferation ability, reduce cell survival ability, reduce cytokine secretion ability, reduce cell killing ability and / or reduce cell damage ability. For example, the determined targets can affect the proportion of activated cells in the cell population, affect the proportion of regulatory cells, affect the proportion of exhausted cells, affect the proportion of central memory cells and / or immature cells, affect the proportion of apoptotic cells and affect the proportion of stem cell-like cells.

[0053] For example, the levels of cytokines expressed by cells can be statistically processed. For example, according to the level of influence on the level of cytokines expressed by cells, the corresponding candidate targets are ranked, and 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, are determined as targets that can affect cells. For example, according to the level of influence on the level of cytokines expressed by cells, the corresponding candidate targets are ranked from the bottom 1 to the top 1000, such as the top 1, top 2, top 3, top 4, top 5, top 6, top 7, top 8, top 9, top 10, top 15, top 20, top 25, top 30, top 35, top 40, top 45, top 50, top 60, top 70, top 80, top 90, top 100, top 200, top 300, top 400, top 500, top 600, top 700, top 800, top 900, or top 1000, as essential targets for cells or targets necessary for secreting the above-mentioned cytokines.

[0054] For example, depending on the effect on the level of cytokines expressed by cells, for example, an increase or decrease of about 0.1% to about 10,000 times, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, %, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 1000-fold, or about 10000-fold of the corresponding candidate target can be used as a target that can affect cells. For example, based on the confidence and / or significance of the level of cytokine expressed by the cell, the logarithm with base 10 is taken, and then the negative number is taken. The resulting value is greater than about 1, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10, which is the corresponding candidate target.

[0055] For example, the interferon family members of the present invention include IFN-γ. For example, the IFN-γ of the present invention may include its functionally active fragments and any domains thereof.

[0056] For example, the tumor necrosis factor family members of the present invention include TNF-α. For example, the TNF-α of the present invention may include its functionally active fragments and / or any of its structural domains.

[0057] For example, the lysosome-associated membrane protein family members of the present invention include CD107a.For example, the CD107a of the present invention may include a functionally active fragment and / or any domain thereof.

[0058] For example, the regulation 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 repeat (CRISPR) system is used to regulate the candidate target. Alternatively, a transcription activator-like effector nuclease (TALEN) system, a zinc finger nuclease system, or a large range of nuclease systems 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.

[0059] For example, the regulation described in the present invention comprises increasing or decreasing the expression or activity of the candidate target at the genomic level. Alternatively, the expression or activity of the candidate target can be transiently increased or decreased at the RNA level. Alternatively, the expression or activity of the candidate target can be increased or decreased at the transcription level, translation level and / or post-translational modification level. In some embodiments, antisense RNA, siRNA, shRNA or short hairpin RNA are used to decrease the expression or activity of the candidate target. In some embodiments, activating RNA is used to increase the expression or activity of the candidate target. For example, the regulation described in the present invention comprises providing a substance that covalently binds and / or non-covalently binds to the candidate target to increase or decrease the expression or activity of the candidate target.

[0060] For example, the method of the present invention can include editing of candidate targets in vivo, in vitro and / or in vitro. For example, the in vivo expression level of the candidate target can be reduced by delivering and editing the gene regulatory system in vivo. For example, by targeting immune cells or their precursor cells, such as bone marrow stem cells, delivering LNPs containing the gene regulatory system or mRNA encoding the gene regulatory system, the in vivo editing of the candidate target 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.

[0061] For example, the modulation of the present invention comprises targeting a specific nucleic acid sequence of the target, introducing single-strand breaks, double-strand breaks and / or mutations within, upstream and / or downstream of the nucleic acid sequence. For example, the modulation of the present invention comprises providing a CRISPR system, a zinc finger nuclease system, a meganuclease system and / or antisense RNA, siRNA, shRNA or short hairpin RNA.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] For example, the regulation 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 guide nucleic acid molecule in the present invention comprises a guide RNA (gRNA). 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. 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 completely 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 cut the candidate target.

[0071] 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.

[0072] 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 can bind to a sequence consisting of about 15 to about 25 nucleotides 3' downstream of the protospacer adjacent motif (PAM) selected from the group consisting of NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, wherein N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] For example, when the gene editing system of the present invention comprises 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.

[0078] For example, in the present invention, in the cell population obtained by the regulation, the proportion of cells expressing the candidate target is reduced to about 95% or less. For example, it can be reduced to at least about 100-1%, such as at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.

[0079] For example, the present invention allows the cell to express an exogenous cell receptor or a functional fragment thereof before or simultaneously with the assay. For example, the exogenous cell receptor in the present invention comprises a T cell receptor or an antigen-binding fragment thereof. For example, the present invention allows the cell to contact a specific antigen of the exogenous cell receptor or a functional fragment thereof before or simultaneously with the assay. For example, the present invention allows the cell to contact the cell expressing the exogenous cell receptor or a functional fragment thereof with a target cell expressing a specific antigen of the exogenous cell receptor or a functional fragment thereof before or simultaneously with the assay. For example, in the contacting of the present invention, the cell to be tested expressing the exogenous cell receptor or a functional fragment thereof and the target cell expressing the specific antigen of the exogenous cell receptor or a functional fragment thereof have a cell ratio of about 1:100 to 100:1. For example, the cell ratio of test cells to target cells is about 1:100, about 1:90, about 1:80, about 1:70, about 1:60, about 1:50, about 1:40, about 1:30, about 1:20, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1::1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1.

[0080] For example, the cells to be tested are contacted with the specific antigen of the exogenous cell receptor or its functional fragment for about 0 to about 72 hours before or simultaneously with the assay. For example, the cells to be tested are contacted with the specific antigen of the exogenous cell receptor or its functional fragment for about at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours. For example, the present invention allows the cell expressing the exogenous cell receptor or its functional fragment to contact the target cell expressing the specific antigen of the exogenous cell receptor or its functional fragment for about 6 hours, about 12 hours, about 16 hours, or 28 hours. For example, before or simultaneously with the determination of the present invention, the cell expressing the exogenous cell receptor or its functional fragment is contacted with the target cell expressing the specific antigen of the exogenous cell receptor or its functional fragment for about 12 hours or about 16 hours.

[0081] For example, in the present invention, the cells to be tested may be contacted with a substance that inhibits cytokine secretion before or during the assay. For example, the substance that inhibits cytokine secretion can inhibit the release of the cytokine from the cell to the extracellular space. For example, the substance that inhibits cytokine secretion can substantially not affect the expression of the cytokine.

[0082] For example, the substance that inhibits cytokine secretion in the present invention comprises Brefeldin A. For example, the substance that inhibits cytokine secretion in the present invention comprises monensin. For example, in the present invention, the cells to be tested are contacted with the substance that inhibits cytokine secretion for about 0 hours to about 72 hours before or simultaneously with the assay.

[0083] For example, in the present invention, the cells to be tested are contacted with a substance that inhibits cytokine secretion for about 4 hours to about 16 hours. For example, in the present invention, the cells to be tested are contacted with a substance that inhibits cytokine secretion for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours. For example, in the present invention, the cells to be tested are contacted with a substance that inhibits cytokine secretion for about 4 hours, about 12 hours, or about 16 hours.

[0084] For example, the present invention allows the cell expressing the exogenous cell receptor or its functional fragment to contact the target cell expressing the specific antigen of the exogenous cell receptor or its functional fragment before or simultaneously with the determination for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about The test cells are contacted with the substance that inhibits cytokine secretion for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 28 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours.

[0085] For example, the determination in the present invention comprises determining the level of the cytokine expressed by the test cells by flow cytometry, Western blotting, or ELISA. For example, the determination in the present invention comprises determining the amount and / or rate of increase in the level of the cytokine expressed by the cells in which the expression or activity of the candidate target is modulated, compared to corresponding cells in which the expression or activity of the candidate target is not modulated, by flow cytometry. For example, compared to the corresponding test cells in which the expression or activity of the candidate target is not modulated, the ratio of positive cells for the cytokine expressed by the test cells in which the expression or activity of the candidate target is modulated is increased (the ratio of positive cells in the test cells after modulation / the ratio of positive cells in the test cells not modulated = the ratio of increase) by about 1.0 times to about 10,000 times, for example, about 1.0 times, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 1000 times, or about 10,000 times. For example, compared to the corresponding test cells in which the expression or activity of the candidate target is not regulated, the increase in the proportion of positive cells expressing the cytokine in the test cells in which the expression or activity of the candidate target is regulated (the proportion of positive cells in the test cells after regulation - the proportion of positive cells in the test cells not regulated = the increase) is about 0.5% to about 100%, for example, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%. , about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.

[0086] For example, the cells to be tested in the present invention include immune cells. For example, the immune cells in the present invention include phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils. For example, the immune cells in the present invention include monocytes, macrophages and / or dendritic cells. For example, the immune cells in the present invention are derived from immune cells differentiated from stem cells. For example, the stem cells in the present invention include induced pluripotent stem cells (iPSC), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells, and / or peripheral blood stem cells. For example, the immune cells in the present invention include B cells, T cells, regulatory T cells, natural killer cells and / or natural killer-like T cells (NKT). For example, the immune cells in the present invention include αβT cells and / or γδT cells. For example, the immune cells in the present invention include tumor infiltrating lymphocytes (TIL). For example, TIL described in the present invention is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, paracancerous tissue fragments, pleural effusion and / or ascites TIL and / or derived from cryopreservation after recovery. Before amplification and genetic modification, the source of cells (for example, immune effector cells (for example, T cells or NK cells)) is obtained from the subject. The term "subject" is intended to include living organisms (for example, mammals) that can induce an immune response therein. The example of the subject includes people, 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 tumor.

[0087] On the other hand, the present invention provides a method for identifying a target, comprising the following steps: (1) regulating the expression or activity of the candidate target in a cell; (2) determining the level of a cytokine expressed by the test cell, wherein the cytokine is selected from the group consisting of an interferon family member, a tumor necrosis factor family member, and a lysosome-associated membrane protein family member; and (3) determining the effect of regulating the candidate target on the cell based on the reading result obtained by the determination.

[0088] In another aspect, the present invention provides a method for identifying a target, the method comprising:

[0089] (1) introducing components of a target regulation system such as 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 simultaneously or sequentially, wherein the target regulation system increases or decreases the expression or activity of the candidate target;

[0090] (1-a) causing the cells to be tested to express an exogenous cell receptor or a functional fragment thereof;

[0091] (1-b) contacting the cells to be tested with the specific antigen of the exogenous cell receptor or its functional fragment;

[0092] (1-c) contacting the cells to be tested (cells that have or are about to be regulated by the candidate target) with a substance that inhibits cytokine secretion;

[0093] (2) measuring the levels of IFN-γ, TNF-α, and / or CD107a expressed by the cells to be tested;

[0094] (3) Determining the effect of regulating the candidate target on the cell through the reading results obtained by the determination; wherein there is no order restriction among step (1), step (1-a), step (1-b), and step (1-c), and during the process of any one of step (1), step (1-a), step (1-b), and step (1-c), another step can be performed simultaneously.

[0095] In another aspect, the present invention provides a method for identifying a target, the method comprising:

[0096] (1-I) introducing a guide nucleic acid of a CRISPR system into the test cell, wherein the guide nucleic acid is capable of inhibiting the expression or activity of the candidate target; four guide nucleic acids are designed for each target gene for different targeting sequences, and the probability of introducing two or more guide nucleic acids into a single cell is at most about 10%;

[0097] (1-a) allowing the cells to be tested to express TCR targeting NYESO-1;

[0098] (1-II) introducing an exogenous nuclease of the CRISPR system or a nucleic acid encoding the exogenous nuclease into the test cell;

[0099] (1-b) contacting the test cell with a target cell expressing the NYESO-1 antigen;

[0100] (1-c) contacting the cells to be tested (cells that have or are about to be regulated by the candidate target) with a substance that inhibits cytokine secretion;

[0101] (2) determining the levels of IFN-γ, TNF-α and / or CD107a expressed by the regulated test cells;

[0102] (3) Determining the effect of regulating the candidate target on the cell through the reading results obtained by the determination; wherein there is no order restriction among step (1-I), step (1-a), step (1-II), step (1-b), and step (1-c), and step (1-I), step (1-a), step (1-II), step (1-b), and step (1-c) can optionally be performed simultaneously.

[0103] On the other hand, the present invention also provides a system for identifying targets, which comprises: a measurement module for measuring the level of cytokines expressed by the cells to be tested and determining the effect of the candidate target on the cells; wherein the cytokines are selected from interferon family members, tumor necrosis factor family members and lysosome-associated membrane protein family members; and before and / or simultaneously with the measurement, the expression or activity of the candidate target in the cells to be tested is regulated.

[0104] In another aspect, the present invention provides a system for identifying a target, the system comprising:

[0105] Module (1) can be used to simultaneously or sequentially introduce components of a target regulatory system such as 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 regulatory system increases or decreases the expression or activity of the candidate target;

[0106] Module (1-a) can be used to make the cells to be tested express an exogenous cell receptor or a functional fragment thereof;

[0107] Module (1-b) can be used to contact the cells to be tested with the specific antigen of the exogenous cell receptor or its functional fragment;

[0108] Module (1-c) can be used to contact the cells to be tested (cells that have or are about to be regulated by the candidate target) with a substance that inhibits cytokine secretion;

[0109] Module (2) can be used to measure the levels of IFN-γ, TNF-α and / or CD107a expressed by the cells to be tested;

[0110] Module (3) can be used to read the results obtained by the determination to determine the effect of regulating the candidate target on the cell; there is no order restriction among modules (1), module (1-a), module (1-b), and module (1-c), and during the process of any module among modules (1), module (1-a), module (1-b), and module (1-c), another module can be arranged in parallel.

[0111] In another aspect, the present invention provides a system for identifying a target, the system comprising:

[0112] Module (1-I) can be used to introduce a guide nucleic acid of the CRISPR system into the test cell, wherein the guide nucleic acid is capable of inhibiting the expression or activity of the candidate target; four guide nucleic acids are designed for each target gene for different targeting sequences, and the probability of introducing two or more guide nucleic acids into a single cell is at most about 10%;

[0113] Module (1-a) can be used to allow the cells to be tested to express TCR targeting NYESO-1;

[0114] Module (1-II) can be used to introduce an exogenous nuclease of the CRISPR system or a nucleic acid encoding the exogenous nuclease into the cell to be tested;

[0115] Module (1-b) can be used to contact the cells to be tested with target cells expressing NYESO-1 antigen;

[0116] Module (1-c) can be used to contact the cells to be tested (cells that have or are about to be regulated by the candidate target) with a substance that inhibits cytokine secretion;

[0117] Module (2) can be used to determine the level of IFN-γ, TNF-α and / or CD107a expressed by the regulated cells to be tested;

[0118] Module (3) can be used to read the results obtained through the determination to determine the effect of regulating the candidate target on the cell; wherein there is no order restriction for the arrangement of modules (1-I), module (1-a), module (1-II), module (1-b), and module (1-c), and modules (1-I), module (1-a), module (1-II), module (1-b), and module (1-c) can be optionally arranged in parallel.

[0119] 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.

[0120] 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.

[0121] Example

[0122] Example 1

[0123] Cell culture

[0124] An exemplary immune cell culture method used in the present invention is as follows: PBMC (peripheral blood mononuclear cells) were obtained from blood samples of healthy donors. PBMC T cells frozen in liquid nitrogen were revived and cultured, and resuspended to 5E5 / ml (5×10 5 T cell TransAct (Miltenyi) was added at a TransAct: cell ratio of 1:100, and recombinant human IL-2 at a concentration of 30 IU / ml was added, and cultured for about 72 hours.

[0125] Cell treatment before transduction

[0126] Exemplary pre-transduction treatment methods for viral transduction in embodiments of the present invention: One day prior to viral transduction, coat a 24- or 6-well suspension culture plate with recombinant human fibrin fragment (Retronectin, Takara) at a final concentration of 15 μg / mL. 250 μL per well of a 24-well plate and 1000 μL per well of a 6-well plate. Protect from light and incubate at 4°C overnight. 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, then discard the plate washer solution.

[0127] Specific receptor transduction

[0128] Method for transduction of exemplary specific receptors used in the present invention: The experimental group was subjected to viral transduction using an appropriate amount of viral dilution. The retroviral vector contained 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), and the lentiviral vector contained a guide RNA library. Centrifuge at 32°C, 2000g for 2 hours. Discard the supernatant from the 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.

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

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

[0131] Generation of Cas9 cells

[0132] The method for producing Cas9 cells used in the embodiment of the present invention is as follows: Cas9 (Kactus Biosystem: Cas9 Nuclease) and cells are electroporated using a Lonza electroporator. After the Cas9 protein is electroporated, according to the cell count density and viability, an appropriate amount of T cell culture medium is added according to the culture medium instructions, and recombinant human IL-2 at a concentration of 300 IU / ml is added to adjust the initial culture cell density to 1×10 6 / ml and continue to culture.

[0133] Example 2 High-throughput genome-wide effector function gene screening process

[0134] 1) Construction of whole genome screening library plasmid

[0135] The in vitro whole-genome screening library design is based on the Brunello library referenced by JG Doench et al. (DOI: 10.1038 / nbt.3437). It contains approximately 80,000 gRNAs targeting approximately 20,000 genes (each gene has four gRNA target sites), as well as 500 control gRNAs that are non-targeting or target non-functional regions as a quality control parameter. The data presented in this paper demonstrate that the Brunello gRNA library has high gene editing efficiency and low off-target effects, making it an ideal library for whole-genome screening.

[0136] First, the oligo sequence of the screening library is synthesized with 25nt homology arms at both ends. A round of PCR amplification is performed based on the homology arm sequence to obtain the dsDNA fragment of the screening library. The dsDNA fragment of the library is then assembled into the designed lentiviral expression vector using the Gibson kit. In addition, the vector also carries a surface marker gene that is easy to stain, which can be used to indicate the subsequent transduction and expression of the library in T cells, and can also be used to enrich transduction-positive cells. The assembled library plasmid is electroporated into Endura competent cells, cultured overnight to amplify the library plasmid, and then the plasmid is extracted to prepare the library plasmid.

[0137] High-throughput NGS sequencing was used to perform quality control on the prepared library plasmids, and the coverage and distribution of gRNA were detected to determine that the library used in the screening platform of the present invention met the library construction standards. Among them, the sequencing data alignment rate of the plasmid library was greater than 70%, the coverage of gRNA was greater than 99%, the gene coverage was 100%, and the gini coefficient of gRNA was less than 0.3.

[0138] 2) Construction of genome-wide knockout cell library

[0139] Lentivirus containing the whole-genome screening library was packaged in HEK293T cells and concentrated 20-fold. The viral titer of the screening library was tested on human PBMC T cells to ensure a biological titer of at least 2E7 / ml, sufficient for subsequent use. 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 efficiency was controlled at 30-40% (the probability of two lentiviruses carrying different gRNAs entering the same cell was controlled to around 10%, reducing the impact on subsequent analysis) and above 70%, respectively. T cells positively transduced by the screening library virus were enriched using a marker protein from the screening library and magnetic beads carrying the corresponding antibody. The Cas9 protein was then electroporated using the Lonza electroporation system for targeted gene editing.

[0140] FIG1 shows the process for constructing a whole genome knockout cell library of the present invention.

[0141] After about a week, once it was confirmed that the T cells had recovered and could proliferate normally, A375 cells expressing the NYESO1 antigen (exemplary amino acid sequence: SLLMWITQC) were co-incubated with the edited T cells to construct a functional screening model.

[0142] 3) Flow cytometry detection of cytokine expression

[0143] For each experimental group, A375 cells expressing NYESO1 antigen were co-incubated with edited T cells. Golgi stop / plug (to inhibit cytokine secretion and facilitate intracellular detection of cytokines by flow cytometry) was added to the co-incubation culture medium in proportion. Flow cytometry was used to detect the levels of effector cytokines produced by TCR-T cells when stimulated by A375.

[0144] Take 200 μL of TCR-T cell suspension from each test group, about 2×10 5Cells were washed once with 200 μL / well PBS per treatment group, centrifuged at 600 g for 3 minutes, and the supernatant discarded. A mixed antibody working solution was prepared for cell surface staining of CD3 / CD8 / TCR-Vb (BD) at an antibody concentration of 1:100 and a cell viability assay dye concentration of 1:10,000. 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube were added for staining and incubated at 2-8°C in the dark for 30 minutes. During the staining process, the reagents required for transcription factor staining were prepared: 4× Fixation / Permeabilization Buffer Set (BD) was diluted with Transcription Factor Buffer Set to 1× Working Solution A; 5× Perm / Wash Buffer (BD) was diluted with double-distilled water to 1× Working Solution B. Both solutions were pre-chilled at 4°C until use. After staining, wash the cells twice with an appropriate amount of PBS (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes), centrifuge at 600 g for 3 minutes, and discard the supernatant. Cell fixation and membrane permeabilization: Resuspend the cells thoroughly and add an appropriate amount of 1× working solution A (100 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes) to fix and permeabilize the membrane. Incubate at 2-8°C in the dark for 40-50 minutes. After fixation and permeabilization, wash the cells with 1× working solution B (200 μL / well for 96-well plates, 2 mL / well for flow cytometry tubes), centrifuge at 2-8°C, centrifuge at 350 g for 6 minutes, and wash twice. Intracellular antibodies (CD107a, GZMB, TNF-α, and IFN-γ, BD / BioLegend) were prepared using 1× Working Solution B at a concentration of 1:100 to 1:200. 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube were added and stained for 30 minutes at 2-8°C in the dark. After staining, cells were washed with 1× Working Solution B (200 μL / well of a 96-well plate and 2 mL / well of a flow cytometry tube) and centrifuged twice at 350 g for 6 minutes at 2-8°C. Cells were resuspended in 100-500 μL of PBS and analyzed by flow cytometry.

[0145] 4) Functional screening model construction and screening process

[0146] The purpose of the functional screening model is to stimulate T cells carrying specific TCRs through tumor cells, and screen out T cells that can produce a large number of signature effector cytokines (markers), thereby discovering target genes that can significantly enhance the effector function of T cells.

[0147] The present invention discovered that the peak production of effector cytokines generally occurs within 48 hours after T cells are stimulated by antigens. Therefore, the present invention designed and tested different effector-target ratios, total stimulation time, and Golgi stop / plug (which inhibits cytokine secretion and facilitates intracellular detection of cytokines using flow cytometry) treatment durations to test the secretion / expression of various cytokines or functional molecules, including CD107a, GZMB, IFN-γ, TNF-α, IL2, and IL6, in NYESO1-TCR-T cells after A375 stimulation.

[0148] In PBMC T cells from multiple donors, the levels of effector cytokines produced by TCR-T cells when stimulated by A375 were determined, and functional markers that can be used in formal effector function gene screening experiments were screened to indicate T cell tumor-specific activation and killing ability, as well as to identify the optimal experimental conditions.

[0149] Using the cell library constructed in this example, after confirming that the enriched and edited T cells have recovered, A375 cells are co-incubated with the edited T cells and subjected to intracellular staining according to the optimal experimental conditions ultimately determined by this invention. T cells expressing IFN-γ and / or TNF-α within the NYESO1-TCR+ positive population are then collected using a flow cytometer. Finally, the genomes of the sorted cells are extracted, and enriched gRNA sequences are captured through high-throughput sequencing and bioinformatics analysis, allowing the identification of potential genes that could enhance T cell effector function.

[0150] FIG2 shows an exemplary high-throughput genome-wide effector function gene screening process of the present invention.

[0151] Example 3 Gene Screening Process Optimization Design

[0152] To optimize the screening process, the present invention tested the expression of different effector molecules after A375 cell stimulation on PBMC T cells from multiple donors to identify the optimal experimental conditions.

[0153] Filter the preferred screening function marker

[0154] Table 1 Irradiated A375 cells stimulate TCR-T cells, and the ratio of positive cell population expressing different markers is increased *: The Mock+A375 vs. Mock ratio data represent the fold increase in the number of cells expressing a specific marker in the TCR-positive cell population that was not gene-edited and co-cultured with A375 (Mock+A375), compared to the TCR-positive cell population that was not gene-edited (Mock). A higher fold increase indicates that the target is more easily used to characterize tumor-specific stimulation. The Gene KO+A375 vs. Gene KO ratio data represent the fold increase in the number of cells expressing a specific marker in the TCR-positive cell population that was edited with a gene known to enhance cell function (such as PD-1 or RASA2) and co-cultured with A375 (Gene KO+A375), compared to the TCR-positive cell population that was edited with a gene known to enhance cell function (such as PD-1 or RASA2) (Gene KO). A higher fold increase indicates that the target is more easily used to characterize functional differences after candidate gene knockout under tumor-specific stimulation conditions.

[0155] The results in Table 1 show that IFN-γ, TNF-α, and CD107a are specifically upregulated in PBMC T cells from multiple donors upon TCR-T stimulation with A375. The upregulation of IFN-γ and TNF-α is more pronounced, particularly in the knockout group. Therefore, these two markers are preferred for screening in this invention.

[0156] Screening efficiency target cell ratio

[0157] In addition, the present invention also tests the expression changes of effector molecules after stimulation with different effector-target cell ratios (E:T).

[0158] Table 2 Irradiated A375 cells with different E:T ratios can stimulate the positive cell population to express effector molecules in TCR-T cells. *: The ratio of the positive cell population of the preferred screening function marker represents IFN-γ + TNF-α - , IFN-γ - TNF-α + IFN-γ + TNF-α + The sum of the cell population proportions. The meaning of the boost factor is the same as that in the notes of Table 1.

[0159] Table 2 shows that in the screening platform of the present invention, different effector-target ratios all have screening effects, and the optimal experimental conditions finally designed can be set to E:T=1:3.

[0160] Screening target cell incubation time and inhibition time of cytokine secretion

[0161] The present invention also tested the effects of different irradiated A375 cell stimulation times and different cytokine secretion inhibition times on expression changes. Golgi stop / plug inhibitors can enclose screening markers in cells, thereby amplifying flow cytometric detection and differentiation. The duration of cytokine secretion inhibition can be controlled by the timing of Golgi stop / plug inhibitor addition. For example, adding the Golgi stop / plug inhibitor 12 hours before the end of the experiment results in a 12-hour cytokine secretion inhibition period.

[0162] Table 3 The increase in the proportion of positive cell populations expressing different effector molecules after irradiated A375 cells stimulated TCR-T cells for different time periods and inhibited cytokine secretion for different time periods *: Preferred screening function marker positive cell population represents IFN-γ + TNF-α - , IFN-γ - TNF-α + IFN-γ + TNF-α + Total cell population. The increase value represents the percentage increase in the positive cell population for a specific marker in a TCR-positive cell population that was edited with a gene known to enhance cell function (such as PD-1 or RASA2) and co-cultured with A375 (Gene KO+A375), compared to the percentage increase in the positive cell population for a gene known to enhance cell function (such as PD-1 or RASA2) (Gene KO).

[0163] Table 3 shows that in the screening platform of the present invention, the optimal experimental conditions are target cell co-incubation time of 12 hours to 16 hours and controlled inhibition of cytokine secretion time of 12 hours to 16 hours.

[0164] According to the optimal experimental conditions determined above, A375 cells were co-cultured with edited T cells in the treatment group and intracellular staining was performed.

[0165] The results in Figures 3A-3B show that the optimal screening function marker positive cell population (IFN-γ + TNF-α - IFN-γ - TNF-α + IFN-γ + TNF-α + The ratio of the total cell population in A375 cells was calculated between stimulated (Figure 3B) and unstimulated (Figure 3A).

[0166] The results in Figures 4A-4B show the ratio of the positive cell population using CD107a as a marker in A375 cells stimulated (Figure 4B) and unstimulated (Figure 4A).

[0167] The results in Figures 5A-5B show the ratio of the positive cell population using GZMB as a marker in A375 cells stimulated (Figure 5B) and unstimulated (Figure 5A).

[0168] The results in Figure 6 show that the optimal screening function marker positive cell population (IFN-γ + TNF-α - IFN-γ - TNF-α + IFN-γ + TNF-α + The ratio of the total cell population was determined when the target cells were incubated for 28 h and the cytokine secretion was inhibited for 4 h under stimulation conditions or without stimulation.

[0169] The results in Figure 7 show that the optimal screening function marker positive cell population (IFN-γ + TNF-α - IFN-γ - TNF-α + IFN-γ + TNF-α + The ratio of the total cell population was determined under the stimulation condition with a target cell incubation time of 16 h and a cytokine secretion inhibition time of 4 h.

[0170] The results showed that compared with the blank control group (untreated group), the specific upregulation ratio of IFN-γ and TNF-α in the treatment group under the preferred experimental conditions of the present invention was significantly increased, proving that the screening platform of the present invention can be used to sensitively screen candidate genes that are potentially related to T cell tumor-specific activation and effector function.

[0171] Example 4 Screening Platform Quality Control Test

[0172] The quality control effect of the screening platform was tested. First, high-throughput NGS sequencing was used to quality control the prepared library plasmids. The results showed that the Gini coefficient of the plasmid library sgRNA was less than 0.25, and the sgRNA coverage was 99.99%. The Gini coefficient of the gene was less than 0.15, and the gene coverage was 100%, demonstrating that the screening platform of the present invention meets the library quality control requirements for target screening.

[0173] 8A-8F show the system quality control results of virus packaging, cell editing and editing efficiency of the screening platform of the present invention.

[0174] In addition, the viral packaging and cell editing systems were optimized. The transduction efficiency of the gRNA library lentivirus screened in human PBMC T cells can be controlled at 30-40% (Figure 8A), and the transduction efficiency of the NYESO-TCR retrovirus can reach over 70% (Figure 8B). The purity of T cells transduced with the marker-enriched library virus using the screening library can reach over 90% (Figure 8C).

[0175] The gene editing efficiency of the screening platform of the present invention was tested. Using the Lonza electroporation system, Cas9 protein was electroporated for PD-1-targeted gene editing. Comparison of the screening marker-positive population with the negative population revealed that, within the 81% screening marker-positive population, the proportion of cells without PD-1 editing was less than 10%, indicating that the protein knockout efficiency of the target gene PD-1 was greater than 80% (Figures 8D-8F).

[0176] The results show that the screening platform of the present invention as a whole meets the requirements for target screening quality control.

[0177] Example 5 Functional verification of the screened targets

[0178] The optimized screening platform of the present invention was used to perform genome-wide Effector function screening. Using the optimized screening model parameters, the enriched edited T cells were co-incubated with A375 cells for 16 hours. In the TCR+ positive population, about 25% could specifically upregulate IFN-γ and / or TNF-α. The subpopulation was collected by flow cytometry and subjected to NGS sequencing analysis. The results showed that the distribution of Safe Harbor guides in the harvested samples did not change significantly. This result shows that the screening platform of the present invention can effectively exclude genes that are not related to cell function.

[0179] Figure 9 shows the gRNA distribution results analyzed by this screening platform.

[0180] In the gRNA distribution results, for example, CD3D and CD3Z showed a large decrease in gRNA expression. These targets are also known in the art to be genes related to T cell proliferation and activation signal transduction, which are beneficial to T cell proliferation. Knocking out these target genes leads to immunodeficiency and inhibits T cell proliferation. For example, knocking out these targets helps maintain cell function, and will result in a decrease in the number of cells in the knockout analysis group.

[0181] In addition, a small number of gRNAs for targets such as FOXO1, RASA2, and BRD4 in the upper right corner were enriched. These targets are also T cell inhibition or exhaustion signal-related targets known in the art, for example, they can potentially inhibit cell function. Knocking out these targets will lead to increased expression of the preferred screening function marker of the knockout analysis group.

[0182] The results showed that the screening platform of the present invention can detect candidate genes related to effector functions with high sensitivity.

[0183] Example 6: Individual verification of the selected targets

[0184] The present invention also designs targeted knockout tools for the screened targets such as RASA2 and BRD4, and independently verifies them.

[0185] Culture of tumor-infiltrating lymphocytes

[0186] 1.1 Tumor tissue receipt and processing

[0187] 1.1.1 Organization reception

[0188] Receive tumor tissue and blood samples from donors, verify and record sample information, and print corresponding sample labels.

[0189] 1.1.2 Tissue processing and culture

[0190] Use 75% alcohol to disinfect the sample tube and blood collection tube and transfer them to a biosafety cabinet. Isolate PBMC cells from the blood sample and freeze them according to the above-mentioned PBMC manual isolation and freezing procedures. Take a culture flask or culture bag with a breathable surface, such as a culture bag (Origen), and add 300 mL of thawed complete culture medium. The complete culture medium can be arbitrarily selected from X-vivo15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and can be supplemented with essential amino acids and antibiotics, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL). Take several 10 cm culture dishes, add an appropriate amount of culture medium, use sterile ophthalmic forceps to remove the tumor tissue from the sample tube into a 10 cm culture dish, wash the tissue and change the culture dish. Use ophthalmic scissors and forceps to perform initial shearing, removing adipose and necrotic tissue. Each tissue block is then minced to approximately 27 cubic millimeters. A non-suspended tumor tissue block is obtained. A 20 mL syringe is used to remove the internal stopcock and connect to a culture bag. Using a pipette, approximately 1 g of tissue is transferred from the syringe into the culture bag. The culture bag is placed in a CO2 incubator for incubation. The scissors and forceps are cleaned and initially disinfected with 75% alcohol. After ultrasonic cleaning, they are sterilized to obtain the first TIL population.

[0191] 1.2 Step (A) In vitro expansion and harvesting

[0192] 1.2.1 Step (A) In vitro amplification

[0193] Depending on the cell growth status, the medium should be replenished or half-replaced every 3-7 days to ensure cell nutrition. Complete culture medium can be arbitrarily selected from X-vivo 15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 (double heron and / or tetracycline) at a concentration of 300-9000 IU / mL (e.g. 1000-9000 IU / mL, such as 6000 IU / mL) can be added. 3-14 days in step (A), for example, samples can be taken and counted on the 13th or 14th day. If the cell number is between 5×10 5 to 5×10 8 During this time, the harvesting step of step (A) is entered.

[0194] 1.2.2 Results of Step (A)

[0195] Collect the cells after in vitro expansion in step (A), centrifuge, discard the culture medium, wash the cells once with PBS or normal saline, obtain the TILs (second TIL population) expanded in vitro in step (A), and take samples for counting and retain about 5×10 5 to 2×10 8 cells into the subsequent in vitro expansion step; about 5×10 5 The remaining cells can be added to the cryopreservation medium and cryopreserved as cryopreserved preREP TIL in vitro cells.

[0196] 1.3 Step (B) TIL activation

[0197] Continue to culture the TILs (second TIL population) expanded in vitro in step (A), or recover the frozen preREP TIL cells in vitro and perform TIL activation in step (B).

[0198] Complete culture medium can be selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi Biotech, etc. Essential amino acids and antibiotics can be added to adjust the cell density to 5×10 5 to 2×10 6Cells are suspended in a 24-well culture plate at a concentration of 1 mL / well. IL-2 is added at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, for example, 6000 IU / mL). T cell activators, such as CD3 agonists and / or CD28 agonists, can be added to the culture medium of each TIL population, for example, approximately 30 ng / mL of CD3 antibody (Miltenyi Biotech, OKT3), approximately 30 ng / mL of CD28 antibody (Merck, 15E8), magnetic beads (Dynabeads, approximately 1 to 10 μm diameter, Thermo Fisher) at a ratio of approximately 1:2-2:1 beads to TILs, and / or transACT (Miltenyi, approximately 100 to 500 nm diameter, TILs) at a ratio of approximately 1:100-1:2000. Culture for approximately 0-4 days to obtain a third TIL population.

[0199] 1.4 Step (C) TIL cell gene editing

[0200] Based on the target gene screened by the present invention, a guide sequence was designed, thawed and added with nuclease-free water to a concentration of about 100 μM. About 2 μL of gRNA (50 μM) was incubated at 95°C for 2 minutes to anneal and then added to P3 buffer, and 0.3-1 μL of Cas9 (such as Kaixia, Ke Rui, Acro, 10 mg / mL) was added and incubated at 25°C for 10 minutes to form a ribonucleoprotein complex (RNP). In P3 buffer (Lonza), the above RNP was mixed with about 1×10 TILs of the third group using a Lonza electroporator. 6 The cells are electroporated. For example, the electroporation procedure can be human T cell stim (EO115). The electroporated cells are cultured for about 0-4 days after gene editing to obtain a fourth TIL population.

[0201] 1.5 Step (D) TIL cell culture after gene editing

[0202] Feeder cells (irradiated healthy donor PBMC T cells) are added to the fourth TIL cell population for culture. The time for contacting TIL with feeder cells needs to be several times T after the TIL is contacted with IL-2 and T cell activator (such as CD3 antibody or a nanomatrix containing CD3 antibody and CD28 antibody, such as transACT) in step (B). n Afterwards (T nThe time period can be from 0 hours to 12 days, for example, 24 hours or 48 hours. First, resuscitate the mixed feeder cells from 1-5 donors; mix the activated TIL cells and feeder cells at a ratio of approximately 1:200, transfer them to a G-Rex100 culture flask or breathable bag, and supplement with complete culture medium. Samples are taken and counted every 1-3 days, and the medium is replenished or half-filled depending on the cell status until the total number of cells is greater than 1×10 9 Alternatively, the in vitro expansion culture of step (D) is carried out for about 5 days to about 14 days, and the in vitro expansion culture of step (D) is terminated.

[0203] 1.6 Harvesting of Tumor-Infiltrating Lymphocytes

[0204] Take the cells amplified in step (D), centrifuge and discard the culture supernatant, and wash three times with PBS or saline or compound electrolyte solution to obtain TILs amplified in step (D) (fifth TIL population). Samples are counted during the third wash. According to the counting results, after the final centrifugation, discard the supernatant and take 3×10 6 The cells were sent for quality control testing; all the remaining cells were added to the freezing solution and the cell density was adjusted to 1-3×10 8 cells / mL for cryopreservation.

[0205] RASA2 Authentication

[0206] Gene knockout detection

[0207] Reagents and materials: DNA extraction solution (QuickExtract DNA extraction solution, Lucigen, QE09050), RNase / DNase free water (Tiangen), EDTA (Shenggong, 0.5 M), Recombinant DNase I (RNase-free, TAKARA).

[0208] Extract genomic DNA: About 2-7 days after tumor-infiltrating lymphocyte T cell knockout, take about 1×10 5 to about 2×10 5 Cells were washed once with PBS, and then the gene-edited cells were resuspended in 44 μL PBS. 6 μL of the prepared nuclease mixture (containing 1 μL DNase I and 5 μL 10× DNase I Buffer) was added and incubated at 37°C for 5 minutes. 2.5 μL of 0.5M EDTA was added to the sample and incubated at 80°C for 10 minutes. After centrifugation and discarding the supernatant, 50 μL of DNA extraction solution was added to the cell pellet. After a brief centrifugation, the following program was run: 75°C-10 minutes; 95°C-5 minutes; 4°C-maintain. The sample can be analyzed using a spectrophotometer (NanoDrop TM) to detect the concentration of DNA samples.

[0209] Sequencing: PCR primers can be designed in the region approximately 100 to 200 nucleotides upstream and downstream of the PAM site. Design the PCR reaction system as follows:

[0210] And amplify according to the following PCR program:

[0211] The PCR products were analyzed by Sanger sequencing.

[0212] Analyzing Crispr Cas9 knockout efficiency

[0213] Crispr Cas9 knockout efficiency was analyzed using the Tracking of Indels by DEcomposition (Tide) method based on Sager sequencing data. For specific methods, see (Brinkman et al, Nucl. Acids Res. (2014) or shinyapps.datacurators.nl / tide / ). Knockout efficiency analysis was performed by inputting the corresponding sgRNA sequence of the present invention, the pre-knockout control sequence, and the test sequence after Crispr Cas9 knockout, with a P-value threshold of 0.001.

[0214] Donor 812 was a patient with oral mucosal melanoma, and donor 904 was a patient with lung cancer. The sgRNA targeting RASA2, in cells derived from donor 812, the knockout efficiency of the CRISPR tool composed of two different sequences of guides targeting RASA2 was 35%, and in cells derived from donor 904, the knockout efficiency was 93.3%. The results show that various gene editing methods of the present invention can achieve a certain proportion of knockout efficiency. Among them, in Figures 10A to 10F, the RA2 group and the RA4 group represent the experimental groups edited with gRNA targeting RASA2 coded as RA2 (SEQ ID NO: 3) and RA4 (SEQ ID NO: 4), respectively.

[0215] Detection of cell proliferation

[0216] Experimental preparation

[0217] For the CD3 antibody group, 30 ng / ml CD3 antibody (Miltenyi Biotech, OKT3) was used to coat a flat-bottom 96-well plate one day in advance at 4°C overnight.

[0218] Starting from the 7th day after gene editing, tumor-infiltrating lymphocytes in each group were re-plated with the same total number of cells. The unstimulated group did not require CD3 antibody stimulation in the expansion efficiency test. The stimulated group was stimulated with 30 ng / mL CD3 antibody (Miltenyi Biotech, OKT3), and the fluorescence of T cells at the time of plating was analyzed using the CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega). The fluorescence of T cells was analyzed using the CTG kit 3 days later. The expansion efficiency of T cells was characterized by the fluorescence on the third day / the fluorescence at the time of plating.

[0219] Figure 10A shows that gene-edited T cells in the no-stimulation group can have significant expansion capacity.

[0220] Figure 10B shows that the gene-edited T cells in the CD3 antibody stimulation group can have a significant expansion capacity. The results show that compared with the control group (NT), the gene-edited T cells of the present invention can have a significant expansion capacity.

[0221] Cell killing ability assay

[0222] Starting six days after gene editing, tumor target cells were plated in 96-well flat-bottom plates. The following day, tumor-infiltrating lymphocytes (TILs) from each group were co-cultured with target cells at varying effector-to-target ratios (E:T). 100 μL of target cells and 100 μL of T cells were added, with triplicate wells set up for each group. A control group containing only target cells was also established.

[0223] According to the instructions for the apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL of the apoptosis detection reagent was added per well, and 25 μL of culture medium diluted with Caspase 3 / 7 Green Dye was added per well. Caspase 3 / 7 activity was recorded using an Incucyte recorder (Sartorius) to analyze T cell cytotoxicity, with recordings every 3 hours for approximately 5 days.

[0224] Figure 10C shows the target cell killing ability of gene-edited T cells. The results show that compared with the control group (NT), gene-edited T cells can have more significant target cell killing ability.

[0225] Cell flow cytometry

[0226] The tumor-infiltrating lymphocyte T cell population obtained on the 8th day after gene editing was used to detect cell expression by flow cytometry.

[0227] Sources of experimental materials for T cell flow cytometry

[0228] V-bottom 96-well plate, manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.

[0229] The flow cytometry antibodies in this example were purchased from BD or Biolegend. 5 to 5×10 5 Add a cell sample to a flow tube or a V-bottom 96-well plate. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, add 1mL / tube to the flow tube and 200μL / well to the 96-well plate, and discard the supernatant. Add the prepared antibody working solution for cell surface staining. The antibody (BD or Biolegend) concentration is 1:100 to 1:200, containing activity detection dye at 1:10000. Stain 100μL / tube of the flow tube and 50μL / well of the 96-well plate, and incubate at 2-8℃ in the dark for 30 minutes. After surface staining, wash the cells once with PBS (200μL / time for 96-well plate and 1mL / time for flow tube), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant after centrifugation. Resuspend the cells in 100-500μL PBS and perform flow cytometry detection.

[0230] Figure 10D shows that gene-edited T cells have a lower proportion of exhausted T cells. For example, exhausted T cells can be CD38-positive and / or CD101-positive cells.

[0231] Cytokine expression flow cytometry

[0232] The cytokine expression of tumor-infiltrating lymphocyte T cell populations obtained on the 7th or 8th day after gene editing was detected by flow cytometry.

[0233] Experimental preparation

[0234] For the CD3 antibody group, 30 ng / ml CD3 antibody (Miltenyi Biotech, OKT3) was used to coat a flat-bottom 96-well plate one day in advance at 4°C overnight.

[0235] Prepare the culture medium required for intracellular factor expression analysis: Take T cell culture medium and add the following volume ratios: Golgistop 0.7:1000, Golgiplug 1:1000, and CD107a antibody 1:500, for a total of 2 μL / mL. Do not add interleukins.

[0236] Detection steps

[0237] After centrifugation, the tumor-infiltrating lymphocytes of each experimental group were resuspended in 600 μL of the culture medium required for the above-mentioned intracellular factor expression detection to a concentration of 1×10 6cells / mL, added into a 96-well plate, 200 μL / well, and incubated in a 37°C incubator overnight.

[0238] After incubation, wash once with 200 μL / well PBS, centrifuge at 600 g for 3 minutes, and discard the supernatant. Prepare a mixed antibody working solution for cell surface staining of CD3 / CD4 / CD8 (BD) at an antibody concentration of 1:100 and a cell viability assay dye concentration of 1:10,000. Stain 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube. Incubate at 2-8°C in the dark for 30 minutes. During the staining process, prepare the reagents required for transcription factor staining: dilute 4× Fixation / Permeabilization Buffer (BD) with Transcription Factor Buffer Set to 1× Working Solution A; dilute 5× Perm / Wash Buffer (BD) with double-distilled water to 1× Working Solution B. Pre-cool at 4°C until use. After staining, wash the cells twice with an appropriate amount of PBS (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes), centrifuge at 600 g for 3 minutes, and discard the supernatant. Cell fixation and membrane permeabilization: Resuspend the cells thoroughly and add an appropriate amount of 1× working solution A (100 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes) to fix and permeabilize the membrane. Incubate at 2-8°C in the dark for 40-50 minutes. After fixation and permeabilization, wash the cells with 1× working solution B (200 μL / well for 96-well plates, 2 mL / well for flow cytometry tubes), centrifuge at 2-8°C, centrifuge at 350 g for 6 minutes, and wash twice. Intracellular antibodies (CD107a, GZMB, TNF-α, and IFN-γ, BD / BioLegend) were prepared using 1× Working Solution B at a concentration of 1:100 to 1:200. 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube were added and stained for 30 minutes at 2-8°C in the dark. After staining, cells were washed with 1× Working Solution B (200 μL / well of a 96-well plate and 2 mL / well of a flow cytometry tube) and centrifuged twice at 350 g for 6 minutes at 2-8°C. Cells were resuspended in 100-500 μL of PBS and analyzed by flow cytometry.

[0239] Figure 10E shows that the gene-edited T cells in the unstimulated group had a higher cytokine expression ratio, for example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression.

[0240] Figure 10F shows that the gene-edited T cells in the stimulated group had a higher cytokine expression ratio, for example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression.

[0241] BRD4 Verification

[0242] A similar experimental method as described above was used to verify BRD4 separately in tumor-infiltrating lymphocytes. Donors 812 were patients with oral mucosal melanoma, and donors 904 were patients with lung cancer. sgRNA targeting BRD4, in cells derived from 812 donors, the knockout efficiencies of the CRISPR tool composed of two different sequences of guides targeting BRD4 were 74.9% and 82.2%, respectively, and in cells derived from 904 donors, the knockout efficiencies were 75.9% and 77.8%, respectively. The results showed that various gene editing methods of the present invention can achieve a certain proportion of knockout efficiency. Among them, in Figures 11A to 11F, the BD3 group and the BD4 group represent the experimental groups edited with gRNA targeting BRD4, code-named BD3 (SEQ ID NO: 5) and BD4 (SEQ ID NO: 6), respectively.

[0243] Figure 11A shows that gene-edited T cells in the no-stimulation group can have significant expansion capacity.

[0244] Figure 11B shows that the gene-edited T cells in the CD3 antibody stimulation group can have a significant expansion capacity. The results show that compared with the control group (NT), the gene-edited T cells of the present invention can have a significant expansion capacity.

[0245] Figure 11C shows that gene-edited T cells have a higher proportion of central memory T cells. For example, central memory T cells can be CD45RO-positive and CD62L-positive cells.

[0246] Figure 11D shows that gene-edited T cells have a lower proportion of exhausted T cells. For example, exhausted T cells can be CD38-positive, CD101-positive, PD-1-positive and / or TIM-3-positive cells.

[0247] Figure 11E shows that the gene-edited T cells in the unstimulated group had a higher cytokine expression ratio, for example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression.

[0248] Figure 11F shows that gene-edited T cells in the stimulated group had higher cytokine expression rates. For example, higher CD107a expression, higher IFN-γ expression, higher TNF-α expression, or higher GZMB expression. These results indicate that gene-edited T cells have more dominant cell subset composition and / or cytokine expression capacity.

[0249] The above-mentioned independent verification results show that the candidate genes related to cell functions that can be screened out by the screening platform of the present invention have ideal application prospects, which proves the advantageous effect of the screening platform of the present invention.

[0250] Example 7 Further screening of the screening platform of the present invention

[0251] By using the optimized screening platform of the present invention, more donor-derived T cells are obtained as cell samples, and candidate genes related to cell function are screened.

[0252] Figure 12 shows the gRNA distribution results analyzed after expanding the cell sample using this screening platform.

[0253] On the one hand, the targets with decreased gRNA numbers such as CD3D, RPS24, VAV1, IL2RG, CD247, RPL35, LCP2, LCK, and LAT are also genes related to T cell proliferation and activation signal transduction known in the art. Knocking out these target genes leads to immunodeficiency and inhibition of T cell proliferation. On the other hand, the screening platform of the present invention also sensitively enriches targets with increased gRNA numbers such as FOXO1, RASA2, BRD4, CYLD, PTPN2, and CBLB. These targets are also targets related to T cell inhibition or exhaustion signals known in the art, for example, potential inhibitory cell function. Knocking out this target will result in increased expression of the preferred screening function marker of the knockout analysis group.

[0254] The above results show that the screening platform of the present invention can detect candidate genes related to cell effector functions with high sensitivity and stability.

[0255] 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.

[0256] Sequence information of the present invention

Claims

1. A method for identifying a target, the method comprising: determining the effect of a candidate target on a cell by measuring the level of a cytokine expressed by the cell to be tested, wherein the cytokine is selected from the group consisting of an interferon family member, a tumor necrosis factor family member, and a lysosome-associated membrane protein family member; and before and / or simultaneously with the determination, the expression or activity of the candidate target in the cell to be tested is regulated.

2. A method for identifying a target, the method comprising the following steps: (1) regulating the expression or activity of the candidate target in a test cell; (2) determining the level of a cytokine expressed by the regulated test cell, wherein the cytokine is selected from the group consisting of an interferon family member, a tumor necrosis factor family member, and a lysosomal-associated membrane protein family member; and (3) determining the effect of regulating the candidate target on the cell based on the reading result obtained by the determination.

3. The method of any one of claims 1-2, wherein the interferon family member comprises IFN-γ.

4. The method of any one of claims 1-3, wherein the tumor necrosis factor family member comprises TNF-α.

5. The method of any one of claims 1-4, wherein the lysosomal-associated membrane protein family member comprises CD107a.

6. The method of any one of claims 1 to 5, wherein the regulation comprises introducing a target regulatory system into the cell to be tested, wherein the target regulatory system increases or decreases the expression or activity of the candidate target.

7. The method of any one of claims 1-6, wherein the modulation comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a meganuclease system and / or an inhibitory RNA.

8. The method of any one of claims 1 to 7, wherein the modulation comprises targeting a specific nucleic acid sequence of the target, introducing single-strand breaks, double-strand breaks and / or mutations into, upstream and / or downstream of the specific nucleic acid sequence.

9. The method of any one of claims 1-8, wherein the modulation comprises increasing or decreasing the expression or activity of the candidate target at the genomic level.

10. The method of any one of claims 1-9, wherein the regulation comprises introducing a target regulation system comprising a guide nucleic acid molecule and a nuclease into the test cell.

11. The method of claim 10, wherein the guide nucleic acid molecule comprises a guide RNA (gRNA) targeting the target.

12. The method of any one of claims 10-11, wherein the nuclease comprises a Cas protein, a Cas protein homolog, or a functionally active fragment thereof.

13. The method of any one of claims 10-12, wherein the nuclease comprises Cas 9 and / or Cas 12.

14. The method according to any one of claims 1 to 13, wherein in the cell population to be tested obtained by the adjustment, the proportion of cells expressing the candidate target is reduced to less than about 95%.

15. The method according to any one of claims 1 to 14, wherein before or simultaneously with the determination, the cells to be tested are allowed to express an exogenous cell receptor or a functional fragment thereof.

16. The method of claim 15, wherein the exogenous cell receptor comprises a T cell receptor.

17. The method according to any one of claims 15 to 16, wherein the test cell expressing an exogenous cell receptor or a functional fragment thereof is contacted with an antigen, wherein the antigen comprises an antigen specific for the exogenous cell receptor or a functional fragment thereof.

18. The method according to any one of claims 15 to 17, wherein before or simultaneously with the determination, the cells to be tested expressing the exogenous cell receptor or a functional fragment thereof are contacted with target cells expressing an antigen, wherein the antigen comprises a specific antigen of the exogenous cell receptor or a functional fragment thereof.

19. The method of any one of claims 15 to 18, wherein in the contacting, the cell ratio of the test cells expressing the exogenous cell receptor or a functional fragment thereof to the target cells expressing the antigen is about 1:100 to about 100:1, and the antigen comprises a specific antigen of the exogenous cell receptor or a functional fragment thereof.

20. The method according to any one of claims 15 to 19, wherein before or simultaneously with the assay, the cells to be tested are allowed to express an exogenous cell receptor or a functional fragment thereof, and the cells to be tested are contacted with a specific antigen of the exogenous cell receptor or a functional fragment thereof for about 0 hours to about 72 hours.

21. The method of any one of claims 15-20, wherein the test cell expressing the exogenous cell receptor or a functional fragment thereof is contacted with the target cell expressing the specific antigen of the exogenous cell receptor or a functional fragment thereof for about 6 hours, about 12 hours, about 16 hours, or 28 hours.

22. The method according to any one of claims 1 to 21, wherein before or simultaneously with the determination, the cells to be tested are contacted with a substance that inhibits cytokine secretion.

23. The method of claim 22, wherein the substance that inhibits cytokine secretion comprises brefeldin A and / or monensin.

24. The method of any one of claims 22-23, wherein the regulated test cells are contacted with a substance that inhibits cytokine secretion for about 0 hours to about 72 hours before or simultaneously with the determination.

25. The method of any one of claims 22-24, wherein the test cells to be regulated are contacted with the substance that inhibits cytokine secretion for about 4 hours, about 12 hours, or about 16 hours.

26. The method of any one of claims 1-25, wherein the determining comprises determining the level of expression of the cytokine by the regulated test cells by flow cytometry, Western blot or ELISA.

27. The method of any one of claims 1-26, wherein the determination comprises determining by flow cytometry the increase in the fold and / or amount of increase in the level of the cytokine expressed by the test cells in which the expression or activity of the candidate target is modulated, compared to the corresponding test cells in which the expression or activity of the candidate target is not modulated.

28. The method of any one of claims 1-27, wherein the test cells to be modulated comprise immune cells.

29. The method of claim 28, wherein the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

30. The method of any one of claims 28-29, wherein the immune cells comprise monocytes, macrophages and / or dendritic cells.

31. The method of any one of claims 28-30, wherein the immune cells comprise B cells, T cells, natural killer cells, regulatory T cells and / or natural killer-like T cells (NKT).

32. The method of any one of claims 28-31, wherein the immune cells comprise αβ T cells and / or γδ T cells.

33. The method of any one of claims 28-32, wherein the immune cells comprise tumor infiltrating lymphocytes (TILs).

34. methods as claimed in claim 33, wherein the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, TILs of fragments of paracancerous tissue, pleural effusion and / or peritoneal effusion and / or TILs revived after cryopreservation.

35. A system for identifying targets, the system comprising: a measurement module for measuring the level of cytokines expressed by cells to be tested, and determining the effect of candidate targets on the cells; wherein the cytokines are selected from interferon family members, tumor necrosis factor family members and lysosome-associated membrane protein family members; and before and / or simultaneously with the measurement, the expression or activity of the candidate target in the cells to be tested is regulated.

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 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 the candidate target; Module (1-a), used for causing the cells to be tested to express an exogenous cell receptor or a functional fragment thereof; Module (1-b), used for contacting the cells to be tested with the specific antigen of the exogenous cell receptor or its functional fragment; Module (1-c), used to contact the cells to be tested (cells that have or are about to be regulated by the candidate target) with a substance that inhibits cytokine secretion; Module (2) is used to measure the level of IFN-γ, TNF-α and / or CD107a expressed by the cells to be tested; Module (3), for determining 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 (1), modules (1-a), modules (1-b) and modules (1-c), and modules (1), modules (1-a), modules (1-b) and modules (1-c) are optionally arranged in parallel.

37. A system for identifying a target, the system comprising: Module (1-I) is used to introduce a guide nucleic acid of a CRISPR system into the cell to be tested, wherein the guide nucleic acid is capable of regulating the expression or activity of the candidate target; 4 guide nucleic acids are designed for each target gene for different targeting sequences, and the probability of introducing two or more guide nucleic acids into a single cell is at most about 10%; Module (1-a), used to make the cells to be tested express TCR targeting NYESO-1; Module (1-II), used to introduce an exogenous nuclease of the CRISPR system or a nucleic acid encoding the exogenous nuclease into the cell to be tested; Module (1-b), used for contacting the cells to be tested with target cells expressing NYESO-1 antigen; Module (1-c), used to contact the cells to be tested (cells that have or are about to be regulated by the candidate target) with a substance that inhibits cytokine secretion; Module (2) is used to determine the level of IFN-γ, TNF-α and / or CD107a expressed by the regulated cells to be tested; Module (3), for determining 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 arrangement of modules (1-I), modules (1-a), modules (1-II), modules (1-b) and modules (1-c), and modules (1-I), modules (1-a), modules (1-II), modules (1-b) and modules (1-c) can be optionally arranged in parallel.

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