Method for screening immune cell and use thereof

Abstract

Provided is a method for screening and preparing an immune cell, which specifically involves contacting an isolated immune cell with a tumor organoid / tumor cell, and screening and preparing a specific cell on the basis of the expression of a specific target. The immune cell obtained by the method exhibits a high specific recognition and killing capacity against tumors, including but not limited to a strong specific cytokine release capacity, a strong tumor-specific killing capacity, and / or high expression levels of tumor-specific recognition and activation markers in the screened immune cell, thereby improving the anti-tumor activity of the cell in cell therapy.

Description

Methods for screening immune cells and their applications Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method and application for screening specific cells. Background Technology

[0002] Currently, immunotherapy is an effective treatment for patients with poor prognoses. However, the immune cells used in immunotherapy have weak anti-tumor capabilities, mainly reflected in the low proportion of tumor-specific immune cells in cell products. Therefore, providing a culture method to enhance the specific recognition and / or killing ability of immune cells against tumor cells holds significant promise for the widespread clinical application of cell therapy.

[0003] However, since immune cell culture methods typically use interleukin-2, CD3 antibodies, and / or PBMCs from healthy donors, the anti-tumor capabilities of the resulting immune cells still need further improvement. Therefore, there is an urgent need in the art for a method to culture immune cells that enhances their specific tumor recognition and killing capabilities in vitro, and for their widespread application in cell therapy. Summary of the Invention

[0004] This invention provides a method and application for screening immune cells. The method of this invention has one or more of the following advantages: the screened immune cells have high specific recognition and killing ability for tumors, including but not limited to strong specific cytokine release ability, strong tumor-specific killing ability, and / or high expression level of tumor-specific recognition activation markers.

[0005] On one hand, the present invention provides a method for screening immune cells, the method comprising contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells, and then screening specific immune cells based on the nucleic acid expression of specific targets.

[0006] On the other hand, the present invention provides a method suitable for screening using single-cell sequencing to improve tumor-specific recognition and / or killing ability, comprising contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells.

[0007] On the other hand, the present invention provides a method for improving tumor-specific recognition and / or killing ability, which is suitable for screening by single-cell sequencing after processing. The method includes contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells.

[0008] On the other hand, the present invention provides a method comprising performing single-cell sequencing on immune cells isolated after contact treatment with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells (e.g., specific immune cells can be screened based on the expression of nucleic acids of specific targets).

[0009] On the other hand, the present invention provides a cell population obtained by the screening method of the present invention.

[0010] On the other hand, the present invention provides a cell population in which the nucleic acid expression level of a specific target is high. For example, the present invention provides the selection of specific targets.

[0011] On the other hand, the present invention provides a pharmaceutical composition comprising a cell population obtained by the screening method of the present invention, and optionally a pharmaceutically acceptable carrier.

[0012] On the other hand, the present invention provides a method for influencing tumor cell growth, comprising applying a cell population obtained by the screening method of the present invention and / or a pharmaceutical composition of the present invention.

[0013] On the other hand, the present invention provides the use of cell populations obtained by the screening method of the present invention and / or pharmaceutical compositions of the present invention in the preparation of pharmaceuticals for the prevention and / or treatment of tumors.

[0014] On the other hand, the present invention provides a medicament for the prevention and / or treatment of tumors, comprising a cell population obtained by the screening method of the present invention and / or a pharmaceutical composition of the present invention as an active ingredient.

[0015] On the other hand, the present invention provides a method for preventing and / or treating tumors, comprising administering to a subject in need a cell population obtained by the screening method of the present invention and / or a pharmaceutical composition of the present invention.

[0016] On the other hand, the present invention provides cell populations obtained by the screening method of the present invention and / or pharmaceutical compositions of the present invention for the prevention and / or treatment of tumors.

[0017] On the other hand, the present invention provides a method for screening tumor-specific TCRs, the method comprising:

[0018] a) Isolate a group of immune cells from a tumor sample;

[0019] b) Co-culture isolated immune cells with tumor cells, three-dimensional aggregates of tumor cells and / or cells derived from three-dimensional aggregates of tumor cells;

[0020] c) Construct a TCR expression library in co-cultured immune cells;

[0021] d) Screen for tumor-specific TCRs based on the TCR expression library.

[0022] In some implementations, the method further includes sequencing a TCR expression library to identify tumor-specific TCRs.

[0023] In some embodiments, the method further includes preparing tumor-specific TCR-T cells.

[0024] In some implementations, the method further includes co-culturing TCR-T cells with tumor cells, three-dimensional aggregates of tumor cells, and / or cells derived from three-dimensional aggregates of tumor cells, and verifying the tumor specificity of TCR by killing target cells.

[0025] On the other hand, the present invention provides a method for screening tumor-specific TCRs, the method comprising:

[0026] a) Isolate a group of immune cells from a tumor sample;

[0027] b) Co-culture the isolated immune cells with tumor-specific peptides;

[0028] c) Construct a TCR expression library in co-cultured immune cells;

[0029] d) Screen for tumor-specific TCRs based on the TCR expression library.

[0030] In some embodiments, the tumor-specific polypeptide is the full-length tumor-specific antigen or a functional fragment thereof.

[0031] In some implementations, the method further includes sequencing a TCR expression library to identify tumor-specific TCRs.

[0032] In some embodiments, the method further includes preparing tumor-specific TCR-T cells.

[0033] In some embodiments, the method further includes co-culturing TCR-T tumor-specific peptides and verifying the tumor specificity of TCRs by killing target cells.

[0034] In some implementations, screening for tumor-specific TCRs involves selecting cells characterized by high expression of tumor reactive targets and identifying them as tumor-specific cell subpopulations.

[0035] In some embodiments, the tumor reactive target is selected from one or more of the following group: (1) CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NOP16, NR4A2, EGR2, LINC00 892,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPINB9,BIRC3,VSIR,IL21R,RE L, MAT2A, ZFP36L1, TAGAP, JARID2, PLEK, and ZBTB32; or (2) CCL4L2, CCL3, CCL4, CSF2, KLRC1, IFNG, MKI67, TUBA1B, RRM2, TYMS, GNLY, STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE2C,TRGV3,FABP5,HMGN2,LINC0 0892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4,GZMA,TOP2A,CSF1,DUSP4,SMC4, MCM7, NKG7, HAVCR2, PKM, CDC20, SLC1A5, MTHFD2, IL2RA, PHLDA1, CAMK1, HNRNPAB, GAPDH, SLC7A5, TMPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2; preferably, tumor reactive targets are TNFRSF9, IFNG, and GZMB.

[0036] In some implementations, the three-dimensional aggregate of target cells comprises tumor organoids (e.g., tumor-derived organoids).

[0037] In some embodiments, the method comprises contacting isolated immune cells with target cells, target cell three-dimensional aggregates and / or cells derived from target cell three-dimensional aggregates, or tumor-specific peptides at a cell ratio of about 1:1 to 60:1.

[0038] In some embodiments, the method comprises contacting isolated immune cells with target cells, target cell three-dimensional aggregates and / or cells derived from target cell three-dimensional aggregates, or tumor-specific peptides for about 4 to 72 hours.

[0039] In some embodiments, the method further includes in vitro expansion of the immune cells before contacting the isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells, or tumor-specific polypeptides.

[0040] In some embodiments, the in vitro expansion includes culturing tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or ascites derived from tumor subjects in an IL-2 culture environment.

[0041] In some embodiments, the method further includes culturing the isolated immune cells in a culture environment containing IL-2 and anti-CD3 and / or anti-CD28 antibodies before, simultaneously and / or after contacting the isolated immune cells with target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells, or tumor-specific peptides.

[0042] In some implementations, the TCR expression library of the immune cells is obtained by single-cell sequencing.

[0043] In some embodiments, the specific immune cells, upon contact with tumor cells from the same subject source, are capable of producing and / or releasing more cytokines (e.g., GZMB, CD107a, TNF-α, or IFN-γ), specifically killing and / or recognizing more tumor cells, and / or expressing more tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107) compared to a population of uncontacted cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells, or tumor-specific peptides.

[0044] In some embodiments, the immune cells include T cells, natural killer cells, and / or natural killer-like T cells.

[0045] In some embodiments, the immune cells comprise αβT cells and / or γδT cells.

[0046] In some implementations, the immune cells comprise tumor-infiltrating lymphocytes.

[0047] In some embodiments, the immune cells comprise cells derived from the subject's tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or peritoneal effusion.

[0048] On the other hand, the present invention provides a cell population comprising tumor-specific TCRs obtained by the method described in any one of the present invention.

[0049] On the other hand, the present invention provides a pharmaceutical composition comprising the cell population described herein, and optionally a pharmaceutically acceptable carrier.

[0050] On the other hand, the present invention provides a method for influencing tumor cell growth, comprising administering the cell population described in the present invention, or the pharmaceutical composition described in the present invention.

[0051] On the other hand, the present invention provides the use of cell populations, and / or the pharmaceutical compositions described herein, in the preparation of medicaments for the prevention and / or treatment of tumors.

[0052] On the other hand, the present invention provides a medicament for the prevention and / or treatment of tumors, comprising the cell population described in the present invention and / or the pharmaceutical composition described in the present invention as active ingredients.

[0053] On the other hand, the present invention provides a method for preventing and / or treating tumors, comprising administering to a subject in need the cell population described in the present invention and / or the pharmaceutical composition described in the present invention.

[0054] On the other hand, the present invention provides the cell population and / or pharmaceutical composition for the prevention and / or treatment of tumors.

[0055] On the other hand, the present invention provides a method for screening immune cells, the method comprising contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and / or cells derived from three-dimensional aggregates of target cells, and then screening specific immune cells based on the expression of nucleic acids of specific targets.

[0056] In some implementations, the target cells comprise tumor cells.

[0057] In some implementations, the three-dimensional aggregate of target cells comprises tumor organoids (e.g., tumor-derived organoids).

[0058] In some embodiments, the method includes contacting isolated immune cells with target cells, three-dimensional aggregates of target cells, and / or cells derived from three-dimensional aggregates of target cells at a cell ratio of about 1:1 to 60:1.

[0059] In some embodiments, the method includes contacting isolated immune cells with target cells, target cell three-dimensional aggregates, and / or cells derived from target cell three-dimensional aggregates for approximately 4 to 72 hours.

[0060] In some embodiments, the method further includes in vitro expansion of the immune cells before contacting the isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells.

[0061] In some embodiments, the in vitro expansion includes culturing tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or ascites derived from tumor subjects in an IL-2 culture environment.

[0062] In some embodiments, the method further includes culturing the isolated immune cells in a culture environment containing IL-2 and anti-CD3 and / or anti-CD28 antibodies before, simultaneously and / or after contacting the isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells.

[0063] In some embodiments, the specific target is selected from one or more of the following group: (1) CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NOP16, NR4A2, EGR2, LINC00892,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPINB9,BIRC3,VSI R, IL21R, REL, MAT2A, ZFP36L1, TAGAP, JARID2, PLEK, and ZBTB32; or (2) CCL4L2, CCL3, CCL4, CSF2, KLRC1, IFNG, MKI67, TUBA1B, R RM2,TYMS,GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE2C,TRGV3 ,FABP5,HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4,GZMA, TOP2A,CSF1,DUSP4,SMC4,MCM7,NKG7,HAVCR2,PKM,CDC20,SLC1A5,MTHFD2,IL2RA,PHLDA1,CAMK1,HNRNPAB,GAPDH,SLC7A5,T MPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2.

[0064] In some implementations, the nucleic acid expression of the specific target of the immune cell is obtained by single-cell sequencing.

[0065] In some embodiments, a population of cells with high levels of nucleic acid expression of the specific target is identified as specific immune cells within the immune cells.

[0066] In some embodiments, the specific immune cells, upon contact with tumor cells from the same subject source, are able to produce and / or release more cytokines (e.g., GZMB, CD107a, TNF-α, or IFN-γ), specifically kill and / or recognize more tumor cells, and / or express more tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107) compared to the original cell population that has not been contacted with the target cells, the target cell three-dimensional aggregates, and cells derived from the target cell three-dimensional aggregates, respectively.

[0067] In some embodiments, the immune cells include T cells, natural killer cells, and / or natural killer-like T cells.

[0068] In some embodiments, the immune cells comprise αβT cells and / or γδT cells.

[0069] In some implementations, the immune cells comprise tumor-infiltrating lymphocytes.

[0070] In some embodiments, the immune cells comprise cells derived from the subject's tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or peritoneal effusion.

[0071] On the other hand, the present invention provides a cell population obtained by any one of the methods described in the present invention.

[0072] On the other hand, the present invention provides a cell population in which the nucleic acid expression level of a specific target is high, wherein the specific target is selected from one or more of the following group: (1) CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SD C4, NOP16, NR4A2, EGR2, LINC00892, BCL2L1, NME1, DDX21, PPAN, LAIR2, SLC7A5, ZBED2, KDM6B, NR4A1, NOLC1, CD82, IL4, NFKBID, SERPINB9, BIRC3, VSIR, IL21R, REL, MAT2A, ZFP36L1, TAGAP, JARID2, PLEK, and ZBTB32; or (2) CCL4L2, CCL3, CCL4, CSF2, KLRC1, IFNG, M KI67,TUBA1B,RRM2,TYMS,GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,U BE2C,TRGV3,FABP5,HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4 ,GZMA,TOP2A,CSF1,DUSP4,SMC4,MCM7,NKG7,HAVCR2,PKM,CDC20,SLC1A5,MTHFD2,IL2RA,PHLDA1,CAMK1,HNRNPAB,GAPDH,SLC7A 5, TMPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2.

[0073] On the other hand, the present invention provides a cell that expresses a T-cell receptor derived from the cell population described in the present invention.

[0074] On the other hand, the present invention provides a pharmaceutical composition comprising the cell population described in the present invention, and / or the cells described in the present invention, and optionally a pharmaceutically acceptable carrier.

[0075] On the other hand, the present invention provides a method for influencing tumor cell growth, comprising administering the cell population, the cells, and / or the pharmaceutical composition described herein.

[0076] The use of the cell population, the cells, and / or the pharmaceutical composition described in this invention in the preparation of a medicament for the prevention and / or treatment of tumors.

[0077] On the other hand, the present invention provides a medicament for the prevention and / or treatment of tumors, comprising the cell population, cells, and / or pharmaceutical composition described in the present invention as active ingredients.

[0078] On the other hand, the present invention provides a method for preventing and / or treating tumors, comprising administering to a subject in need the cell population, cells, and / or pharmaceutical composition described herein.

[0079] The cell populations, cells, and / or pharmaceutical compositions described in this invention are used for the prevention and / or treatment of tumors.

[0080] Other aspects and advantages of the invention will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the invention are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this invention enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention. Accordingly, the descriptions in the accompanying drawings and specification are merely exemplary and not restrictive. Attached Figure Description

[0081] A brief description of the attached figures is as follows:

[0082] Figure 1 illustrates an exemplary process for the expansion of tumor-infiltrating lymphocytes, the establishment of tumor organoids, and co-incubation according to the present invention.

[0083] Figure 2 shows CD8 + The cellular UMAP map delineates specific tumor-specific cell subpopulations.

[0084] Figure 3 shows the proportion of TCR-positive cells in the TCR-T cells to be validated after transduction of TCRs from two donors.

[0085] Figures 4A and 4B show the results of the secretion fold of IFN-γ and GZMB and the expression fold of CD137 after co-incubation of the TCR-T to be verified in this invention with matched PDO.

[0086] Figure 5 shows CD4 + The cellular UMAP map delineates specific tumor-specific cell subpopulations. Detailed Implementation

[0087] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0088] Terminology Definition

[0089] In this invention, the term "organoid" generally refers to a three-dimensional aggregate of one or more cell types. For example, the organoid is formed in vitro and exhibits a three-dimensional organ structure. For example, the organoid is capable of mimicking the surface appearance, actual structure, or function of a tissue or organ.

[0090] In this invention, the term "immune cell" generally refers to cells involved in innate and adaptive immune responses. For example, this may include, but is not limited to, lymphocytes (such as T cells (including thymocytes) and B cells), natural killer (NK) cells, NKT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and mast cells. In some embodiments, T cells include, for example, CD4-positive T cells, CD8-positive T cells (also known as cytotoxic T cells or CTLs), regulatory T cells (Tregs), Th1 cells, Th2 cells, Th17 cells, αβ T cells, and / or γδ T cells. In some embodiments, immune cells also include modified immune effector cells, such as chimeric antigen receptor (CAR) modified immune effector cells (e.g., CAR-T cells, CAR-NK cells) and T cell receptor (TCR) modified immune effector cells (e.g., TCR-T cells).

[0091] In this invention, the term "chimeric antigen receptor (CAR)" generally refers to an engineered antigen receptor. For example, a CAR may comprise an extracellular antigen-binding domain fused to a cytoplasmic domain comprising a signal transduction domain via a hinge and transmembrane domain. In some embodiments, the CAR extracellular domain may bind to an antigen expressed by a target cell in an MHC-independent manner, thereby leading to cell activation and proliferation. In some embodiments, the CAR extracellular domain may recognize a tag fused to an antibody or its antigen-binding fragment. For example, a single CAR construct may be able to target a variety of different antigens by replacing one antibody with another. In some embodiments, the CAR extracellular domain may comprise an antigen-binding fragment derived from an antibody. Antigen-binding domains that can be used in this disclosure may include, for example, scFv, antibodies, antigen-binding regions of antibodies, variable regions of heavy / light chains, and / or single-chain antibodies.

[0092] In this invention, the term "T-cell receptor (TCR)" generally refers to an engineered antigen receptor. For example, a TCR may comprise TCRα and / or TCRβ chains that have been isolated and cloned from a population of T cells that recognize a specific target antigen. For example, the TCRα and / or TCRβ genes (i.e., TRAC and TRBC) may be cloned from a population of T cells isolated from an individual with a specific malignancy or from a population of T cells isolated from humanized mice immunized with specific tumor antigens or tumor cells. Engineered TCRs can recognize antigens through the same mechanism as their endogenous counterparts (e.g., by recognizing their homologous antigens presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of target cells), thereby leading to the activation and proliferation of TCR-engineered cells.

[0093] In this invention, the term "encoding" generally refers to the ability to directly or indirectly infer the structural or compositional information of another class of molecules from the structural or compositional information of one molecule, based on substantially established rules. For example, the nucleotide sequence can be inferred from the amino acid sequence, such as based on the property of DNA transcription of complementary nucleic acids, including nucleic acids capable of translation into polypeptides. For example, DNA can encode RNA transcribed from DNA. Similarly, DNA can encode polypeptides translated from RNA transcribed from DNA.

[0094] In this invention, the term "NK cell" is also called "natural killer cell," which generally refers to a cell with large granules in its cytoplasm. NK cells develop from bone marrow lymphoid stem cells and can differentiate and develop dependent on the bone marrow or thymus microenvironment. In this invention, the proportion of NK cells in TIL cells can be altered using the method of this invention.

[0095] In this invention, "CD4"+ "Cell" usually refers to CD4-positive cells, such as T cells. The term "CD4" is used to describe this. + "Cells" and "CD4-positive cells" can be used synonymously. These cells can be identified by methods known in the art, such as staining the cells with a fluorescently labeled antibody against CD4 and sorting the cells using fluorescence-activated cell sorting.

[0096] In this invention, "CD8" + "Cells" usually refers to CD8-positive cells, such as T cells. The term "CD8" is used to describe... + "Cells" and "CD8-positive cells" can be used synonymously. These cells can be identified by methods known in the art, such as staining the cells with a fluorescently labeled antibody against CD8 and sorting the cells using fluorescence-activated cell sorting.

[0097] In this invention, the term "tumor-infiltrating lymphocytes" or "TIL" generally refers to a cell population initially obtained as leukocytes that has left the subject's bloodstream and migrated into the tumor. TILs may include, but are not limited to, CD8+. + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs can include primary TILs and secondary TILs. "Primary TILs" can be those TIL cells obtained from a subject's tissue sample, and "secondary TILs" can be any TIL population that has been expanded or amplified in this invention. In some embodiments, the tumor-infiltrating lymphocytes of this invention can be unisolated and unpurified, or can be interinfiltrating with tumor cells. For example, the TILs of this invention can refer to TIL populations.

[0098] In this invention, the term "stage" in the terms "a phase of in vitro expansion," "single-stage in vitro expansion," or "first-stage in vitro expansion," generally refers to a segment of the expansion process that TILs undergo in vitro. In one embodiment, each stage can be defined by a change in the number of TIL cells. In one embodiment, TIL cells are considered to have entered the next stage of in vitro expansion when the number of TIL cells increases by at least about 1 time. In some embodiments, TIL cells are considered to have entered the next stage of in vitro expansion when the number of TIL cells increases by at least about 1 to 50 times, for example, at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times. In one implementation, each stage can be defined by the conditions of TIL cell culture. In one implementation, TIL cells are considered to have entered the next stage of in vitro expansion after the addition or supplementation of T cell activators and / or T cell growth factors to the cell culture medium. In one implementation, TIL cells are considered to have entered the next stage of in vitro expansion after centrifugation and / or cell washing. In one implementation, each stage can also be defined by the number of days of TIL cell culture. In one implementation, after TIL cells have been cultured in vitro for approximately 1 to 100 days, such as approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 30 days, approximately 40 days, approximately 50 days, or approximately 100 days, the TIL cells can be considered to have entered the next stage of in vitro expansion.

[0099] In this invention, the term "first-stage in vitro expansion" generally refers to the stage of expansion using T-cell growth factors after obtaining primary TILs from tissue. In one embodiment, the tissue of this invention may be selected from the group consisting of tumor tissue and pleural effusion, wherein the pleural effusion of this invention may be the pleural effusion of a patient with metastatic cancer. In one embodiment, the expansion of this invention may be autologous or allogeneic in vivo expansion, or it may be in vitro expansion. The first-stage in vitro expansion of this invention may also be referred to as the preREP (pre-rapid expansion) stage. For example, TILs derived from tumor tissue that have not been expanded in vitro may be referred to as the first TIL group. For example, TILs obtained through the first-stage in vitro expansion in a two-step TIL culture method may be referred to as the second TIL group.

[0100] In this invention, the term "second-stage in vitro expansion" generally refers to a stage where tissue taken from a subject and expanded, followed by further expansion, is performed. In one embodiment, the number of TILs expanded in the second stage of this invention is increased compared to those expanded in the first stage, for example, by at least about 10 times (or at least about 20, 30, 40, 50, 60, 70, 80, or 90 times), or in one embodiment, by at least about 100 times. In one embodiment, the culture conditions for the second-stage in vitro expansion may differ from those for the first-stage in vitro expansion, for example, the culture medium added may be different. For example, in the two-step culture method of this invention, the second-stage in vitro expansion may also be referred to as the REP (Rapid Expansion) stage. For example, in the two-step TIL culture method, the TILs obtained after the second-stage in vitro expansion may be referred to as the third TIL population.

[0101] In this invention, the term "tumor-specific cell" generally refers to cells that can specifically recognize and / or kill tumors. Tumor-specific cells may possess specific tumor-specific recognition and killing capabilities, express tumor-specific recognition activation markers, or have the ability to release tumor-specific cytokines. For example, tumor-specific cells can be identified by co-culturing with a specific tumor and detecting cytokine expression, production and / or release, and / or tumor cell apoptosis. Tumor-specific cells may exhibit a more specific ability to inhibit tumor growth compared to ordinary cells.

[0102] In this invention, the term "single-cell sequencing" generally refers to a method that allows the identification of multiple coding elements in a single cell. This includes sequencing genomic elements such as nuclear or organelle DNA, their transcripts, or a combination of both. Typically, coding elements in a single cell can be physically, spatially, or via cell-specific barcodes that allow for the correct cellular allocation of elements after sequencing. For example, single-cell RNA sequencing (scRNA-seq) is used to identify expression patterns and / or variable sequences of immune receptors. For example, the scRNA-seq method allows for the parallel identification of mRNA from at least 1000 cells using droplet-based sorting techniques such as 10X Genomicschromium. Other alternative methods for characterizing cell expression patterns and / or TCR sequences include GeoMx™ or CosMx™ from NanoString Technologies, Visium spatial gene expression from 10X Genomics, or ZipSeq, which can correlate this information through spatial distribution in samples such as FFPE (formalin-fixed paraffin embedding).

[0103] In this invention, the term "isolation" generally refers to alteration or removal from a natural state. For example, cells, nucleic acids, or peptides naturally present in living animals are not "isolated," but cells, nucleic acids, or peptides in the same state that are partially or completely isolated are "isolated." Isolated cells, nucleic acids, or proteins may exist in substantially purified forms or may exist in non-natural environments, such as delivery carriers.

[0104] In this invention, the term "treatment" generally refers to treatment and / or prevention. The therapeutic effect is achieved by suppressing, alleviating, or eradicating a disease state.

[0105] In this invention, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the active ingredient. For example, a pharmaceutically acceptable carrier may not interfere with the biological activity of the active ingredient; for example, a pharmaceutically acceptable carrier may not interfere with the effectiveness of the biological activity possessed by the active ingredient. Such carriers may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable carriers may also contain compatible solid or liquid fillers, diluents, or encapsulating substances suitable for human administration. Other contemplated carriers, excipients, and / or additives that may be used in the formulations described herein may include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming ions (such as sodium), etc. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art. In this invention, "pharmaceuticalally acceptable carrier" can be understood as a carrier that does not contain a nucleic acid form used in genetic engineering.

[0106] In this invention, the term "tumor tissue" generally refers to a sample of tissue from a tumor in the object, including any solid tumor and / or non-solid tumor in the object. The term "tumor sample" generally refers to a sample or sample library obtained from a patient's tumor. The tumor may also contain metastases or a collection of metastases.

[0107] In this invention, the terms "about" and "approximately" generally refer to a statistically significant range of values. Such a range may be within an order of magnitude of a given value or range, and may include within 50%, preferably within 20%, more preferably within 10%, and most preferably within 5%. The permissible variations included in the terms "about" or "approximately" may depend on the specific system under investigation and will be readily understood by those skilled in the art.

[0108] The terms “above,” “below,” “at most,” and “at least” include the stated number.

[0109] Invention Details

[0110] The content of this invention is partly based on the following findings: In the process of screening for tumor-specific immune cells, cell surface markers of immune cells are typically used as sorting markers. For example, after co-incubating immune cells with tumor cells, cell populations positive for cell surface proteins (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107) are sorted as tumor-specific cells. However, the specific immune cells obtained by this method have weak cell function; for example, their cytokine release capacity and specific killing ability need to be improved. Furthermore, this method of sorting cells first and then performing cell / TCR sequencing easily leads to the loss of true positives or the introduction of false positives, affecting the accuracy of screening and sequencing. In addition, the accuracy of the specific immune cells obtained by this method is not high; most of the specific immune cells, after verification, cannot specifically recognize tumor cells.

[0111] This invention unexpectedly discovered that by contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells, specific immune cells can be screened based on their T-cell receptor (TCR) expression or the nucleic acid expression of specific targets. For example, after immune cells contact target cells, or three-dimensional aggregates of target cells, or cells derived from three-dimensional aggregates of target cells, or tumor-specific polypeptides (e.g., full-length antigens or their functional fragments), a TCR expression library can be constructed using single-cell sequencing. Based on the mRNA expression of specific targets, specific immune cell populations can be clustered to obtain specific immune cell populations. For example, by using TCR sequencing, the nucleic acid and / or protein sequences of the TCR (T-cell receptor) in the aforementioned immune cell population can be obtained. For example, by expressing the TCR (T-cell receptor) sequence in the aforementioned immune cell population, tumor-specific TCR-T cells can be obtained. The positive rate of tumor-specific TCRs obtained by screening through single-cell sequencing after contact with target cells, contact with three-dimensional aggregates of target cells, contact with cells derived from three-dimensional aggregates of target cells, or contact with tumor-specific peptides in this invention can exceed 50%, for example, exceeding 50%, exceeding 55%, exceeding 60%, exceeding 65%, exceeding 70%, exceeding 75%, exceeding 80%, exceeding 85%, exceeding 90%, exceeding 95%, or exceeding 99%. Among the three tests of IFN-γ secretion, GZMB secretion, and CD137 expression, if two of the following folds (the difference between the numerical result of TCR-T after co-incubation with matched PDO and the numerical result after culture in culture medium, and the difference between the numerical result of untransduced T cells after co-incubation with matched PDO and the numerical result after culture in culture medium, and the fold between the two differences) are greater than or equal to 1.0 (e.g., greater than 1.0, greater than 1.1, greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, greater than 10, greater than 100), the TCR-T is considered validated and is a positive tumor-specific TCR.

[0112] On one hand, the present invention provides a method for screening tumor-specific TCRs, the method comprising:

[0113] a) Isolate a group of immune cells from a tumor sample;

[0114] b) Co-culture isolated immune cells with tumor cells, three-dimensional aggregates of tumor cells and / or cells derived from three-dimensional aggregates of tumor cells;

[0115] c) Construct a TCR expression library in co-cultured immune cells;

[0116] d) Screen for tumor-specific TCRs based on the TCR expression library.

[0117] In some implementations, the method further includes sequencing a TCR expression library to identify tumor-specific TCRs. Any suitable TCR sequencing method and TCR expression library construction known in the art can be used for sequencing and library construction in this method.

[0118] In some embodiments, the method further includes preparing tumor-specific TCR-T cells. In some embodiments, the method further includes co-culturing TCR-T cells with tumor cells, three-dimensional aggregates of tumor cells, and / or cells derived from three-dimensional aggregates of tumor cells, and verifying the tumor specificity of TCRs by killing target cells.

[0119] On the other hand, the present invention also provides a method for screening tumor-specific TCRs, the method comprising:

[0120] a) Isolate a group of immune cells from a tumor sample;

[0121] b) Co-culture the isolated immune cells with tumor-specific peptides;

[0122] c) Construct a TCR expression library in co-cultured immune cells;

[0123] d) Screen for tumor-specific TCRs based on the TCR expression library.

[0124] In some embodiments, the tumor-specific polypeptide is the full-length tumor-specific antigen or a functional fragment thereof. Any tumor-specific antigen well-known in the art can be used in the method described in this invention.

[0125] In some embodiments, the method further includes co-culturing TCR-T tumor-specific peptides and verifying the tumor specificity of TCRs by killing target cells.

[0126] In some embodiments, screening for tumor-specific TCRs includes selecting cells characterized by high expression of tumor-reactive targets to identify a tumor-specific cell subpopulation. In some embodiments, the tumor-reactive targets are selected from one or more of the following group: (1) CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NOP16, NR4A2, EGR2, LINC00892,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPINB9,BIRC3,VSI R, IL21R, REL, MAT2A, ZFP36L1, TAGAP, JARID2, PLEK, and ZBTB32; or (2) CCL4L2, CCL3, CCL4, CSF2, KLRC1, IFNG, MKI67, TUBA1B, R RM2,TYMS,GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE2C,TRGV3 ,FABP5,HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4,GZMA, The target tumor markers are TOP2A, CSF1, DUSP4, SMC4, MCM7, NKG7, HAVCR2, PKM, CDC20, SLC1A5, MTHFD2, IL2RA, PHLDA1, CAMK1, HNRNPAB, GAPDH, SLC7A5, TMPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2. Preferably, the tumor-reactive targets are TNFRSF9, IFNG, and GZMB.

[0127] In some implementations, the TCR expression library of the immune cells is obtained by single-cell sequencing.

[0128] In some embodiments, compared to a population of uncontacted cells selected from target cells, target cell three-dimensional aggregates, and cells derived from target cell three-dimensional aggregates, or tumor-specific peptides, the specific immune cells, upon contact with tumor cells from the same subject, are capable of producing and / or releasing more cytokines (e.g., GZMB, CD107a, TNF-α, or IFN-γ), specifically killing and / or recognizing more tumor cells, and / or expressing more tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107).

[0129] On one hand, the present invention provides a method for screening immune cells, the method comprising contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells, and then screening specific immune cells based on the nucleic acid expression of specific targets.

[0130] For example, the method includes contacting isolated immune cells with target cells (e.g., tumor cells) and then screening for specific immune cells based on the expression of specific target nucleic acids. For example, the method includes contacting isolated immune cells with target cell three-dimensional aggregates (tumor organoids, tumor-derived organoids) and then screening for specific immune cells based on the expression of specific target nucleic acids. For example, the method includes contacting isolated immune cells with isolated cells after digestion of target cell three-dimensional aggregates (tumor organoids, tumor-derived organoids) and then screening for specific immune cells based on the expression of specific target nucleic acids. For example, the cells derived from the three-dimensional cell aggregates include cells dissociated from the three-dimensional cell aggregates. For example, the extracellular matrix of the three-dimensional cell aggregates can be disrupted using a commercially available kit (cell digestion solution or collagen hydrolysate), causing the cells in the three-dimensional cell aggregates to dissociate.

[0131] For example, the contact method described in this invention may include co-culturing tumor organoids with immune cells, the method comprising the step of mixing organoids or tumor-specific peptides as described herein with immune cells in in vitro culture. Mixing may include sequentially layering T cells and organoids into the same wells in a multi-well plate, or may include sequentially pipetting T cells and organoids into a gel. For example, cells may be digested and dissociated from organoids and co-cultured with the immune cells.

[0132] For example, in the method, isolated immune cells are contacted with target cells, three-dimensional aggregates of target cells, and / or cells derived from three-dimensional aggregates of target cells, or tumor-specific polypeptides, at a cell ratio of about 1:1 to 100:1, preferably about 1:1 to 60:1. For example, the method includes contacting the isolated immune cells with cells from dissociated three-dimensional aggregates of cells at a cell ratio of about 1:100, about 1:50, about 1:20, about 1:10, about 1:5, 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 50:1, or about 100:1.

[0133] For example, in the method, isolated immune cells are contacted with target cells, three-dimensional aggregates of target cells, and / or cells derived from three-dimensional aggregates of target cells, or tumor-specific polypeptides for approximately 1 hour to 7 days, preferably approximately 4 hours to 72 hours. For example, the method includes contact for approximately 1 hour to 7 days. For example, the method includes contact for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 15 hours, approximately 18 hours, approximately 20 hours, approximately 22 hours, approximately 24 hours, approximately 26 hours, approximately 28 hours, approximately 30 hours, approximately 32 hours, approximately 34 hours, approximately 36 hours, approximately 38 hours, approximately 40 hours, approximately 42 hours, approximately 44 hours, approximately 46 hours, approximately 48 hours, approximately 50 hours, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days.

[0134] For example, a method for preparing organoids includes culturing tumor-derived cells in an organoid culture medium to prepare the at least one organoid. For example, the organoid culture medium (optionally including any extracellular matrix) is removed from the at least one organoid before mixing it with immune cells. The extracellular matrix can be destroyed using a commercially available kit (cell digestion solution or collagen hydrolysate). For example, an alternative matrix can be used instead of the removed matrix.

[0135] For example, the method of the present invention further includes in vitro expansion of the immune cells before contacting the isolated immune cells with tumor organoids and / or cells derived from tumor organoids. The co-culture of immune cells with material derived from tumor organoids can be in vitro and / or ex vivo.

[0136] For example, the organoids of the present invention may comprise autologous cells (i.e., cells obtained from the same patient) or be composed of allogeneic cells (i.e., cells obtained from different patients). For example, organoids may be obtained by culturing tumor cells. For example, the organoids of the present invention comprise organoids in a culture medium containing interleukins (such as IL-2, IL-7, IL-15, or IL-21). For example, at least one of the organoids comprises or is composed of mammalian cells. For example, organoids may be isolated into populations sharing one or more genotypes, phenotypes, and / or epigenetic markers before being mixed with immune cells. For example, organoids that are partially matched with the immune cells of the present invention (where partially matched means that the immune cells and the organoids may have at least one identical HLA type) may be used in the methods of the present invention.

[0137] For example, organoid culture media can be used to prepare organoids for co-culture, for instance, by promoting growth, division (expansion), structural tissue formation, or other development to produce organoids suitable for co-culture. Suitable organoid culture media for different tissues are known in the art, for example. Preferred organoid culture media contain Wnt agonists, mitotic growth factors (e.g., selected from EGF, FGF, HGF, and BDNF), and / or BMP inhibitors. For example, organoid culture media may also contain TGF-β inhibitors.

[0138] For example, organoid culture media may contain one or more basal media (such as DMEM / F12 medium, Gibco), Wnt ligands, Wnt agonists, BMP inhibitors, EGF and TGF-β inhibitors, and optionally also contain one or more of the following: p38 MAPK inhibitors, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or antimicrobial agents (such as primary cell antibiotics (primocin)).

[0139] For example, a co-culture medium for immune cells and tumor organoid-derived cells may comprise a portion of immune cell culture medium (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) and a portion of organoid cell culture medium (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). For instance, in a preferred embodiment, the co-culture medium comprises approximately 50% immune cell culture medium (such as T cell culture medium, e.g., RPMI 1640 (Gibco)) and approximately 50% organoid culture medium.

[0140] For example, by providing biomaterials or synthetic materials that interact with cell membrane proteins, cells for forming organoids can be cultured, mimicking cellular niches. For instance, the extracellular matrix (ECM) used for culturing organoids comprises any biomaterial or synthetic material, or a combination thereof. For example, one type of ECM is secreted by epithelial cells, endothelial cells, luminal endodermal-like cells, and connective tissue cells. This ECM includes various polysaccharides, water, elastin, and glycoproteins, wherein glycoproteins include collagen, entactin / nidogen, fibronectin, and laminin. For example, in some embodiments, collagen is used as the ECM. Different types of ECM are known, including various compositions containing different types of glycoproteins and / or different combinations of glycoproteins.

[0141] Examples of commercially available extracellular matrix (ECM) include extracellular matrix proteins (Invitrogen) and basement membrane formulations derived from EHS mouse sarcoma cells (e.g., Cultrex basement membrane extract (Trevigen) or Matrigel (BD Biosciences)). For example, the ECM is Matrigel (BD Biosciences) containing laminin, nestin, and type IV collagen. In some embodiments, the ECM contains laminin, nestin, type IV collagen, and heparan sulfate proteoglycan. For example, the ECM can be a synthetic ECM. For example, a synthetic ECM (such as ProNectin) can be used. In another example, the ECM can be a plastic (such as polyester) or a hydrogel. In some embodiments, the synthetic ECM can be coated with biological materials, such as one or more glycoproteins, like collagen or laminin.

[0142] For example, the method further includes in vitro expansion of the immune cells prior to the contact. For example, the method includes in vitro expansion of the isolated immune cells for approximately 2 hours to 28 days, such as approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 10 days, approximately 14 days, or approximately 28 days, prior to contacting the isolated immune cells with a three-dimensional aggregate of cells and / or cells derived from the three-dimensional aggregate of cells.

[0143] For example, the in vitro expansion includes culturing tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion and / or ascites derived from tumor subjects in a culture environment of IL-2 at a concentration of 300 to 9000 IU / mL.

[0144] For example, the method further includes culturing the immune cells in a culture environment containing IL-2 at a concentration of 300 to 9000 IU / mL and anti-CD3 and / or anti-CD28 antibodies before and / or after the contact.

[0145] For example, the method of the present invention includes: (A) contacting a first TIL group derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion and / or ascites, and not expanded in vitro, with one or more T cell growth factors (such as IL-2), wherein a second TIL group is obtained by step (A); (B) contacting the second TIL group with a T cell activator (such as anti-CD3 antibody and / or anti-CD28 antibody) and / or T cell growth factors, and optionally performing gene editing, wherein a third TIL group is obtained by step (B); (C) co-culturing the third TIL group with feeder cells, wherein a fourth TIL group is obtained by step (C).

[0146] For example, before step (A), between step (A) and step (B), between step (B) and step (C), and / or after step (C) of the present invention, the isolated immune cells may be contacted with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells. For example, for the first TIL group, the second TIL group, the third TIL group, and / or the fourth TIL group obtained by the present invention, the isolated immune cells may be contacted with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells. For example, step (A) is cultured for approximately 3-14 days. For example, step (B) is cultured for approximately 3-14 days. For example, step (C) is cultured for approximately 3-14 days.

[0147] For example, the method of the present invention includes: (A) contacting a first group of TILs derived from tumor tissue, tumor-associated lymphoid tissue with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion and / or ascites, and not expanded in vitro, with one or more T cell growth factors (such as IL-2), wherein a second group of TILs is obtained after approximately 10 to 14 days following step (A); (B) contacting the second group of TILs with a T cell activator (such as anti-CD3 antibody and / or anti-CD28 antibody) and / or T cell growth factors, and optionally performing gene editing, wherein a third group of TILs is obtained after approximately 6 hours to 8 days following step (B); (C) co-culturing the third group of TILs with feeder cells, wherein a fourth group of TILs is obtained after approximately 10 to 14 days following step (C); and (D) contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells, wherein a fifth group of TILs is obtained after approximately 6 hours to 8 days following step (D). Optionally, from the fifth TIL population obtained, single-cell sequencing can be used to obtain a cell population with high levels of nucleic acid expression of specific targets, which is then identified as a specific immune cell (population).

[0148] For example, when immune cells come into contact with target cells, three-dimensional aggregates of target cells, or cells derived from three-dimensional aggregates of target cells, or with tumor-specific peptides, single-cell sequencing can be used to obtain the nucleic acid expression status of the specific target on the immune cells after contact. For example, single-cell expression library sequencing can be used to obtain the nucleic acid expression status of the specific target on the immune cells after contact. For example, while obtaining the nucleic acid expression status of the specific target on the immune cells after contact through single-cell expression library sequencing, TCR sequencing can be performed to obtain the TCR sequence in the corresponding cells.

[0149] For example, downstream analysis of sequencing data from expression libraries can yield single-cell expression matrices. Alternatively, TCR clonality, TCR sequence, and VDJ genotyping can be obtained from sequencing data of TCR libraries corresponding to individual cell barcodes. For example, cluster analysis can be performed based on the nucleic acid expression levels (expression matrix) of each gene in a single cell to obtain cell populations (also known as clusters) with high levels of nucleic acid expression for specific targets. For example, the clustering method of this invention can include unsupervised clustering. For example, the clustering method can include Louvain modularization algorithm, SLM algorithm, Leiden algorithm, k-means clustering, fuzzy k-means clustering algorithm, or Jarvis-Patrick clustering. For example, the method of this invention first standardizes the nucleic acid expression levels of each gene in a single cell, performs dimensionality reduction analysis using PCA, and further obtains two-dimensional projections and individual clusters from the PCA dimensionality reduction results using UMAP (Uniform Manifold Approximation and Projection) and clustering algorithms. For example, the dimensionality reduction method of the present invention may also include t-SNE, Sammon mapping, curve component analysis, random neighborhood embedding, isometric mapping, maximum variance expansion, local linear embedding, or Laplacian eigenmaps. For example, among the obtained clusters, clusters with the characteristic of high expression of tumor reactive targets provided by the present invention are identified and confirmed as tumor-specific cell subpopulations.

[0150] For example, among the immune cells, a cell population with a high level of nucleic acid expression of the specific target is identified as a specific immune cell. For example, the cell population with a high level of nucleic acid expression of the specific target may refer to a cell population selected in this invention whose expression level of the specific target nucleic acid is increased by 1% or more, for example, from about 1% to 10,000 times, compared to the ungrouped original cell population. For example, an increase of approximately 10,000 times, approximately 1,000 times, approximately 100 times, approximately 50 times, approximately 40 times, approximately 30 times, approximately 20 times, approximately 10 times, approximately 9 times, approximately 8 times, approximately 7 times, approximately 6 times, approximately 5 times, approximately 4 times, approximately 3 times, approximately 2 times, approximately 1 time, approximately 99%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%.

[0151] For example, the cell population with high nucleic acid expression level of the specific target can refer to the cell population selected in this invention that has a 1% or higher increase in the nucleic acid expression level of the specific target compared to the remaining cell population (i.e., the original cell population that has come into contact with the target cells, the three-dimensional aggregates of the target cells and the cells derived from the three-dimensional aggregates of the target cells, excluding the cell population selected in this invention). For example, the increase is about 1% to 10,000 times. For example, an increase of approximately 10,000 times, approximately 1,000 times, approximately 100 times, approximately 50 times, approximately 40 times, approximately 30 times, approximately 20 times, approximately 10 times, approximately 9 times, approximately 8 times, approximately 7 times, approximately 6 times, approximately 5 times, approximately 4 times, approximately 3 times, approximately 2 times, approximately 1 time, approximately 99%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%.

[0152] For example, among the immune cells, a cell population with high levels of nucleic acid expression of the specific target is identified as specific immune cells. For example, the cell population with high levels of nucleic acid expression of the specific target can refer to a cell population selected in this invention whose specific target nucleic acid expression level is increased by 1% or more, for example, from about 1% to 10,000 times, relative to a reference cell population (e.g., a primitive cell population that has not been in contact with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells). For example, an increase of approximately 10,000 times, approximately 1,000 times, approximately 100 times, approximately 50 times, approximately 40 times, approximately 30 times, approximately 20 times, approximately 10 times, approximately 9 times, approximately 8 times, approximately 7 times, approximately 6 times, approximately 5 times, approximately 4 times, approximately 3 times, approximately 2 times, approximately 1 time, approximately 99%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%.

[0153] For example, the specific targets described in this invention are selected from the following group: CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NOP16, NR4A2, EGR2, LINC00892 ,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPINB9,BIRC3,VSIR,IL21R ,REL,MAT2A,ZFP36L1,TAGAP,JARID2,PLEK,ZBTB32; or CCL4L2,CCL3,CCL4,CSF2,KLRC1,IFNG,MKI67,TUBA1B,RRM2,TYMS, GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE2C,TRGV3,FABP5, HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4,GZMA,TOP2A The ingredients are CSF1, DUSP4, SMC4, MCM7, NKG7, HAVCR2, PKM, CDC20, SLC1A5, MTHFD2, IL2RA, PHLDA1, CAMK1, HNRNPAB, GAPDH, SLC7A5, TMPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2. For example, preferably selected from one or more of the following group: CCL4L2, CSF2, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, GZMB, FABP5, CSF1, LINC00892, and SLC7A5. More preferably selected from one or more of the following group: TNFRSF9, IFNG, and GZMB.

[0154] For example, compared to the original cell population that has not been in contact with cells selected from target cells, target cell three-dimensional aggregates, and cells derived from target cell three-dimensional aggregates, the specific immune cells, upon contact with tumor cells from the same subject, are able to produce and / or release more cytokines (e.g., GZMB, CD107a, TNF-α, or IFN-γ), specifically kill more tumor cells (e.g., by detecting target cell death through co-incubation with target cells, such as by target cell counting, continuous recording of target cell apoptosis using incucyte, live cell imaging, etc.), and / or express more tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107).

[0155] For example, the immune cells comprise tumor-infiltrating lymphocytes isolated and cultured from tissue samples of melanoma, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, or kidney cancer. In embodiments of the invention, the immune cells comprise tumor-infiltrating lymphocytes isolated and cultured from tissue samples of advanced, metastatic, and / or recurrent tumors. In embodiments of the invention, tumor tissue includes, but is not limited to, tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or ascites.

[0156] For example, immune cells may comprise cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, natural killer T-like (NKT) cells, tumor-infiltrating lymphocytes (TILs), or lymphokine-activated killer (LAK) cells. For example, immune cells may comprise αβT cells and / or γδT cells. For example, immune cells may comprise CAR cells and / or TCR cells. The methods described herein can be used to treat a variety of diseases, including cancer, infectious diseases, and immunodeficiency. In one embodiment, the immune cells are autologous to the patient. In this case, the cells transferred into the patient comprise autologous cells to prevent rejection or adverse immune responses to the administered cells.

[0157] On the other hand, immune cell populations are isolated from fluid tissues containing immune cells, such as bone marrow or ascites. Immune cells, such as lymphocytes (e.g., TILs, CTLs, NK cells, and LAK cells), can be isolated using various methods known in the art. For example, in one method of isolating CTLs, allogeneic restricted CTLs are generated by in vitro stimulation of native spleen cells with a suitable antigen. For example, blood samples containing cell precursors from mammals can be purified to obtain PBLs (peripheral blood lymphocytes) and incubated with cells stimulated by a specific antigenic peptide. Human primary NK cells can be expanded in the presence of bone marrow cell lines that have been genetically engineered to express NK cell-specific molecules. LAK cells can be generated, for example, by treating a patient's monocytes with interleukin-2. Monocytes can be collected, for example, by repeated lymphocyte ablation using a continuous flow cell separator. In some implementations, immune cells, such as lymphocytes (e.g., TILs, CTLs, NK cells, or LAK cells), are isolated using affinity purification steps such as FACS (fluorescence-activated cell sorting), MACS (magnetic-activated cell sorting), or batch purification using antibodies against appropriate surface antigens. In some cases, the obtained immune cells, such as lymphocytes (e.g., TILs, CTLs, NK cells, or LAK cells), comprise a proliferative population. In other cases, the obtained cell population may not have clonal capacity or unlimited proliferative capacity.

[0158] For example, methods for culturing tumor-infiltrating lymphocytes (TILs) include: obtaining TILs from a subject's tissue sample, which can be an in situ tumor sample or a metastatic tumor sample obtained during patient surgery, weighing at least about 1 g, or multiple tissue samples combined. Tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or peritoneal effusion are transported at approximately 2-8°C in a sample transport medium, such as commercially available tumor tissue transport solutions, tumor tissue preservation solutions, or tumor tissue transport solutions, and processed within 48 hours. Tissue blocks can be mechanically broken down to approximately 1-27 cubic millimeters each, transferred into a breathable culture bag or Grex, and cultured in serum-free cell culture medium and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL) for approximately 3-14 days. Cells are collected from the culture medium and transferred into a breathable culture bag, or a Grex or Xuri device. The serum-free culture medium can be supplemented with the CD28 antibody, CD3 antibody, and CD28 antibody of the present invention, magnetic beads (e.g., Dynabeads) containing CD3 antibody and CD28 antibody, and / or nanomatrix (e.g., transACT) containing CD3 antibody and CD28 antibody, IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL), and optionally, the expression of the target gene in the cell population is edited (e.g., by transduction gene editing using a ribonucleoprotein complex (RNP) carrying gRNA and Cas protein, or an LNP containing gRNA and Cas protein, or an LNP containing nucleic acid encoding gRNA and Cas protein). After activating the TIL of the present invention for a certain period of time, three-dimensional cell aggregates or cells derived from three-dimensional cell aggregates are added (TIL to cells derived from three-dimensional cell aggregates at a ratio of about 1:1 to about 60:1), and the culture is expanded for about 3-14 days. Cells can be collected from the culture medium using a cell processing system, washed, cryopreserved, and analyzed. The final product can have a CD3 content greater than 80%, a cell viability greater than 50%, and cells with a CD3 content greater than 80% can be considered memory effector cells and effector cells. Upon stimulation, they can secrete IFN-γ and / or exhibit an upregulated proportion of activated cells. For example, between the various steps in the above method, steps such as enrichment of CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1, contact with target cells, three-dimensional aggregates of target cells, or cells derived from three-dimensional aggregates of target cells, and / or single-cell sequencing can be performed to screen specific immune cells based on the expression of specific target nucleic acids.

[0159] Suitable conditions for cell culture include appropriate culture media (e.g., minimum essential medium or RPMI 1640) that may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFP, and TNF-α, or any other additives known to the technician for cell growth.

[0160] On the other hand, the present invention provides a cell population obtained by the method of the present invention.

[0161] On the other hand, the present invention provides a cell population in which the nucleic acid expression level of a specific target is high. For example, the specific target of the present invention is selected from the group consisting of: CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NOP16, NR4A2, EGR2, LINC00892. ,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPINB9,BIRC3,VSIR,IL21R ,REL,MAT2A,ZFP36L1,TAGAP,JARID2,PLEK,ZBTB32; or CCL4L2,CCL3,CCL4,CSF2,KLRC1,IFNG,MKI67,TUBA1B,RRM2,TYMS, GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE2C,TRGV3,FABP5, HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4,GZMA,TOP2A The specific targets described in this invention are selected from one or more of the following groups: CCL4L2, CSF2, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, GZMB, FABP5, CSF1, LINC00892, and SLC7A5.For example, the specific targets described in this invention are selected from one or more of the following group: TNFRSF9, IFNG, GZMB.

[0162] For example, the cell products of the present invention, after contact with cell three-dimensional aggregates or cells derived from cell three-dimensional aggregates, or tumor-specific peptides, and screening based on the expression of nucleic acids of specific targets, can be used for the discovery of specific TCRs. For example, the type or sequence of antigen-binding receptors in the screened cells can be determined. For example, TCRs derived from the screened cells can be used to develop engineered TCR cells. For example, compared to cell populations that have not been contacted with cell three-dimensional aggregates or cells derived from cell three-dimensional aggregates, TCRs obtained from the cell populations screened according to the present invention, when used to engineer cells, show an increase in tumor specificity of approximately 10,000 times to approximately 1%, for example, an increase of approximately 10,000 times, approximately 1,000 times, approximately 100 times, approximately 50 times, approximately 40 times, approximately 30 times, approximately 20 times, approximately 10 times, approximately 9 times, approximately 8 times, approximately 7 times, approximately 6 times, approximately 5 times, etc. Approximately 4-fold, approximately 3-fold, approximately 2-fold, approximately 1-fold, approximately 99%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%. For example, tumor specificity can be detected by measuring the levels of tumor-specific cytokines such as IFN-γ produced and / or released after exposure to specific matched tumor cells, the expression of specific activating molecular markers, and / or the number of tumor cells and / or the level of apoptosis after exposure to specific matched tumor cells.

[0163] On the other hand, the present invention provides a TCR comprising a T cell receptor derived from the specific immune cells described in the present invention. On the other hand, the present invention provides a nucleic acid molecule encoding a T cell receptor derived from the specific immune cells described in the present invention. On the other hand, the present invention provides a nucleic acid vector comprising a nucleic acid molecule encoding a T cell receptor derived from the specific immune cells described in the present invention. On the other hand, the present invention provides a cell expressing a T cell receptor derived from the specific immune cells described in the present invention. On the other hand, the present invention provides a pharmaceutical composition comprising the cell population of the present invention, and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a suspension of cells in a sterile buffer. Cells expanded using the present invention may be administered via any suitable route known in the art. In some embodiments, cells may be administered by a single intra-arterial or intravenous infusion lasting approximately 30 to 60 minutes. Other suitable routes of administration may include intraperitoneal, intrathecal, and intralymphatic administration.

[0164] For example, compared to the original cell population that has not been in contact with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells, TCRs derived from specific immune cells identified in this invention, when expressed in T cells, are able to produce and / or release more cytokines (e.g., GZMB, IFN-γ, CD107a, TNF-α), specifically kill and / or recognize more tumor cells, and / or express more tumor-specific recognition markers (e.g., selected from CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107) upon contact with tumor cells from the same subject.

[0165] In one embodiment, a TCR derived from the contacted immune cells is expressed in T cells, and the TCR-T then produces IFN-γ. For example, compared to a primitive cell population that has not been contacted with cells derived from or representing cellular three-dimensional aggregates, a TCR derived from the specific immune cells identified in this invention, when expressed in T cells, produces and / or releases IFN-γ at an amount increased by approximately 100,000-fold to approximately 1% upon contact with tumor cells from the same subject, for example, by approximately 100,000-fold, approximately 10,000-fold, approximately 1,000-fold, approximately 100-fold, approximately 50-fold, approximately 40-fold, approximately 30-fold, approximately 20-fold, etc. Approximately 10 times, approximately 9 times, approximately 8 times, approximately 7 times, approximately 6 times, approximately 5 times, approximately 4 times, approximately 3 times, approximately 2 times, approximately 1 time, approximately 99%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%. For example, cytokine production and / or release can be detected by cytokine assay kits and / or flow cytometry.

[0166] For example, compared to a primitive cell population that has not been in contact with cells that have come into contact with or are derived from cellular three-dimensional aggregates, when TCRs derived from specific immune cells identified in this invention are expressed in T cells, the proportion of cells expressing tumor-specific recognition markers selected from CD39, CD103, CD137, OX40, ICOS, CD25, CD69, or PD-1 is increased by approximately 10,000-fold to approximately 1% of the total cells after contact with tumor cells from the same subject, for example, by approximately 10,000-fold, approximately 1,000-fold, or approximately 100-fold. Approximately 50 times, approximately 40 times, approximately 30 times, approximately 20 times, approximately 10 times, approximately 9 times, approximately 8 times, approximately 7 times, approximately 6 times, approximately 5 times, approximately 4 times, approximately 3 times, approximately 2 times, approximately 1 time, approximately 99%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%. For example, the expression rate of cell markers can be detected by flow cytometry.

[0167] For example, compared to a primitive cell population that has not been in contact with cells derived from or associated with cellular three-dimensional aggregates, when TCRs derived from specific immune cells identified in this invention are expressed in T cells, the TCR-T cells, upon contact with tumor cells derived from the same subject, exhibit a tumor cell apoptosis level that is approximately 10,000-fold to approximately 1%, for example, an increase of approximately 10,000-fold, approximately 1,000-fold, approximately 100-fold, approximately 50-fold, approximately 40-fold, approximately 30-fold, approximately 20-fold, approximately 10-fold, and approximately 9-fold. Approximately 8 times, approximately 7 times, approximately 6 times, approximately 5 times, approximately 4 times, approximately 3 times, approximately 2 times, approximately 1 time, approximately 99%, approximately 95%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%.

[0168] On the other hand, the present invention provides a method for influencing tumor cell growth, comprising administering the cell population of the present invention, the cells of the present invention, and / or the pharmaceutical composition of the present invention. For example, after obtaining the above-mentioned cells, a cell culture and expansion step can be performed to obtain the therapeutically required amount of cells. For example, after obtaining the above-mentioned cells, a cell culture and expansion step and / or a cell reinfusion step can be performed to prevent and / or treat tumors.

[0169] On the other hand, the present invention provides the use of the cell population of the present invention, the cells of the present invention, and / or the pharmaceutical composition of the present invention in the preparation of a medicament for the prevention and / or treatment of tumors.

[0170] On the other hand, the present invention provides a medicament for the prevention and / or treatment of tumors, comprising the cell population of the present invention, the cells of the present invention, and / or the pharmaceutical composition of the present invention as active ingredients.

[0171] On the other hand, the present invention provides a method for preventing and / or treating tumors, comprising administering to a subject in need the cell population of the present invention, the cells of the present invention, and / or the pharmaceutical composition of the present invention.

[0172] On the other hand, the present invention provides cell populations, cells, and / or pharmaceutical compositions of the present invention for the prevention and / or treatment of tumors.

[0173] In some implementations, any suitable dose of cells can be administered. In some implementations, for example when the tumor is melanoma, approximately 1 × 10⁻⁶ cells can be administered. 9 Approximately 13.7 × 10 10 1 cell, or approximately 2.3 × 10 9 Approximately 13.7 × 1010 Cells. In some implementations, approximately 1 × 103 cells may be applied. 9 Approximately 12×10 10 Cells. In some implementations, approximately 1.2 × 10⁻⁶ cells may be applied. 10 Approximately 4.3 × 10 10 Cells. In some implementations, approximately 3 × 10⁶ cells may be applied. 10 Approximately 12×10 10 Cells. In some implementations, approximately 4 × 10⁶ cells may be applied. 10 Approximately 10×10 10 Cells. In some implementations, approximately 5 × 10⁶ cells may be applied. 10 Approximately 8×10 10 Cells. In some implementations, approximately 6 × 10⁶ cells may be applied. 10 Approximately 8×10 10 Cells. In some implementations, approximately 7 × 10⁶ cells may be applied. 10 Approximately 8×10 10 Cells. In some embodiments, the effective therapeutic dose can be approximately 1 × 10⁻⁶ cells. 9 Approximately 13.7 × 10 10 1 cell, or approximately 2.3 × 10 9 Approximately 13.7 × 10 10 In some implementations, the effective therapeutic dose may be approximately 1 × 10⁻⁶. 9 Approximately 12×10 10 Cells. In some embodiments, the therapeutically effective dose can be approximately 1.2 × 10⁻⁶ cells. 10 Approximately 4.3 × 10 10 Cells. In some embodiments, the effective therapeutic dose can be approximately 3 × 10⁻⁶ cells. 10 Approximately 12×10 10 Cells. In some embodiments, the effective therapeutic dose can be approximately 4 × 10⁻⁶ cells. 10 Approximately 10×10 10 Cells. In some embodiments, the effective therapeutic dose can be approximately 5 × 10⁻⁶ cells. 10 Approximately 8×10 10 Cells. In some embodiments, the effective therapeutic dose can be approximately 6 × 10⁻⁶. 10 Approximately 8×10 10 Cells. In some embodiments, the effective therapeutic dose can be approximately 7 × 10⁻⁶ cells. 10 Approximately 8×10 10 Each cell.

[0174] In some embodiments, cells can be administered in a single dose. This administration can be by injection, for example, intravenous injection. In some embodiments, cells can be administered in multiple doses. The dose can be once, twice, three times, four times, five times, six times, or more than six times per year. The dose can be once a month, once every two weeks, once a week, or once every two days. In some embodiments, cell administration can be continuous.

[0175] On one hand, the present invention provides a method for influencing cell (such as tumor cells) growth, which may include administering the cells of the present invention and / or the pharmaceutical composition of the present invention to a subject. In some embodiments, influencing tumor growth may include reducing the volume of the tumor to about 99-0.1% of its pre-administration state, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0176] On one hand, the present invention provides the use of the cells of the present invention and / or the pharmaceutical compositions of the present invention in the preparation of medicaments, which can be used to prevent and / or treat diseases and / or symptoms. For example, the diseases and / or symptoms of the present invention may include tumors. In some embodiments, the tumors of the present invention are selected from solid tumors. In some embodiments, the tumors of the present invention may be selected from one or more of the group consisting of: melanoma, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer. For example, the tumors of the present invention may be advanced solid tumors.

[0177] On one hand, the present invention provides a method for preventing and / or treating diseases and / or symptoms, which may include administering the cells of the present invention and / or the pharmaceutical composition of the present invention to a subject. For example, the diseases and / or symptoms of the present invention may include tumors. In some embodiments, the tumor of the present invention is selected from solid tumors. In some embodiments, the tumor of the present invention may be selected from one or more of the following groups: melanoma, ovarian cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer. For example, the tumor of the present invention may be an advanced solid tumor.

[0178] On one hand, the present invention provides a cell and / or a pharmaceutical composition of the present invention, which can be used to prevent and / or treat diseases and / or symptoms. For example, the diseases and / or symptoms of the present invention may include tumors. In some embodiments, the tumor of the present invention is selected from solid tumors. In some embodiments, the tumor of the present invention may be selected from one or more of the following groups: melanoma, ovarian cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer. For example, the tumor of the present invention may be an advanced solid tumor.

[0179] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the methods and uses of the present invention and are not intended to limit the scope of the invention.

[0180] Example

[0181] Example 1: Expansion of tumor-infiltrating lymphocytes, establishment of tumor organoids, and co-incubation 1.1 Receiving and processing of tumor tissue

[0182] Receive tumor tissue from the donor, verify and record the sample information, and print the corresponding sample label.

[0183] 1.2 Tissue processing, TIL culture and expansion, tumor single-cell digestion and cryopreservation

[0184] Add an appropriate amount of rewarmed complete culture medium to a culture dish, and add IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, or 6000 IU / mL). Using sterile ophthalmic forceps, remove tumor tissue from the sample tube into the culture dish, wash the tissue, and replace the culture dish. Use ophthalmic scissors and forceps to perform initial cutting, removing adipose and necrotic tissue. Use a disposable scalpel to further chop each tissue block into pieces approximately 1-27 cubic millimeters in size.

[0185] Using pipettes, 60-80% of the tumor tissue was transferred to 90% of the non-suspended tumor tissue blocks at approximately 0.1-0.2 g / well into 6-well culture plates pre-filled with 3 ml of complete culture medium. The remaining 20-40% of the tumor tissue was resuspended in X-vivo medium using a tissue digestion enzyme combination (Mitteni), and digested in a single-cell suspension preparer (RWD) according to the corresponding tissue digestion protocol. After digestion, the cells were filtered to obtain a tumor-derived single-cell suspension, counted, and cryopreserved (subpopulation a). The culture plate was placed in a CO2 incubator, and medium was added or half-replaced according to the cell state until prePRP was harvested, yielding the preREP TILs population (subpopulation b). After harvesting preREP TILs, the cells were counted, resuspended in complete culture medium, and the cell concentration was adjusted to 2 × 10⁶ cells / well. 6After stimulating the cells with OKT3 (30 ng / ml) for 24-48 hours, add feeder cells at a ratio of 1:50-200. Place the culture plate in a CO2 incubator and replenish or replace half of the medium according to the cell status until the PRP is harvested to obtain the REP TILs population (c subpopulation).

[0186] 1.3 PDO Model Cultivation

[0187] The remaining 10% of non-suspension tumor tissue blocks from the TILs culture in step 1.2 above were further minced with a scalpel and added to a special tissue digestion solution (Organoid Dissociation Solution, BioGenous). Digestion was carried out at 37°C for 2-5 minutes. After digestion, 5 volumes of medium containing 10% FBS were added to terminate the digestion. The tissue suspension was filtered through a 100μm filter, washed, and thoroughly mixed with Martigel on ice. The mixture was then spot-spread onto the bottom of a cell culture plate, which was placed in a 37°C incubator. After the Martigel had fully solidified, complete culture medium was carefully added for further culture. Once the organoids grew to approximately the size of a soybean, they were passaged or cryopreserved to obtain the PDO (Patient-Derived Organoid) model. Continuous passage was performed to obtain the PDO (d subset).

[0188] 1.4 Co-incubation of REP TILs with PDO

[0189] The PDO(d subset) amplified in step 1.3 was digested and counted, with approximately 2.5 × 10⁻⁶ cells retained. 5 Up to 2×10 6 One PDO cell was resuspended in 1 ml of T cell culture medium, and the remaining PDO cells were frozen. The REP TILs population (c subset) obtained in step 1.2 was harvested, counted, and approximately 1 × 10⁻⁶ cells were retained. 7 Up to 2×10 7 Each cell was resuspended in 9 ml of T cell culture medium. The PDO single-cell suspension was mixed with the TILs cell suspension and added to 1-2 wells of a 6-well culture plate for co-incubation. The ratio of TILs to PDO-derived tumor single cells was approximately 1:1 to 60:1, for example, 1:1-10:1. The culture plate was placed in a CO2 incubator for incubation. The co-incubation time for TILs and tumor single cells was approximately 4 to 72 hours, for example, 6, 8, 10, 12, 20, 24, 36, or 48 hours.

[0190] Figure 1 illustrates an exemplary process for the expansion of tumor-infiltrating lymphocytes, the establishment of tumor organoids, and co-incubation according to the present invention.

[0191] Example 2: Construction and sequencing of single-cell expression libraries and TCR libraries

[0192] Collect REP TILs (e subset) after co-incubation with PDO in step 1.4 above, wash with sequencing buffer (PBS + 0.04% BSA), resuspend and count. Take the cell suspension corresponding to the number of cells loaded, prepare the reaction system according to the official operating procedure provided by 10x Genomics, and then inject the sample and paraffin oil into the corresponding well of the chip (10x Genomics) to complete the sample loading. According to the operating procedure, load the chip into the droplet builder (10x Genomics) and complete the droplet construction according to the standard guideline. Next, also according to the official operating procedure provided by Genomics, complete the droplet demulsification, nucleic acid recovery, cDNA amplification, expression library construction and quality control, V(D)J amplification, library construction and quality control, etc., to finally obtain the expression library and TCR library of PDO-stimulated TILs. Sequencing of the TILs expression library and TCR library was performed according to the sequencing adapters added to the library sequence during library construction (Illumina).

[0193] Example 3: Bioinformatics Analysis and Identification of Tumor-Specific TCR Subgroups

[0194] The sequencing data obtained in Example 2 above were used to perform downstream analysis on the sequencing data of TIL expression libraries and TCR libraries using the default parameters and default workflow of CellRanger (e.g., ver. 7.0.0). The Unique Molecular Identifier (UMI) readings corresponding to a single cell barcode were calculated to obtain the single-cell expression matrix file of TILs and the TCR clonal type corresponding to a single cell barcode, including TCR sequence, VDJ genotyping and other information.

[0195] Cluster analysis of TILs single-cell expression matrix files was performed using the R package Seurat (e.g., R package ver. 4.2.3; Seurat ver. 4.1.3). The expression matrix was first filtered to remove low-quality cells with fewer than 200 detected genes and a mitochondrial gene proportion greater than 15%, as well as genes expressed in fewer than 3 cells. The filtered expression matrix was first normalized using LogNormalize, followed by normalization and PCA analysis using highly variable genes. For multiple batches and samples, Harmony was used to batch-correct the PCA results, and UMAP was used for dimensionality reduction analysis. The Louvain algorithm was used to obtain the individual clusters of the single-cell RNA sequencing data. Among the obtained clusters, those exhibiting high expression of the tumor reactive markers provided in this invention were identified as tumor-specific cell subpopulations.

[0196] Figure 2 shows the CD8 + Cells are delineated in the UMAP map to identify specific tumor-specific cell subpopulations. Figure 5 shows the effect on CD4. + Cells were delineated in UMAP maps to identify specific tumor-specific cell subpopulations. TNFRSF9, IFNG, and GZMB were used as tumor reactive markers to delineate these subpopulations.

[0197] By combining cell barcode analysis with dimensionality reduction clustering of single-cell expression matrices and TCR library analysis, TCR clonal types present in the defined tumor-specific cell subpopulations were identified as tumor-specific TCRs. TCRs with a clone number greater than or equal to 3 (meaning at least three cells in the defined tumor-specific cell subpopulation express this TCR) were determined as preferred tumor-specific TCRs.

[0198] Example 4: Validation of the efficacy of the tumor-specific TCR determined by the present invention

[0199] 4.1 TCR sequence synthesis, viral packaging, and TCR-T construction

[0200] Based on the tumor-specific TCRs V(D)J sequence obtained according to the procedure of the present invention, the nucleic acid encoding the TCR is obtained and its efficacy is verified. The nucleic acid of the TCR to be verified is transferred into T cells from various sources, such as peripheral blood or stem cell differentiation, preferably PBMC cells, according to methods known in the art, such as viral transduction, LNP transfection, or electroporation, thereby obtaining the TCR-T cells to be verified.

[0201] For example, T cell transduction can be performed using a retroviral vector containing a nucleic acid fragment encoding a tumor-specific TCR V(D)J sequence to be validated. One day prior to transduction, 250 μL of recombinant human fibrin fragment (Takara) at a final concentration of 15 μg / mL was coated onto each 24-well suspension plate and left overnight. The coated 24-well plates were then removed, and 500 μL of blocking buffer containing 2% BSA was added for blocking at room temperature for 30 minutes. The blocking buffer was discarded, and the plates were washed twice. Then, 1–100 μL of retroviral buffer was added to each well, and the plates were centrifuged at 32°C, 2000 g, for 2 hours. The supernatant from the 24-well plates was discarded, and 500–1000 μL of resuscitated and activated T cells were added to each well, resulting in a cell concentration of approximately 5 × 10⁻⁶ cells. 5 Cells / mL, centrifuged at 30-32℃, 1000g for 10 minutes. After centrifugation, the culture plate was placed in a 37℃, 5% CO2 incubator to obtain transduced cells. After transduction, the cells were cultured for approximately 0-4 days to obtain the TCR-T cell population to be validated. The TCR expression ratio was detected by flow cytometry (Beckman-Coulter).

[0202] Figure 3 shows the proportion of TCR-positive cells in the TCR-T cells to be validated after transduction of TCRs from two donors.

[0203] In TCR-T cells, all TCRs to be verified were expressed normally. Cell counts and viability were performed every 1–3 days. Based on the count results, T cell culture medium was added, along with recombinant human IL-2 at a concentration of 30–100 IU / ml, to adjust the initial culture cell density to 0.5–2 × 10⁶ cells / ml. 6 Cells / ml, continue culturing.

[0204] Example 5: Validation of TCR-T cell tumor specificity

[0205] The PDO or tumor single cells obtained and cryopreserved in Example 1.3 were thawed, cultured overnight with IFN-γ (20 ng / ml, Biolegend), and then PDO cells were digested and counted or tumor cells were directly counted. After washing and resuspending, they were co-incubated with the TCR-T cell population obtained in 4.1 at a ratio of 4:1 or 1:1 at 37°C for 6 hours. After co-incubation, the supernatant was collected, and the secretion of IFN-γ and GZMB in the culture supernatant was detected by cytometric bead array (BD). The expression of CD137 in the TCR-T cell population was detected by flow cytometry (Beckman Coulter), as shown in Figure 5.

[0206] Figures 4A and 4B show the results of the secretion fold of IFN-γ and GZMB and the expression fold of CD137 after co-incubation of the TCR-T to be verified in this invention with matched PDO (Tables 1 and 2).

[0207] Among them, if two of the three tests of IFN-γ secretion, GZMB secretion, and CD137 expression show a fold increase (the difference between the numerical result of TCR-T after co-incubation with matched PDO and the numerical result after culture in culture medium, and the difference between the numerical result of control untransduced T cells after co-incubation with matched PDO and the numerical result after culture in culture medium, and the fold increase between the two differences) is greater than or equal to 1.2 times, the TCR-T is considered validated and is a validated positive tumor-specific TCR.

[0208] Table 1. IFN-γ secretion, GZMB secretion and CD137 expression

[0209] Table 2. IFN-γ secretion, GZMB secretion and CD137 expression

[0210] The results show that the method of the present invention can be used to obtain tumor-specific cells and tumor-specific TCRs. The tumor-specific TCRs obtained by transducing cells, after co-incubation with corresponding tumor cells, exhibit significantly enhanced cytokine secretion capacity and increased expression ratio of the CD137 tumor reactivity marker.

[0211] 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 variations of the embodiments described herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

Claims

1. A method for screening tumor-specific TCRs, the method comprising: A group of immune cells was isolated from a tumor sample; The isolated immune cells were co-cultured with tumor cells, three-dimensional aggregates of tumor cells, and / or cells derived from three-dimensional aggregates of tumor cells. Construct a TCR expression library in co-cultured immune cells; Tumor-specific TCRs were screened based on the TCR expression library.

2. The method according to claim 1, further comprising sequencing a TCR expression library to identify tumor-specific TCRs.

3. The method according to claim 1 or 2, wherein the method further comprises preparing tumor-specific TCR-T cells.

4. The method according to claim 3, further comprising co-culturing TCR-T cells with tumor cells, three-dimensional aggregates of tumor cells and / or cells derived from three-dimensional aggregates of tumor cells, and verifying the tumor specificity of TCR by killing target cells.

5. A method for screening tumor-specific TCRs, the method comprising: A group of immune cells was isolated from a tumor sample; The isolated immune cells were co-cultured with tumor-specific peptides; Construct a TCR expression library in co-cultured immune cells; Tumor-specific TCRs were screened based on the TCR expression library.

6. The method according to claim 5, wherein the tumor-specific polypeptide is the full-length tumor-specific antigen or a functional fragment thereof.

7. The method according to claim 5 or 6, wherein the method further comprises sequencing a TCR expression library to identify tumor-specific TCRs.

8. The method according to any one of claims 5-7, wherein the method further comprises preparing tumor-specific TCR-T cells.

9. The method according to claim 8, further comprising co-culturing TCR-T tumor-specific peptides and verifying the tumor specificity of TCR by killing target cells.

10. The method according to any one of claims 1-9, wherein screening for tumor-specific TCRs comprises selecting cells characterized by high expression of tumor reactive targets and identifying them as tumor-specific cell subpopulations.

11. The method of claim 10, wherein the tumor reactive target is selected from one or more of the group consisting of: (1) CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NOP16, NR4 A2,EGR2,LINC00892,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPINB9,BI RC3, VSIR, IL21R, REL, MAT2A, ZFP36L1, TAGAP, JARID2, PLEK, and ZBTB32; or (2) CCL4L2, CCL3, CCL4, CSF2, KLRC1, IFNG, MKI67, TUB A1B,RRM2,TYMS,GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE2C,T RGV3,FABP5,HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4,GZM A,TOP2A,CSF1,DUSP4,SMC4,MCM7,NKG7,HAVCR2,PKM,CDC20,SLC1A5,MTHFD2,IL2RA,PHLDA1,CAMK1,HNRNPAB,GAPDH,SLC7A5, TMPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2; Preferably, the tumor reactive targets are TNFRSF9, IFNG, and GZMB.

12. The method according to claims 1-11, wherein the three-dimensional aggregate of target cells comprises tumor organoids (e.g., tumor-derived organoids).

13. The method according to any one of claims 1-12, wherein the method comprises contacting isolated immune cells with target cells, target cell three-dimensional aggregates and / or cells derived from target cell three-dimensional aggregates, or tumor-specific polypeptides at a cell ratio of about 1:1 to 60:

1.

14. The method according to any one of claims 1-13, wherein the method comprises contacting isolated immune cells with target cells, target cell three-dimensional aggregates and / or cells derived from target cell three-dimensional aggregates, or tumor-specific polypeptides for about 4 to 72 hours.

15. The method according to any one of claims 1-14, wherein the method further comprises, prior to contacting the isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells, or tumor-specific polypeptides, the immune cells are expanded in vitro.

16. The method of claim 15, wherein the in vitro expansion comprises culturing tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion and / or ascites derived from a tumor subject in an IL-2 culture environment.

17. The method according to any one of claims 1-16, further comprising culturing the isolated immune cells in a culture environment containing IL-2 and anti-CD3 and / or anti-CD28 antibodies before, simultaneously and / or after contacting the isolated immune cells with target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells, or tumor-specific polypeptides.

18. The method according to any one of claims 1-17, wherein the TCR expression library of the immune cells is obtained by single-cell sequencing.

19. The method according to any one of claims 1-18, wherein, compared to a population of uncontacted cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells, or tumor-specific polypeptides, the specific immune cells, upon contact with tumor cells of the same subject origin, are capable of producing and / or releasing more cytokines (e.g., GZMB, CD107a, TNF-α, or IFN-γ), specifically killing and / or recognizing more tumor cells, and / or expressing more tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107).

20. The method of any one of claims 1-19, wherein the immune cells comprise T cells, natural killer cells, and / or natural killer-like T cells.

21. The method according to any one of claims 1-20, wherein the immune cells comprise αβT cells and / or γδT cells.

22. The method according to any one of claims 1-21, wherein the immune cells comprise tumor-infiltrating lymphocytes.

23. The method according to any one of claims 1-22, wherein the immune cells comprise cells derived from the subject's tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or ascites.

24. A cell population comprising tumor-specific TCRs selected by the method of any one of claims 1-23.

25. A pharmaceutical composition comprising the cell population of claim 24, and optionally a pharmaceutically acceptable carrier.

26. A method of influencing tumor cell growth, comprising administering the cell population of claim 24, or the pharmaceutical composition of claim 25.

27. The use of the cell population of claim 24 and / or the pharmaceutical composition of claim 25 in the preparation of a medicament for the prevention and / or treatment of tumors.

28. A medicament for the prevention and / or treatment of tumors, comprising the cell population of claim 24 and / or the pharmaceutical composition of claim 25 as an active ingredient.

29. A method for preventing and / or treating tumors, comprising administering to a subject in need the cell population of claim 24 and / or the pharmaceutical composition of claim 25.

30. The cell population of claim 24, and / or the pharmaceutical composition of claim 25, for the prevention and / or treatment of tumors.

31. A method for screening immune cells, the method comprising contacting isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells, and then screening specific immune cells based on the expression of nucleic acids of specific targets.

32. The method of claim 31, wherein the target cells comprise tumor cells.

33. The method of claim 31, wherein the three-dimensional aggregate of target cells comprises tumor organoids (e.g., tumor-derived organoids).

34. The method according to any one of claims 31-33, wherein the method comprises contacting isolated immune cells with target cells, three-dimensional aggregates of target cells and / or cells derived from three-dimensional aggregates of target cells at a cell ratio of about 1:1 to 60:

1.

35. The method according to any one of claims 31-34, wherein the method comprises contacting isolated immune cells with target cells, target cell three-dimensional aggregates and / or cells derived from target cell three-dimensional aggregates for about 4 hours to 72 hours.

36. The method according to any one of claims 31-35, wherein the method further comprises, prior to contacting the isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells, the immune cells are expanded in vitro.

37. The method of claim 36, wherein the in vitro expansion comprises culturing tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion and / or ascites derived from a tumor subject in an IL-2 culture environment.

38. The method according to any one of claims 31-37, further comprising culturing the immune cells in a culture environment containing IL-2 and anti-CD3 and / or anti-CD28 antibodies before, simultaneously and / or after contacting the isolated immune cells with cells selected from target cells, three-dimensional aggregates of target cells and cells derived from three-dimensional aggregates of target cells.

39. The method according to any one of claims 31-38, wherein the specific target is selected from one or more of the group consisting of: (1) CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NOP16, NR4A2,EGR2,LINC00892,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPINB9, BIRC3, VSIR, IL21R, REL, MAT2A, ZFP36L1, TAGAP, JARID2, PLEK, and ZBTB32; or (2) CCL4L2, CCL3, CCL4, CSF2, KLRC1, IFNG, MKI67, T UBA1B,RRM2,TYMS,GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE2C, TRGV3,FABP5,HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4,GZ MA,TOP2A,CSF1,DUSP4,SMC4,MCM7,NKG7,HAVCR2,PKM,CDC20,SLC1A5,MTHFD2,IL2RA,PHLDA1,CAMK1,HNRNPAB,GAPDH,SLC7A5, TMPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2.

40. The method according to any one of claims 31-39, wherein the nucleic acid expression of the specific target of the immune cell is obtained by single-cell sequencing.

41. The method according to any one of claims 31-40, wherein in the immune cells, a population of cells with high levels of nucleic acid expression of the specific target is identified as specific immune cells.

42. The method according to any one of claims 31-41, wherein, compared to the original cell population that has not been contacted with cells selected from target cells, three-dimensional aggregates of target cells, and cells derived from three-dimensional aggregates of target cells, the specific immune cells, upon contact with tumor cells of the same subject origin, are capable of producing and / or releasing more cytokines (e.g., GZMB, CD107a, TNF-α, or IFN-γ), specifically killing and / or recognizing more tumor cells, and / or expressing more tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107).

43. The method of any one of claims 31-42, wherein the immune cells comprise T cells, natural killer cells, and / or natural killer-like T cells.

44. The method according to any one of claims 31-43, wherein the immune cells comprise αβT cells and / or γδT cells.

45. The method according to any one of claims 31-44, wherein the immune cells comprise tumor-infiltrating lymphocytes.

46. ​​The method according to any one of claims 31-45, wherein the immune cells comprise cells derived from the subject's tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, adjacent normal tissue, pleural effusion, and / or ascites.

47. A cell population obtained by the method of any one of claims 31-46.

48. A cell population having high levels of nucleic acid expression of a specific target, said specific target being selected from one or more of the following group: (1) CCL4L2, XCL2, CSF2, CCL1, XCL1, CCL4, IFNG, CRTAM, CCL3L1, CCL3, TNFRSF9, LTA, TNF, GZMB, RGCC, FABP5, DUSP2, IL13, BCL2A1, GADD45B, PIM3, MIR155HG, CSF1, IER3, MYC, ICAM1, FASLG, SDC4, NO P16,NR4A2,EGR2,LINC00892,BCL2L1,NME1,DDX21,PPAN,LAIR2,SLC7A5,ZBED2,KDM6B,NR4A1,NOLC1,CD82,IL4,NFKBID,SERPI NB9, BIRC3, VSIR, IL21R, REL, MAT2A, ZFP36L1, TAGAP, JARID2, PLEK, and ZBTB32; or (2) CCL4L2, CCL3, CCL4, CSF2, KLRC1, IFNG, MKI6 7,TUBA1B,RRM2,TYMS,GNLY,STMN1,LAG3,CCL3L1,IKZF2,HMGB2,H2AFZ,CTSW,TRGV8,GZMB,TNFRSF9,AFAP1L2,PRF1,KLRD1,UBE 2C,TRGV3,FABP5,HMGN2,LINC00892,PCLAF,ENTPD1,CENPF,UBE2S,APBB2,ASPM,TUBB,TPX2,ITGA2,MYO1E,H2AFX,HOPX,ATP8B4, GZMA,TOP2A,CSF1,DUSP4,SMC4,MCM7,NKG7,HAVCR2,PKM,CDC20,SLC1A5,MTHFD2,IL2RA,PHLDA1,CAMK1,HNRNPAB,GAPDH,SLC7A5 , TMPO, CXCL8, MIR181A1HG, NUSAP1, LAYN, CCNB1, KPNA2, MRPL52, CDK6, RANBP1, DUSP5, KLRC2, CRTAM, TRGC2, PHGDH, RAN, and CCNA2.

49. A cell that expresses a T-cell receptor derived from the cell population of claim 47 or 48.

50. A pharmaceutical composition comprising the cell population of claim 47 or 48, and / or the cells of claim 49, and optionally a pharmaceutically acceptable carrier.

51. A method for influencing tumor cell growth, comprising administering the cell population of claim 47 or 48, the cells of claim 49, and / or the pharmaceutical composition of claim 50.

52. The use of the cell population of claim 47 or 48, the cells of claim 49, and / or the pharmaceutical composition of claim 50 in the preparation of a medicament for the prevention and / or treatment of tumors.

53. A medicament for the prevention and / or treatment of tumors, comprising the cell population of claim 47 or 48, the cells of claim 49, and / or the pharmaceutical composition of claim 50 as active ingredients.

54. A method for preventing and / or treating tumors, comprising administering to a subject in need the cell population of claim 47 or 48, the cells of claim 49, and / or the pharmaceutical composition of claim 50.

55. The cell population of claim 47 or 48, the cells of claim 49, and / or the pharmaceutical composition of claim 50, for the prevention and / or treatment of tumors.

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