Enrichment method and application of tumor specific cells

Abstract

The invention provides a method for enriching tumor specific cells, and particularly relates to a method for obtaining tumor specific cells aiming at specific tumor species by enriching specific markers. The invention also provides application of the tumor specific cell. The separated immune cells have strong release ability to specific cytokines of lung tumors and cervical tumors, have strong specific killing ability to advanced tumors, or have high expression level of tumor specific recognition markers in a corresponding time window after tumor antigen stimulation. The sorting step reduces the amount of cells required for cell therapy or the in vitro culture time required for cell therapy.

Description

A method for enriching tumor-specific cells and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for enriching tumor-specific cells and its application. Background Art

[0002] Currently, immunotherapy is an effective treatment for patients with a poor prognosis. However, the immune cells used in immunotherapy have weak anti-tumor capabilities, primarily manifested in a low proportion of immune cells with tumor-specific killing capabilities in cell products. Therefore, the enrichment and isolation of specific types of tumor-specific immune cells through magnetic bead separation or cell sorting of tumor-specific cell surface markers holds great promise for the widespread clinical application of cell therapy.

[0003] However, solid tumors are highly heterogeneous between different tumor types and even between different patients with the same tumor type. This heterogeneity is reflected not only in the tumor itself but also in the differences in immune infiltration, immunosuppressive mechanisms, and immune cell status within its tumor microenvironment. Therefore, tumor-specific immune cells corresponding to different tumor types may have different types of tumor-specific cell surface markers. For example, Celine ML et al. mentioned in their abstract (Celine ML et al., Clin Cancer Res 2021; 27:4089–100) that in ovarian cancer, CD39, CD103, and PD-1 are tumor-specific markers of lymphocytes; Zheng et al. mentioned in their summary (Zheng et al., 2022, Cancer Cell 40, 410–423) that in bile duct and pancreatic cancer, CXCL13 and GZMA are tumor-specific markers of lymphocytes.

[0004] Therefore, there is an urgent need in this field for a tumor-specific surface marker targeting a specific tumor type, which can be used to enrich tumor-specific immune cells or discover tumor-specific TCRs and be widely used in cell therapy.

[0005] Summary of the Invention

[0006] The present invention provides a method for enriching tumor-specific cells and its application. The method of the present invention has one or more of the following advantages: the sorted immune cells have a strong ability to release specific cytokines for lung tumors and cervical tumors, the sorted immune cells have a strong ability to specifically kill lung tumors and cervical tumors, the sorted immune cells have a strong ability to specifically kill advanced tumors, or the sorted immune cells have a high expression level of tumor-specific recognition markers (such as 4-1BB or CD69) within a corresponding time window after tumor antigen stimulation, such as within 12-36 hours, or the sorting step of the present invention reduces the amount of cells required for cell therapy or the in vitro culture time required for cell therapy.

[0007] The present invention is based in part on the discovery that, in specific tumor tissue samples, such as those from advanced ovarian cancer, enriched CD137-positive immune cells, isolated using CD137 as a surface marker, did not show enhanced specific cytotoxicity or cytokine release against advanced ovarian cancer cells. However, in lung and cervical cancer tissue samples, the present invention surprisingly discovered that enriched CD137-positive immune cells, isolated using CD137 as a surface marker, showed specific cytotoxicity and cytokine production and / or release against lung and cervical cancer cells, respectively.

[0008] In one aspect, the present invention provides a cell population comprising CD137-positive cells derived from lung tumors and / or cervical tumors. In another aspect, the present invention provides a method for identifying tumor-specific killer cells, comprising determining the presence, number, and / or proportion of CD137-positive cells in a tumor sample from a patient with lung cancer and / or cervical cancer.

[0009] In another aspect, the present invention provides a method for obtaining tumor-specific killer cells, comprising isolating CD137-positive cells from a tumor sample of a patient with a lung tumor and / or a cervical tumor.

[0010] On the other hand, the present invention provides a method for improving tumor-specific killing, thereby increasing the number or activity of CD137-positive cells in cell therapy products.

[0011] On the other hand, the present invention provides a method for reducing tumor-specific killer cells, thereby reducing the number or activity of CD137-positive cells in a cell therapy product, for example, for constructing a stable disease model of lung cancer or cervical cancer.

[0012] For example, after obtaining the above cells, they can be cultured and expanded to obtain the required amount of cells for treatment. For example, after obtaining the above cells, they can be cultured and expanded and / or reinfused to prevent and / or treat lung tumors and / or cervical tumors.

[0013] In one aspect, the present invention provides a method for enriching tumor-specific cells, comprising isolating autologous CD137-positive cells derived from a subject with lung tumor and / or cervical tumor.

[0014] In another aspect, the present invention provides a cell obtained by the method of the present invention.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0016] In another aspect, the present invention provides a method of influencing cell growth comprising administering a cell of the present invention and / or a pharmaceutical composition of the present invention.

[0017] In another aspect, the present invention provides use of the cells of the present invention and / or the pharmaceutical composition of the present invention in the preparation of a medicament for preventing and / or treating diseases and / or symptoms comprising lung tumors and / or cervical tumors.

[0018] Those skilled in the art will readily appreciate other aspects and advantages of the present invention from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present invention. As will be appreciated by those skilled in the art, the disclosure of the present invention enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the descriptions in the drawings and specification of the present invention are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are briefly described as follows:

[0020] 1A-1B show a flow chart of the cell culture method of the present invention.

[0021] FIG2 shows the results of CD137 expression in lung cancer, ovarian cancer, and cervical cancer-derived tissues after pre-culture.

[0022] FIG3 shows the results of CD137 expression in lung cancer and cervical cancer-derived tissues after preREP amplification.

[0023] Figures 4A and 4B show the results of tumor-specific responses (A: 4-1BB / B: CD69 expression) of CD137-positive cells sorted from lung cancer tumor tissues.

[0024] Figures 5A and 5B show the results of tumor-specific responses (A: 4-1BB / B: CD69 expression) of CD137-positive cells sorted from cervical cancer tumor tissues.

[0025] Figures 6A and 6B show the results of sorting CD137-positive cells for tumor-specific response (A: 4-1BB / B: CD69 expression) from preREP TILs derived from cervical cancer tumor tissue.

[0026] FIG7 shows the results of tumor-specific IFN-γ secretion by CD137-positive cells sorted from lung cancer tumor tissues.

[0027] FIG8 shows the results of tumor-specific IFN-γ secretion by CD137-positive cells sorted from cervical cancer tumor tissues.

[0028] FIG9 shows the results of tumor-specific IFN-γ secretion by CD137-positive cells sorted from ovarian cancer tumor tissues.

[0029] FIG10 shows the results of tumor-specific IFN-γ secretion by sorting CD137-positive cells from preREP TILs derived from cervical cancer tumor tissue.

[0030] FIG11 shows the results of tumor-specific IFN-γ secretion by sorting CD137-positive cells from preREP TILs derived from lung cancer tumor tissue. DETAILED DESCRIPTION

[0031] The following describes the embodiments of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0032] Definition of terms

[0033] In the present invention, the term "immune cell" generally refers to cells involved in innate and adaptive immune responses. For example, it 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 such as CD4 positive T cells, CD8 positive T cells (also referred to as cytotoxic T cells or CTL), regulatory T cells (Treg), Th1 cells, Th2 cells, Th17 cells α β T cells and / or γ δ T cells. In some embodiments, immune cells also include modified immune effector cells, such as chimeric antigen receptor (CAR) modified immune effector cells (such as CAR-T cells, CAR-NK cells), T cell receptor (TCR) modified immune effector cells (such as TCR-T cells).

[0034] In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to an engineered antigen receptor. For example, CAR may include an extracellular antigen binding domain fused to a cytoplasmic domain comprising a signaling domain via a hinge and a transmembrane domain. In some embodiments, the CAR extracellular domain can bind to an antigen expressed by a target cell in an MHC-independent manner, thereby leading to activation and proliferation of the cell. In some embodiments, the extracellular domain of CAR can recognize a tag fused to an antibody or its antigen-binding fragment. For example, a single CAR construct can be made to target a variety of different antigens by replacing another antibody with one antibody. In some embodiments, the extracellular domain of CAR may include an antigen-binding fragment derived from an antibody. Antigen binding domains that can be used in the present disclosure may include, for example, scFv, antibodies, antigen-binding regions of antibodies, variable regions of heavy chains / light chains, and / or single-chain antibodies.

[0035] In the present invention, the term "T cell receptor (TCR)" generally refers to an engineered antigen receptor. For example, a TCR may comprise TCR α and / or TCR β chains that have been isolated and cloned from a T cell population that recognizes a specific target antigen. For example, TCR α and / or TCR β genes (i.e., TRAC and TRBC) can be cloned from a T cell population isolated from an individual with a specific malignancy or from a T cell population isolated from a humanized mouse immunized with a specific tumor antigen or tumor cell. Engineered TCRs can recognize antigens (e.g., by recognizing their cognate antigens presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of target cells) by the same mechanism as their endogenous counterparts, thereby leading to activation and proliferation of TCR engineered cells.

[0036] In the present invention, the term "encode" generally refers to the ability to directly or indirectly infer, based on essentially defined rules, the structure or composition of one molecule from the structure or composition of another related class of molecules. For example, the nucleotide sequence can be inferred from the amino acid sequence, or from the properties of a deoxyribonucleic acid that transcribes complementary nucleic acids, including nucleic acids that can be translated into polypeptides. For example, a deoxyribonucleic acid can encode an RNA transcribed from the deoxyribonucleic acid. Similarly, a deoxyribonucleic acid can encode a polypeptide translated from the RNA transcribed from the deoxyribonucleic acid.

[0037] In the present invention, the term "NK cell," also known as "natural killer cell," generally refers to a type of cell with large granules in its cytoplasm. NK cells develop from bone marrow lymphoid stem cells and can differentiate and develop in either the bone marrow or thymic microenvironment. In the present invention, the proportion of NK cells in TIL cells can be altered using the methods of the present invention.

[0038] In the present invention, "CD4+ Cells" generally refer to CD4-positive cells, such as T cells. The term "CD4 + These cells can be identified by methods known in the art, such as by staining the cells with fluorescently labeled antibodies against CD4 and using fluorescence-activated cell sorting.

[0039] In the present invention, "CD8 + Cells” generally refer to CD8-positive cells, such as T cells. The term “CD8 + These cells can be identified by methods known in the art, such as by staining the cells with fluorescently labeled antibodies against CD8 and using fluorescence-activated cell sorting.

[0040] In the present invention, the term "tumor infiltrating lymphocytes" or "TIL" generally refers to a population of cells originally obtained as leukocytes that have left the bloodstream of a subject and migrated into a tumor. TIL may include, but is 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 the present invention. In some embodiments, the tumor-infiltrating lymphocytes of the present invention may not be isolated and purified, or may be infiltrating with tumor cells. For example, the TILs of the present invention may refer to a TIL population.

[0041] In the present invention, the term "stage" in "one stage of in vitro expansion", "single stage of in vitro expansion", or "first stage of in vitro expansion" generally refers to a period of expansion process that TIL undergoes in vitro. In one embodiment, each stage can be divided by the change in the number of TIL cells. In one embodiment, when the number of TIL cells increases by at least about 1 times, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In some embodiments, when the number of TIL cells increases by at least about 1-50 times, for example, at least about 1 times, 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, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the conditions of TIL cell culture. In one embodiment, when T cell activators and / or T cell growth factors are added or supplemented to the cell culture medium, the TIL cells can be considered to have entered the next stage of in vitro expansion. In one embodiment, when the TIL cells are centrifuged and / or washed, the TIL cells can be considered to have entered the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the number of days of TIL cell culture. In one embodiment, after the TIL cells are cultured in vitro for about 1-100 days, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days or about 100 days, the TIL cells can be considered to have entered the next stage of in vitro expansion.

[0042] In the present invention, the term "T cell activator" generally refers to a substance that binds to the corresponding binding receptor on the T cell and mediates the T cell co-stimulatory response. A T cell activator can be a substance other than an antigen receptor required for a T cell to produce an effective immune response. A T cell activator can refer to a T cell co-stimulatory molecule. For example, the T cell activator of the present invention can include its variant, homolog or any substance comprising its functionally active fragment. T cell activators can include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activation receptors, BTLA (the gene GeneID encoding it can be 151888), Toll ligand receptor, OX40 (the gene GeneID encoding it can be 7293), CD2 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 924), CD27 (the gene GeneID encoding it can be 939), CD28 (the gene GeneID encoding it can be 939), CD29 (the gene GeneID encoding it can be 941), CD30 (the gene GeneID encoding it can be 942), CD31 (the gene GeneID encoding it can be 943), CD32 (the gene GeneID encoding it can be 944), CD33 (the gene GeneID encoding it can be 945), CD34 (the gene GeneID encoding it can be 946), CD35 (the gene GeneID encoding it can be 947), CD36 (the gene GeneID encoding it can be 948), CD37 (the gene GeneID encoding it can be 949), CD38 (the gene GeneID encoding it can be 949), CD39 (the gene GeneID encoding it can be 950), CD40 (the gene GeneID encoding it can be 951), CD41 (the gene GeneID encoding it can be Its gene GeneID can be 940), CD30 (the gene encoding it can be 943), CD40 (the gene encoding it can be 958), CDS, ICAM-1 (the gene encoding it can be 3383), LFA-1 (CD11a / CD18) (the gene encoding it can be 3689), 4-1BB (CD137) (the gene encoding it can be 3604), B7-H3 (the gene encoding it can be 80381), ICOS ( CD278) (the gene encoding it may be 29851), GITR (the gene encoding it may be 8784), BAFFR (the gene encoding it may be 115650), LIGHT (the gene encoding it may be 8740), HVEM (LIGHTR) (the gene encoding it may be 8764), KIRDS2, SLAMF7 (the gene encoding it may be 57823), NKp80 (KLRF1) (the gene encoding it may be 8784), 51348), NKp44 (the gene encoding it may be 9436), NKp30 (the gene encoding it may be 259197), NKp46 (the gene encoding it may be 9437), CD19 (the gene encoding it may be 930), CD4 (the gene encoding it may be 920), CD8α (the gene encoding it may be 925), CD8β (the gene encoding it may be 926), IL-2Rβ, IL-2Rγ,IL7Rα (the gene encoding it may be GeneID 3676), ITGA4 (the gene encoding it may be GeneID 3676), VLA1 (the gene encoding it may be GeneID 3672), CD49a (the gene encoding it may be GeneID 3672), IA4 (the gene encoding it may be GeneID 3732), CD49D (the gene encoding it may be GeneID 3676), ITGA6 (the gene encoding it may be GeneID 3655), VLA-6 (the gene encoding it may be GeneID 3655), CD49f (the gene encoding it may be GeneID 3655), ITGAD (the gene encoding it may be GeneID 3676), The gene encoding it may be 3681), CD11d (the gene encoding it may be 3681), ITGAE (the gene encoding it may be 3682), CD103 (the gene encoding it may be 3682), ITGAL (the gene encoding it may be 3683), CD11a (the gene encoding it may be 3683), LFA-1 (the gene encoding it may be 3683), ITGAM (the gene encoding it may be 3684), CD11b (the gene encoding it may be 3684), ITGAX (the gene encoding it may be 3685). The gene GeneID of the gene encoding the leukemia cell line may be 3687), CD11c (the gene GeneID of the gene encoding the leukemia cell line may be 3687), ITGB1 (the gene GeneID of the gene encoding the leukemia cell line may be 3688), CD29 (the gene GeneID of the gene encoding the leukemia cell line may be 3688), ITGB2 (the gene GeneID of the gene encoding the leukemia cell line may be 3689), CD18 (the gene GeneID of the gene encoding the leukemia cell line may be 3689), LFA-1 (the gene GeneID of the gene encoding the leukemia cell line may be 3689), ITGB7 (the gene GeneID of the gene encoding the leukemia cell line may be 3695), NKG2D (the gene GeneID of the gene encoding the leukemia cell line may be 22914), NKG2C (the gene GeneID of the gene encoding the leukemia cell line may be 22915), The gene GeneID may be 3822), TNFR2 (the gene encoding it may be 7133), TRANCE / RANKL (the gene encoding it may be 8600), DNAM1 (CD226) (the gene encoding it may be 10666), SLAMF4 (CD244, 2B4) (the gene encoding it may be 51744), CD84 (the gene encoding it may be 8832), CD96 (Tactile) (the gene encoding it may be 10225), CEACAM1 (the gene encoding it may be 634),CRTAM (the gene encoding it may be 56253 in GeneID), Ly9 (CD229) (the gene encoding it may be 4063 in GeneID), CD160 (BY55) (the gene encoding it may be 11126 in GeneID), PSGL1 (the gene encoding it may be 6404 in GeneID), CD100 (SEMA4D) (the gene encoding it may be 10507 in GeneID), CD69 (the gene encoding it may be 969 in GeneID), SLAMF6 (NTB-A, Ly108) (the gene encoding it may be 114836 in GeneID), SLAM (SLAMF1, CD150, IPO-3) (the gene encoding it may be 6504 in GeneID), BLAME (SLAMF 8) (the gene encoding it may be 56833), SELPLG (CD162) (the gene encoding it may be 6404), LTBR (the gene encoding it may be 4055), LAT (the gene encoding it may be 27040), GADS (the gene encoding it may be 9402), SLP-76 (the gene encoding it may be 3937), PAG / Cbp (the gene encoding it may be 55824), CD19a, and a ligand that specifically binds to CD3, a ligand that specifically binds to CD28, a ligand that specifically binds to HVEM, a ligand that specifically binds to CD40L, a ligand that specifically binds to OX40, and a ligand that specifically binds to 4-1BB. The co-stimulatory intracellular signaling domain may refer to the intracellular portion of a T cell activator. The intracellular signaling domain may comprise an intact intracellular portion of a molecule derived therefrom or an intact native intracellular signaling domain or a functional fragment thereof. 、

[0043] In the present invention, the term "T cell growth factor" generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation. For example, the T cell growth factor of the present invention may include its variants, homologs, or any substance containing its functionally active fragments. In one embodiment, the T cell growth factor can be selected from one or more of the following groups: IL-2 (the gene encoding it may be 3558), IL-4 (the gene encoding it may be 3565), IL-6 (the gene encoding it may be 3569), IL-7 (the gene encoding it may be 3574), IL-10 (the gene encoding it may be 3586), IL-12 (the gene encoding it may be 3592 or 3593), IL-15 (the gene encoding it may be 3600), IL-21 (the gene encoding it may be 59067), TNF-α (the gene encoding it may be 100137091), interferon γ (the gene encoding it may be 3458), GZMB (the gene encoding it may be 3002), CD107a (the gene encoding it may be 6499), etc.

[0044] In the present invention, the term "first stage in vitro expansion" generally refers to the stage of amplification using T cell growth factors after primary TILs are obtained from tissues. In one embodiment, the tissue of the present invention can be selected from the following groups: tumor tissue and pleural effusion, and the pleural effusion of the present invention can be pleural effusion of a patient with metastatic cancer. In one embodiment, the amplification of the present invention can be in vivo amplification performed by autologous or allogeneic means, or it can be in vitro amplification. The first stage in vitro amplification of the present invention can also be called the preREP (pre-rapid amplification) stage. For example, TILs derived from tumor tissue and not amplified in vitro can be called the first TIL group. For example, TILs obtained through the first stage in vitro amplification in the culture method of the present invention divided into two steps can be called the second TIL group.

[0045] In the present invention, the term "second stage in vitro expansion" generally refers to the stage in which the tissue removed from the subject is expanded and then expanded again. In one embodiment, the number of TIL cells expanded in vitro in the second stage of the present invention is increased compared to the TIL expanded in vitro in the first stage, for example, it can be increased by at least about 10 times (or at least about 20, 30, 40, 50, 60, 70, 80 or 90 times), or in one embodiment, the number of cells can be increased by at least about 100 times. In one embodiment, the culture conditions of the second stage in vitro expansion can be different from those of the first stage in vitro expansion, for example, the culture substances added can be different. For example, in the culture method of the present invention divided by the two-step method, the second stage in vitro expansion can also be called the REP (rapid expansion) stage. For example, in the culture method of the present invention divided by the two-step method, the TIL obtained by the second stage in vitro expansion can be called the third TIL population.

[0046] In the present invention, the term "tumor-specific cells" generally refers to cells that can specifically inhibit tumor growth. Tumor-specific cells may possess specific tumor-killing capabilities or tumor-specific cytokine release capabilities. For example, tumor-specific cells can be identified by co-culturing with specific tumors and detecting cytokine expression, production, and / or release, and / or tumor cell apoptosis. Tumor-specific cells may possess more specific anti-tumor growth capabilities than standard cells.

[0047] As used herein, the term "isolated" generally refers to altering or removing from its natural state. For example, a cell, nucleic acid, or peptide naturally present in a living animal is not "isolated," but a partially or completely separated cell, nucleic acid, or peptide of the same state is "isolated." An isolated cell, nucleic acid, or protein can exist in a substantially purified form or can exist in a non-natural environment, such as a delivery vehicle.

[0048] In the present invention, the term "CD137" generally refers to a member of the TNFR family. CD137 is also known as 4-1BB or TNFSFR9. The CD137 of the present invention can also encompass functionally active fragments thereof, and is not limited to substances containing functionally active fragments of CD137 produced after processing and / or modification in cells. For example, the CD137 of the present invention can include functionally active fragments thereof and any extracellular domain, transmembrane domain, or intracellular domain of CD137.

[0049] In the present invention, the term "treatment" generally refers to treatment and / or prevention. The therapeutic effect is achieved by inhibiting, alleviating or eradicating the disease state.

[0050] As used herein, 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 typically 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 materials suitable for human administration. Other contemplated carriers, excipients, and / or additives that may be used in the formulations described herein may include, for example, flavorings, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (e.g., serum albumin, gelatin, casein), salt-forming counterions (e.g., sodium), and the like. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art. As used herein, "pharmaceutically acceptable carrier" is understood to mean a vector that does not include nucleic acid forms used in genetic engineering.

[0051] In the present invention, the term "tumor tissue" generally refers to a sample from a tumor in a subject, including any solid tumor and / or any tissue that is not a solid tumor in a subject.

[0052] In the present invention, the terms "about" and "approximately" generally refer to a statistically significant numerical range. Such a range can be within an order of magnitude of a given value or range, can be included within 50%, preferably included within 20%, more preferably included within 10%, and most preferably included within 5%. The permissible variations encompassed by the terms "about" or "approximately" may depend on the specific system under study and can be readily understood by those of ordinary skill in the art.

[0053] The terms "above," "below," "at most," and "at least" are inclusive.

[0054] Detailed Description of the Invention

[0055] In one aspect, the present invention provides a cell population that is CD137 positive. For example, the cell population is derived from an autologous subject with a lung tumor and / or a cervical tumor. In one aspect, the present invention provides a cell population that is CD69 positive. For example, the cell population is derived from an autologous subject with a lung tumor and / or a cervical tumor.

[0056] The present invention is based in part on the discovery that, in specific tumor tissue samples, such as those from advanced ovarian cancer, enriched CD137-positive immune cells, isolated using CD137 as a surface marker, did not show enhanced specific cytotoxicity or cytokine release against advanced ovarian cancer cells. However, in lung and cervical cancer tissue samples, the present invention surprisingly discovered that enriched CD137-positive and / or CD69-positive immune cells, isolated using CD137 and / or CD69 as surface markers, showed specific cytotoxicity and cytokine production and / or release, respectively, against lung and cervical cancer cells.

[0057] For example, the present invention provides the use of CD137 and / or CD69 as markers to identify and separate naturally occurring tumor-specific tumor infiltrating lymphocytes (TIL). In one embodiment, CD137 and / or CD69 are markers for selectively enriching tumor-specific TIL colonies to develop adoptive immunotherapy. In one embodiment, of the present invention, CD137 positive and / or CD69 positive cell colonies include separating and culturing CD137 positive and / or CD69 positive cell colonies from lung cancer or cervical cancer tissue samples. In such embodiments, CD137 positive and / or CD69 positive cell colonies can include tumor-specific T cells.

[0058] For example, the present invention provides methods for enriching and amplifying CD137-positive and / or CD69-positive cells. In one embodiment, CD137-positive and / or CD69-positive cells are selectively isolated from a tumor sample. In another embodiment, CD137-positive and / or CD69-positive cells are selectively isolated from a TIL population co-cultured with an HLA-matched lung cancer or cervical cancer tumor cell line.

[0059] In one embodiment, the CD137-positive and / or CD69-positive cells produce IFN-γ after contact with HLA-matched lung or cervical cancer tumor cells. For example, compared to non-tumor specific cells or a primary cell population that has not been sorted for CD137 and / or CD69, the tumor specific cells produce and / or release IFN-γ at an amount that is increased by about 100,000-fold to about 1%, such as about 100,000-fold, about 10,000-fold, about 1000-fold, about 50-fold, about 40-fold, about 30-fold, about 20-fold, about 10-fold, about 9-fold, about 8-fold, about 7-fold, about 6-fold, about 5-fold, about 4-fold, about 3-fold, about 2-fold, about 1-fold, 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-fold. %, 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%, or about 1%.

[0060] In one embodiment, CD137-positive and / or CD69-positive cells are able to express more tumor-specific recognition markers after contact with HLA-matched lung cancer or cervical cancer tumor cells. For example, compared to non-tumor-specific cells or original cell populations that have not been sorted by CD137 and / or CD69, the tumor-specific cells, after contact with tumor cells derived from the same subject, express tumor-specific recognition markers selected from 4-1BB or CD69. The proportion of cells in the total cells is increased by about 100,000 times to about 1%, for example, by about 100,000 times, about 10,000 times, about 1,000 times, about 100 times, about 50 times, about 40 times, about 30 times, about 20 times, about 10 times. %, about 9 times, about 8 times, about 7 times, about 6 times, about 5 times, about 4 times, about 3 times, about 2 times, about 1 times, 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%, or about 1%. For example, for CD4 positive cells, the tumor specific recognition marker can be CD69; for example, for CD8 positive cells, the tumor specific recognition marker can be CD137.

[0061] For example, the present invention provides a method for culturing isolated CD137-positive and / or CD69-positive cells in an in vitro expansion environment. In some cases, the CD137-positive and / or CD69-positive cells of the present invention are administered to a subject in need after culture. Compared to prior art methods that do not enrich CD137-positive and / or CD69-positive cells, the shortened culture duration of the present invention enriches and expands the proportion of tumor specificity, thereby increasing the yield and / or effect of immunotherapy for lung tumors and / or cervical tumors.

[0062] For example, the present invention includes a method for treating a patient's tumor, comprising administering to a patient in need thereof an effective amount of a CD137-positive and / or CD69-positive cell population. In embodiments of the present invention, CD137-positive and / or CD69-positive cells can be isolated and cultured from a melanoma, cervical tumor, lung tumor, bladder tumor, breast tumor, head and neck tumor, pancreatic tumor, liver tumor, gastric tumor, colorectal tumor, bile duct tumor, or kidney tumor tissue sample. In embodiments of the present invention, CD137-positive and / or CD69-positive cells can be isolated and cultured from a late, metastatic, and / or recurrent tumor tissue sample. In embodiments of the present invention, tumor tissue includes, but is not limited to, tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, paracancerous tissue, pleural effusion, and / or peritoneal effusion.

[0063] For example, CD137 positive and / or CD69 positive cells can include immune cells.For example, CD137 positive and / or CD69 positive cells can include cytotoxic T lymphocytes (CTL), natural killer (NK) cells, natural killer T cell-like (NKT) cells, tumor infiltrating lymphocytes (TIL) or lymphokine-activated killer (LAK) cells.For example, CD137 positive and / or CD69 positive cells can include αβT cells and or γδT cells.For example, CD137 positive and / or CD69 positive cells can include 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, CD137 positive and / or CD69 positive cells are patient's own. In this case, the cells transferred into the patient's body include the patient's own cells, to prevent rejection or adverse immune response to the cells administered.

[0064] On the other hand, CD137 positive and / or CD69 positive cell group is separated from the liquid tissue comprising immune cells, such as bone marrow or ascites. Immune cells such as lymphocytes (such as, TIL, CTL, NK cells and LAK cells) can be separated using various methods known in the art. For example, allogeneic restricted CTL is produced by stimulating natural splenocytes in vitro with suitable antigens. For example, a blood sample containing cell precursors taken from a mammal can be used, PBL (peripheral blood lymphocyte, peripheral blood lymphocytes) is obtained after purification, and is incubated with the stimulatory cells of specific antigen peptides. Human primary NK cells can be expanded in the presence of bone marrow cell lines, and the cell line is genetically modified to express NK cell-specific molecules. LAK cells can be produced by, for example, treating the patient's mononuclear lymphocytes with interleukin-2. For example, mononuclear lymphocytes can be collected by repeated lymphocyte separation using a continuous flow cell separator. In some embodiments, immune cells such as lymphocytes (e.g., TIL, CTL, 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 to appropriate surface antigens. In some cases, the immune cells such as lymphocytes (e.g., TIL, CTL, NK cells or LAK cells) obtained comprise a colony with clonal capacity. In other cases, the cell population obtained may not have clonal capacity or may not have unlimited clonal capacity.

[0065] In one embodiment, tumor-specific cells are isolated from lung tumor and / or cervical tumor tissue containing CD137-positive and / or CD69-positive cell populations. In most cases, tumor tissue contains a heterogeneous mixture of cells and cell types, including cancer cells and CD137-positive and / or CD69-positive cell populations containing tumor-specific cells. Tumor tissue may also include tumor antigens. In one embodiment, the tumor antigens have been exposed to tumor-specific cells and the tumor-specific cells have been stimulated. Before selectively isolating CD137-positive and / or CD69-positive cells, tumor tissue is removed from the patient. By culturing CD137-positive and / or CD69-positive cells in an in vitro cell culture environment, CD137-positive and / or CD69-positive cells can be further enriched for tumor-specific cells. For example, by further isolating cells with other biomarkers, CD137-positive and / or CD69-positive cells can be further enriched for tumor-specific T cells.

[0066] Another embodiment includes obtaining cells from tumor tissue. The tumor tissue may include cancer cells. T cells can be isolated from a large amount of tumor tissue before culture or expansion, for example, by flow cytometry, negative or positive selection, or other methods. Another embodiment includes obtaining cells from tumor tissue after in vitro expansion. Although the prior art shows that immune cell surface markers may undergo substantial changes after in vitro expansion in an artificial environment, and the original surface markers may be unusable for sorting tumor-specific cells, the present invention unexpectedly discovered that after the preREP stage of TIL field treatment, tumor-specific cells can still be enriched by enriching CD137-positive and / or CD69-positive cells. For example, the preREP stage of the present invention may include the following steps: culturing the tumor tissue containing tumor cells and immune cells in an in vitro culture environment, such as in a culture medium containing IL-2 at a concentration of 300-9000 IU / mL, for about 3-14 days.

[0067] On the other hand, the method of culturing tumor infiltrating lymphocytes (TIL) of the present invention comprises: (A) contacting a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and / or peritoneal effusion and not expanded in vitro with one or more T cell growth factors, wherein a second TIL population is obtained by step (A); (B) contacting the second TIL population with a T cell activator and / or T cell growth factor, and optionally performing gene editing, wherein a third TIL population is obtained by step (B); (C) co-culturing the third TIL population with feeder cells, wherein a fourth TIL population is obtained by step (C). For example, before step (A) of the present invention, between step (A) and step (B), between step (B) and step (C), and / or after step (C), CD137 and / or CD69 may be enriched. For example, the first TIL population obtained, the second TIL population obtained, the third TIL population obtained, and / or the fourth TIL population obtained in the present invention can be enriched for CD137 and / or CD69.

[0068] On the other hand, the method of culturing tumor-infiltrating lymphocytes (TILs) of the present invention comprises: (A) enriching a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion, and / or peritoneal effusion and not expanded in vitro for CD137 and / or CD69, and then contacting the first TIL population with one or more T cell growth factors, wherein a second TIL population is obtained by step (A); (B) contacting the second TIL population with a T cell activator and / or a T cell growth factor, and optionally performing gene editing, wherein a third TIL population is obtained by step (B); (C) co-culturing the third TIL population with feeder cells, wherein a fourth TIL population is obtained by step (C). Step (A) is performed for about 7 to about 14 days; step (B) is performed for about 0 to about 8 days; and step (C) is performed for about 5 to about 14 days.

[0069] On the other hand, the method of culturing tumor-infiltrating lymphocytes (TILs) of the present invention comprises: (A) contacting a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion, and / or peritoneal effusion and not expanded in vitro with one or more T cell growth factors, wherein a second TIL population is obtained by step (A); (B) enriching the second TIL population for CD137 and / or CD69, and then contacting the second TIL population with a T cell activator and / or a T cell growth factor, and optionally performing gene editing, wherein a third TIL population is obtained by step (B); (C) co-culturing the third TIL population with feeder cells, wherein a fourth TIL population is obtained by step (C). Step (A) is performed for about 7 to about 14 days; step (B) is performed for about 0 to about 8 days; and step (C) is performed for about 5 to about 14 days.

[0070] On the other hand, the method of culturing tumor-infiltrating lymphocytes (TILs) of the present invention comprises: (A) contacting a first TIL population derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion, and / or peritoneal effusion and not expanded in vitro with one or more T cell growth factors, wherein a second TIL population is obtained by step (A); (B) contacting the second TIL population with a T cell activator and / or a T cell growth factor, and optionally performing gene editing, wherein a third TIL population is obtained by step (B); (C) enriching the third TIL population for CD137 and / or CD69 and then co-culturing with feeder cells, wherein a fourth TIL population is obtained by step (C). Step (A) is performed for about 7 to about 14 days; step (B) is performed for about 0 to about 8 days; and step (C) is performed for about 5 to about 14 days.

[0071] For example, the present invention provides a method for culturing tumor-infiltrating lymphocytes (TILs) comprising obtaining TILs from a tissue sample from a subject, which can be an in situ tumor sample or a metastatic tumor sample obtained during surgery. The sample can weigh at least about 1 g, or multiple tissue pieces can be combined. Tumor tissue, tumor-associated lymph nodes with or without metastasis, metastatic lesions, paracancerous tissue, pleural effusion, and / or peritoneal effusion are transported in a sample transport medium, such as a commercially available tumor tissue transport medium, tumor tissue preservation medium, or tumor tissue transport medium, at approximately 2-8°C and processed within 48 hours. The tissue pieces can be mechanically broken into pieces of approximately 1-27 cubic millimeters in size, transferred into a breathable culture bag or Grex, and cultured for approximately 3-14 days with 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). The cells in the culture medium are collected and transferred into a breathable culture bag, or Grex, or Xuri device. The serum-free culture medium of the cells can be supplemented with CD28 antibodies, CD3 antibodies, and CD28 antibodies of the present invention, magnetic beads comprising CD3 antibodies and CD28 antibodies (e.g., Dynabeads), and / or nanomatrices comprising CD3 antibodies and CD28 antibodies (e.g., transACT), IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL), and optionally editing the expression of the target gene in the cell population (e.g., by transducing gene editing with a ribonucleoprotein complex (RNP) carrying gRNA and Cas protein, or LNP comprising gRNA and Cas protein, or LNP comprising a nucleic acid encoding gRNA and Cas protein). After activating the TIL of the present invention for a certain period of time, irradiated PBMCs are added (TIL and PBMC at a ratio of about 1:40 to about 1:400), and the culture is expanded for about 3-14 days. Cells can be collected from the culture medium using a cell processing system, washed, frozen, and tested. The final product can have a CD3 ratio greater than 80% and a cell viability greater than 50%. Cells greater than 80% can be memory and effector cells. Upon stimulation, they can secrete IFN-γ and / or exhibit an increased proportion of activated cells. For example, enrichment for CD137 and / or CD69 can be performed between the various steps in the above method.

[0072] Any cell separation method can be used to separate the cells of the present invention from a biological sample. For example, antibodies that bind CD137 and / or CD69 can be bound to a physical support, such as magnetic beads, magnetic particles, magnetic nanomaterials, microbeads, columns, adsorption columns, and adsorption membranes. Conjugating antibodies to physical supports is well known in the art.

[0073] For example, CD137 positive and / or CD69 positive cells can be isolated from HLA-matched tumor tissue. For example, CD137 positive and / or CD69 positive cells can be selectively isolated and co-cultured with HLA-matched tumor cell lines. Examples of HLA-matched tumor cell lines can include allogeneic cancer cell lines, HLA-matched lung or cervical tumor cell lines, autologous cancer cells, and any other HLA cells matched with CD137 positive and / or CD69 positive cells. In another embodiment, CD137 positive and / or CD69 positive cells produce IFN-γ after exposure to HLA-matched tumor cell lines (e.g., autologous tumor cells).

[0074] Suitable conditions for cell culture include an appropriate culture medium (e.g., minimal essential medium or RPMI medium 1640), which 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 for cell growth known to those of skill.

[0075] In another aspect, the present invention provides a method for identifying tumor-specific killer cells, comprising determining the presence, number and / or ratio of CD137-positive and / or CD69-positive cells in a tumor sample of a lung cancer and / or cervical cancer patient.

[0076] In another aspect, the present invention provides a method for obtaining tumor-specific killer cells, comprising isolating CD137-positive and / or CD69-positive cells from a tumor sample of a patient with a lung tumor and / or a cervical tumor.

[0077] On the other hand, the present invention provides a method for improving tumor-specific killing, thereby increasing the number or activity of CD137-positive and / or CD69-positive cells in cell therapy products.

[0078] On the other hand, the present invention provides a method for reducing tumor-specific killer cells, thereby reducing the number or activity of CD137-positive and / or CD69-positive cells in a cell therapy product, for example, for constructing a stable disease model of lung cancer or cervical cancer.

[0079] For example, after obtaining the above cells, they can be cultured and expanded to obtain the required amount of cells for treatment. For example, after obtaining the above cells, they can be cultured and expanded and / or reinfused to prevent and / or treat lung tumors and / or cervical tumors.

[0080] In one aspect, the present invention provides a cell, which can be cultured according to the culture method of the present invention. In one embodiment, the cell provided by the present invention can include one cell or one batch of cells cultured according to the culture method of the present invention. In one embodiment, the cell provided by the present invention can include multiple cells or multiple batches of cells cultured according to the culture method of the present invention, combined in any proportion.

[0081] In some embodiments, the cells expanded using the methods of the present invention can be administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition can be a suspension of cells in a sterile buffer. Cells expanded using the PBMCs of the present invention can be administered by any suitable route known in the art. In some embodiments, the cells can be administered as a single intra-arterial or intravenous infusion, and the infusion can last for about 30 to 60 minutes. Other suitable routes of administration can include intraperitoneal, intrathecal, and intralymphatic administration.

[0082] For example, after enrichment, the cell product of the present invention increases the proportion of CD137-positive and / or CD69-positive cells. For example, compared to a cell population not enriched based on CD137 and / or CD69, the proportion of CD137-positive and / or CD69-positive cells in the enriched cell population of the present invention increases by about 100,000 times to about 1%, for example, by about 100,000 times, about 10,000 times, about 1,000 times, about 100 times, about 50 times, about 40 times, about 30 times, about 20 times, about 10 times, about 9 times, about 8 times, about 7 times, about 6 times, about 5 times, about 10 times, about 1 ... 4 times, about 3 times, about 2 times, about 1 times, 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%, or about 1%.

[0083] For example, after enrichment, the cell product of the present invention has an increased proportion of CD137-positive and / or CD69-positive cells. For example, relative to a cell population not enriched for CD137 and / or CD69, the proportion of CD137-positive and / or CD69-positive cells in the enriched cell population of the present invention is about 99.99% to 0.1%, such as about 99.99%, about 99.9%, 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%.

[0084] For example, the cell product of the present invention has enhanced tumor-specific cytokine release ability after enrichment of CD137-positive and / or CD69-positive cells. For example, compared with a cell population that is not enriched based on CD137 and / or CD69, or screened out based on CD137 and / or CD69, in the cell population enriched according to the present invention, after contacting specific matching tumor cells, the production and / or release level of tumor-specific cytokines such as IFN-γ is increased by about 100,000 times to about 1%, for example, by about 100,000 times, about 10,000 times, about 1,000 times, or about 1,000 times. About 100 times, about 50 times, about 40 times, about 30 times, about 20 times, about 10 times, about 9 times, about 8 times, about 7 times, about 6 times, about 5 times, about 4 times, about 3 times, about 2 times, about 1 times, 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%, or about 1%.

[0085] For example, the cell product of the present invention has enhanced tumor-specific killing ability after enrichment of CD137-positive and / or CD69-positive cells. For example, compared with a cell population that is not enriched based on CD137 and / or CD69, or screened out based on CD137 and / or CD69, in the cell population enriched according to the present invention, the level of tumor cell apoptosis is increased by about 100,000 times to about 1% after contact with specific matching tumor cells, for example, by about 100,000 times, about 10,000 times, about 1,000 times, about 100 times, about 50 times, about 40 times, about 30 times, about 20 times, about 10 times, about 10 times, about 2 ... 9 times, about 8 times, about 7 times, about 6 times, about 5 times, about 4 times, about 3 times, about 2 times, about 1 times, 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%, or about 1%.

[0086] For example, the cell product of the present invention can be used for the discovery of specific TCRs after enrichment of CD137-positive and / or CD69-positive cells. For example, the type or sequence of the antigen-binding receptors of the enriched cells is determined. For example, the TCRs derived from the enriched cells can be used to develop engineered TCR cells. For example, compared to a cell population that is not enriched based on CD137 and / or CD69, or after CD137 and / or CD69 is screened out, the tumor specificity of the TCR obtained from the enriched cell population of the present invention is increased by about 100,000 times to about 1%, for example, by about 100,000 times, about 10,000 times, about 1000 times, about 100 times, about 50 times, about 40 times, about 30 times, about 20 times, about 10 times, about 9 times, about 8 times, about 7 times, about 8 times, about 9 times, about 8 ... In some embodiments, the expression of a novel antibody or antigen in a human cell may be expressed in an amount greater than about 1%, about 6 times, about 5 times, about 4 times, about 3 times, about 2 times, about 1 times, 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%, or about 1%. For example, tumor specificity can be determined by measuring the level of production and / or release of tumor-specific cytokines, such as IFN-γ, after contact with specifically matched tumor cells, and / or the number and / or level of apoptosis of tumor cells after contact with specifically matched tumor cells.

[0087] In some embodiments, any suitable dose of cells may be administered. In some embodiments, for example, when the tumor is a melanoma, about 2.3×10 9 to about 13.7×10 10 In some embodiments, about 1×10 9 to about 12×10 10 In some embodiments, about 1.2×10 10 to about 4.3×10 10 In some embodiments, approximately 3×10 10 to about 12×10 10 In some embodiments, approximately 4×10 10 to about 10×10 10 In some embodiments, about 5×10 10 to about 8×10 10 In some embodiments, about 6×10 10 to about 8×10 10 In some embodiments, about 7×10 10 to about 8×10 10 In some embodiments, the therapeutically effective dose can be about 2.3×10 9 to about 13.7×10 10 In some embodiments, the therapeutically effective dose may be about 1×10 9 to about 12×10 10 In some embodiments, the therapeutically effective dose can be about 1.2×10 10 to about 4.3×10 10 In some embodiments, the therapeutically effective dose can be about 3×10 10 to about 12×10 10 In some embodiments, the therapeutically effective dose can be about 4×10 10 to about 10×10 10 In some embodiments, the therapeutically effective dose can be about 5×10 10 to about 8×10 10 In some embodiments, the therapeutically effective dose can be about 6×10 10 to about 8×10 10 In some embodiments, the therapeutically effective dose can be about 7×10 10 to about 8×10 10 cells.

[0088] In some embodiments, the cells can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, the cells can be administered in multiple doses. The 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, the cells can be administered continuously.

[0089] In one aspect, the present invention provides a pharmaceutical composition, which in some embodiments may comprise the cells of the present invention and a pharmaceutically acceptable carrier.

[0090] In one aspect, the present invention provides a kit that may include a cell activator, a cell growth factor, and / or feeder cells for the cell culture method of the present invention, and instructions describing the steps of the cell culture method of the present invention. In another aspect, the present invention provides a kit that may include the cells of the present invention and / or the pharmaceutical composition of the present invention.

[0091] In one aspect, the present invention provides a method of affecting the growth of cells, such as tumor cells, which can include administering to a subject a cell of the present invention and / or a pharmaceutical composition of the present invention. In some embodiments, affecting tumor growth can include reducing the volume of the tumor to about 99-0.1% of the volume before administration, such as 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%.

[0092] In one aspect, 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 a medicament, which can be used to prevent and / or treat a disease and / or symptom. For example, the disease and / or symptom of the present invention can include a tumor. In some embodiments, the tumor of the present invention is selected from a solid tumor. In some embodiments, the tumor of the present invention can be selected from one or more of the following groups: melanoma, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and renal cancer. For example, the tumor of the present invention can be an advanced solid tumor.

[0093] In one aspect, the present invention provides a method for preventing and / or treating a disease and / or symptom, which may comprise administering to a subject a cell of the present invention and / or a pharmaceutical composition of the present invention. For example, the disease and / or symptom of the present invention may comprise a tumor. In some embodiments, the tumor of the present invention is selected from a solid tumor. 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 renal cancer. For example, the tumor of the present invention may be an advanced solid tumor.

[0094] In one aspect, the present invention provides a TIL of the present invention and / or a pharmaceutical composition of the present invention, which can be used to prevent and / or treat a disease and / or symptom. For example, the disease and / or symptom of the present invention can include a tumor. In some embodiments, the tumor of the present invention is selected from a solid tumor. In some embodiments, the tumor of the present invention can 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 renal cancer. For example, the tumor of the present invention can be an advanced solid tumor.

[0095] Without intending to be bound by any theory, the following examples are merely intended to illustrate the methods and uses of the present invention and are not intended to limit the scope of the present invention.

[0096] Example

[0097] Example 1 Sorting and expansion of tumor-infiltrating lymphocytes

[0098] 1.1 Tumor tissue receipt and processing

[0099] 1.1.1 Organization reception

[0100] Receive tumor tissue from the donor, verify and record the sample information, and print the corresponding sample label.

[0101] 1.1.2 Tissue processing, digestion, and pre-culture

[0102] Take several 10 cm culture dishes and add an appropriate amount of rewarmed complete culture medium. The complete culture medium can be arbitrarily selected from X-vivo 15 medium or other commercial T cell culture medium, such as T cell culture medium from brands such as Stem Cell, Lonza, Thermo, and Miltenyi Biopharmaceuticals. Essential amino acids and antibiotics can be added, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL) can be added. Use sterile ophthalmic forceps to remove the tumor tissue from the sample tube and place it in a 10 cm culture dish. Wash the tissue and change the culture dish. Use ophthalmic scissors and ophthalmic forceps to perform preliminary shearing to remove fat tissue and necrotic tissue. Use a disposable scalpel to continue mincing each tissue block to a size of approximately 1-27 cubic millimeters (preferably 1-3 cubic millimeters). Non-suspended tumor tissue pieces were collected at a rate of 1 g per tube and transferred to a single-cell digestion tube (RWD or Miltenyi Biotech) pre-filled with 4 ml of complete medium. The appropriate dose of digestive enzyme (Miltenyi Biotech) was added. The single-cell digestion tube was placed in a single-cell suspension preparation instrument (RWD or Miltenyi Biotech) and digestion was performed using the appropriate tissue digestion program. After digestion, the tissue suspension was collected and filtered through a 70-μm sterile mesh (Miltenyi Biotech) to remove incompletely digested tissue. The single-cell suspension was collected and placed in a 15 ml centrifuge tube (JET or ThermoFisher), washed with 10 ml of complete medium, and centrifuged at 500-600 g for 10 minutes at 25°C. After centrifugation, the supernatant was discarded, the cells were resuspended in complete medium, and counted. Based on the count results, the single cells obtained from the tumor digestion were cultured in 6-well culture plates (ThermoFisher) at a density of 2.0E6 / ml, with a culture volume of 3 ml per well. The culture plate is placed in a carbon dioxide incubator for pre-culture for 0-48 hours, preferably 0-24 hours, to obtain a pre-cultured cell population (as "subpopulation a").

[0103] 1.1.3 Tissue processing and culture

[0104] Take several 10 cm culture dishes and add an appropriate amount of rewarmed complete culture medium. The complete culture medium can be X-vivo 15 medium or other commercial T cell culture medium, such as those from Stem Cell, Lonza, Thermo, Miltenyi Biotech, etc., and can be supplemented with essential amino acids and antibiotics, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL). Use sterile ophthalmic forceps to remove the tumor tissue from the sample tube and place it in a 10 cm culture dish. Wash the tissue and change the culture dish. Use ophthalmic scissors and ophthalmic forceps to perform preliminary shearing to remove fat tissue and necrotic tissue. Use a disposable scalpel to further mince each tissue block to approximately 1-27 cubic millimeters in size. Use a pipette to transfer the non-suspended tumor tissue blocks at approximately 0.1-0.2 g / well to a 6-well culture plate pre-filled with 3 ml of complete culture medium. The culture plate is placed in a carbon dioxide incubator, and the fluid is replenished or half-replaced according to the cell status until preREP is harvested to obtain a preREP TILs population (as "subpopulation b"). The culture process of PreREP is briefly as follows: the cell population is added to a serum-free culture medium and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, e.g., 6000 IU / mL) and cultured for about 3-14 days. Until prePRP is harvested, a preREP TILs population is obtained.

[0105] 1.2 Step (A) Sorting and expansion of CD137-positive and CD3-positive cells in pre-cultured cells

[0106] 1.2.1 Step (A) Pre-culture of CD137-positive cells with magnetic beads

[0107] Single cells obtained from tumor digestion were pre-cultured for 22-24 hours, harvested, counted, washed with pre-chilled cell sorting buffer (PBS + 0.5% BSA + 2mM EDTA), and centrifuged at 500g for 10 minutes at 4°C. Seventy percent of the pre-cultured cell population (subpopulation a) (referred to as "subpopulation c") was taken and the corresponding volume of Anti-CD137 PE (Miltenyi Biotec or Biolegend) antibody was added according to the cell number, with a volume of 100ul / 1.0×10 7 Total cells were incubated at 2-8℃ in the dark for 30 minutes. After staining, they were washed with pre-cooled cell sorting solution and centrifuged at 4℃, 300g for 10 minutes. 7 The ratio of total cells was determined by resuspending single cells with cold cell sorting buffer and adjusting the ratio to 20ul / 1.0×10 7Add Anti-PE magnetic beads (Miltenyi Biotec) to the total cells, mix thoroughly and incubate at 2-8°C in the dark for 15 minutes. After incubation, wash with pre-cooled cell sorting solution and centrifuge at 4°C, 300g for 10 minutes. 7 Resuspend the cells in pre-cooled cell sorting buffer to determine the proportion of total cells.

[0108] 1.2.2 Step (A) Magnetic bead separation of CD137-positive and -negative cells from pre-cultured cells

[0109] Select a magnetic separation column (Miltenyi Biotec) according to the number of cells and place the separation column in the corresponding magnet (Miltenyi Biotec) slot. Use the corresponding volume of cell sorting solution to wash the separation column. After washing, add the single cell suspension containing magnetic bead-labeled cells to the separation column. After it is completely dripped, add a certain volume of sorting solution for washing. The single cell suspension that flows through the separation column and drips is marked as CD137 negative cells. The separated CD137 negative cells (as "d subpopulation") are centrifuged and quantified according to 1-2×10 6 Remove the separation column from the magnetic slot and place it in a suitable 15ml centrifuge tube (JET or ThermoFisher). Add the corresponding volume of separation buffer and use the matching piston to flush out the magnetic bead-bound cells in the separation column and mark them as CD137-positive cells (as "e subset"), count them, and temporarily store them at 2-8°C.

[0110] 1.2.3 Step (A) Magnetic bead labeling of CD3-positive cells

[0111] Take 25% of the pre-cultured cell population (subpopulation a) in step 1.2.1 (A) (referred to as "subpopulation f") and centrifuge. Discard the supernatant. Add Anti-CD3 magnetic beads (Miltenyi Biotec) and cell sorting solution according to the cell number. Mix thoroughly and incubate at 2-8°C in the dark for 15 minutes. After incubation, wash with pre-cooled cell sorting solution and centrifuge at 4°C, 300g, for 10 minutes. 7 Resuspend the cells in pre-cooled cell sorting buffer to determine the proportion of total cells.

[0112] 1.2.4 Step (A) Magnetic bead separation of CD3 positive and negative cells

[0113] Select a magnetic separation column (Miltenyi Biotec) based on the number of cells and place the separation column in the corresponding magnet (Miltenyi Biotec) slot. Use the corresponding volume of cell sorting solution to wash the separation column. After washing, add the single cell suspension containing CD3 magnetic bead-labeled cells to the separation column. After it is completely dripped, add a certain volume of sorting solution for washing. The single cell suspension that flows through the separation column and drips is marked as CD3-negative cells. The separated CD3-negative cells (as "g subpopulation") are centrifuged according to 1-2×106 Remove the separation column from the magnetic slot and place it in a suitable 15ml centrifuge tube. Add the corresponding volume of separation buffer and use the matching piston to flush out the CD3 magnetic bead-bound cells in the separation column and mark them as CD3-positive cells (as "h subset"). After counting, store them temporarily at 2-8°C.

[0114] 1.2.5 Step (A) Expansion and harvesting of CD137-positive and CD3-positive cells obtained by magnetic bead sorting

[0115] Complete culture medium can be selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL) can be added. According to the counting results, the CD137-positive cells and CD3-positive cells obtained by sorting are counted at a concentration of 1-5.0×10 5 The cells were distributed at a density of 4-5 × 10 / well into G-rex 24-well culture plates (Wilson Wolf) and cultured separately. 6 Feeder cells (irradiated healthy donor PBMC T cells) were added to each sorted cell culture well at a ratio of 1:1 / well. Depending on the cell status, the medium was replenished or replaced halfway until harvest. After 14-21 days of expansion, the cells that had completed in vitro expansion in step (A) were collected, centrifuged, the culture medium was discarded, and the cells were washed once with PBS or saline to obtain CD137-positive sorted cells (as "subpopulation i") and CD3-positive sorted cells (as "subpopulation j") after in vitro expansion in step (A), and samples were counted and retained, leaving approximately 5×10 6 to 1×10 7 The remaining cells were added to the freezing solution and the cell density was adjusted to 1-5×10 7 cells / mL for cryopreservation.

[0116] 1.3 Step (B) Isolation and Expansion of CD137-Positive Cells from preREP TILs

[0117] 1.3.1 Step (B) Magnetic Bead Labeling of CD137-Positive Cells in preREP TILs

[0118] The preREP TILs population (subpopulation b) obtained by amplification in step 1.1.3 was harvested and counted, and about 5×10 6 to 1×10 7The cells (as "k subpopulation") were washed with pre-cooled cell sorting solution (PBS + 0.5% BSA + 2mM EDTA) and centrifuged at 500g for 10 minutes at 4°C. The corresponding volume of Anti-CD137 PE (Miltenyi Biotec or Biolegend) antibody was added according to the number of cells, with a volume of 100ul / 1.0×10 7 Total cells were incubated at 2-8℃ in the dark for 30 minutes. After staining, they were washed with pre-cooled cell sorting solution and centrifuged at 4℃, 300g for 10 minutes. 7 The ratio of total cells was determined by resuspending single cells with cold cell sorting buffer and adjusting the ratio to 20ul / 1.0×10 7 Add Anti-PE magnetic beads (Miltenyi Biotec) to the total cells, mix thoroughly and incubate at 2-8°C in the dark for 15 minutes. After incubation, wash with pre-cooled cell sorting solution and centrifuge at 4°C, 300g for 10 minutes. 7 Resuspend the cells in pre-cooled cell sorting buffer to determine the proportion of total cells.

[0119] 1.3.2 Step (B) Isolation of CD137-positive and CD137-negative cells from preREP TILs

[0120] Select a magnetic separation column (Miltenyi Biotec) based on the number of cells and place the separation column in the corresponding magnet (Miltenyi Biotec) slot. Use the corresponding volume of cell separation solution to wash the separation column. After washing, add the single cell suspension containing the magnetic bead-labeled k subpopulation to the separation column. After it is completely dripped, add a certain volume of separation solution for washing. The single cell suspension that flows through the separation column and drips is marked as CD137-negative cells. The separated CD137-negative cells (as "l subpopulation") are centrifuged and sieved according to 1-2×10 6 Remove the separation column from the magnetic slot and place it in a suitable 15ml centrifuge tube. Add the corresponding volume of separation buffer and use the matching piston to flush out the magnetic bead-bound cells in the separation column and mark them as CD137-positive cells (as "m subpopulation"). After counting, store them temporarily at 2-8°C.

[0121] 1.3.3 Step (B) Expansion and harvesting of CD137-positive and unsorted preREP TILs obtained by magnetic bead sorting

[0122] Complete culture medium can be selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi, etc., and essential amino acids and antibiotics can be added, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL) can be added. According to the counting results, the CD137 positive cells (m subpopulation) obtained by sorting and 1-1.5×10 6 Unsorted preREP TILs (subpopulation b) were 1-5.0×10 5 The cells were distributed at a density of 4-5 × 10 / well into G-rex 24-well culture plates (Wilson Wolf) and cultured separately. 6 Feeder cells (irradiated healthy donor PBMC T cells) were added to each sorted cell culture well at a ratio of 1:1 / well, and the medium was replenished or half-replaced according to the cell status until harvest. After 14 days of expansion, the cells that completed in vitro expansion in step (B) were collected, centrifuged, the culture medium was discarded, and the cells were washed once with PBS or saline to obtain CD137-positive sorted cells (as "n subpopulation") and unsorted TILs (as "o subpopulation") after in vitro expansion in step (B), and samples were counted and retained for approximately 5×10 6 to 1×10 7 The remaining cells were added to the freezing solution and the cell density was adjusted to 1-5×10 7 cells / mL for cryopreservation.

[0123] The flowchart of the embodiment of the present invention may be performed with reference to any one of the examples in FIG. 1A-1B .

[0124] Example 2 Flow cytometry detection of CD137 expression at magnetic bead sorting time points

[0125] Five percent of the pre-cultured cell population (subpopulation a) was collected (as “subpopulation p”) or (subpopulation b) preREP TILs were harvested (1-5×10 5 The cells (as "q subpopulation") were washed with pre-cooled PBS and centrifuged at 4°C, 500g, for 10 minutes. Flow cytometry was used to detect CD137 expression on the surface of TIL cells before sorting to provide data support for magnetic bead sorting of CD137-positive cells. Sources of the main reagents and materials for flow cytometry assays: V-bottom 96-well plates, manufacturer Corning, catalog number 3894; flow cytometry tubes, manufacturer Corning, catalog number 352052; flow cytometry antibodies were purchased from BD or Biolegend. 1-5×10 5Add a cell sample to a flow tube or a V-bottom 96-well plate. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, add 1mL / tube to the flow tube and 200μL / well to the 96-well plate, and discard the supernatant. Add the prepared antibody working solution for cell surface staining. The antibody (BD or Biolegend) concentration is 1:100 to 1:200, containing activity detection dye at 1:10000. Stain 100μL / tube of the flow tube and 50μL / well of the 96-well plate, and incubate at 2-8℃ in the dark for 30 minutes. After surface staining, wash the cells once with PBS (200μL / time for 96-well plate and 1mL / time for flow tube), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant after centrifugation. Resuspend the cells in 100-500μL PBS and perform flow cytometry detection.

[0126] FIG2 shows that CD3+CD4+ or CD3+CD8+ TILs in tissues derived from lung cancer, ovarian cancer, and cervical cancer have clear CD137 expression after pre-culture.

[0127] Figure 3 shows that preREP CD3+CD4+ or CD3+CD8+ TILs after expansion from lung cancer and cervical cancer tissues clearly expressed CD137.

[0128] Example 3 Detection of tumor-specific recognition and killing functions of each subpopulation after amplification

[0129] The CD137-positive sorted cells (i subpopulation) and CD3-positive sorted cells (j subpopulation) after sorting and expansion of the pre-cultured cell population (a subpopulation) or the CD137-positive sorted cells (n subpopulation) and unsorted TILs (o subpopulation) after sorting and expansion of the preREP TILs population (b subpopulation) were collected, and each group of TIL cells was co-cultured with the target cells, i.e., the recovered autologous CD3-negative cells (g subpopulation) in a round-bottom 96-well culture plate at an effector-target ratio (T cells: target cells, E:T=5:1-10:1). The target cells should be pretreated with IFN-γ (20 ng / ml) for 12-18 hours. 100 μL each of target cells and T cells, two replicate wells for each group, and a TransACT stimulation group as a positive control group, adding transACT (diameter of about 100 to 500 nm, Miltenyi) to make the transACT working solution concentration 1:200 (v / v); the non-stimulation group as a negative control group, only adding the same volume of cell culture medium. Incubate in a 37°C incubator for 12-18 hours. After incubation, collect the supernatant and freeze at -20 or -80°C for testing, and collect the remaining cells in the wells for tumor-specific recognition markers (4-1BB / CD69) detection. Sources of the main reagents and materials for flow cytometry: V-bottom 96-well plates, manufacturer Corning, product number 3894; flow tubes, manufacturer Corning, product number 352052; flow cytometry antibodies purchased from BD or Biolegend. 1-5×10 5 Add a cell sample to a flow tube or a V-bottom 96-well plate. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, add 1mL / tube to the flow tube and 200μL / well to the 96-well plate, and discard the supernatant. Add the prepared antibody working solution for cell surface staining. The antibody (BD or Biolegend) concentration is 1:100 to 1:200, containing activity detection dye at 1:10000. Stain 100μL / tube of the flow tube and 50μL / well of the 96-well plate, and incubate at 2-8℃ in the dark for 30 minutes. After surface staining, wash the cells once with PBS (200μL / time for 96-well plate and 1mL / time for flow tube), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant after centrifugation. Resuspend the cells in 100-500μL PBS and perform flow cytometry detection.

[0130] Figures 4A and 4B show that the tumor-specific response (A: 4-1BB / B: CD69 expression) of sorted CD137-positive cells in lung cancer tumor tissues was clearly higher than that of CD3-positive cells.

[0131] Figures 5A and 5B show that the tumor-specific response (A: 4-1BB / B: CD69 expression) of sorted CD137-positive cells in cervical cancer tumor tissues was clearly higher than that of CD3-positive cells.

[0132] Figures 6A and 6B show that the tumor-specific response (A: 4-1BB / B: CD69 expression) of sorted CD137-positive cells in preREP TILs derived from cervical cancer tumor tissue was significantly higher than that of unsorted preREP TILs. The CD3-positive sorted and expanded TILs group was a test group that was only sorted for T cell markers (CD3) but not CD137.

[0133] Example 4 Flow cytometry detection of tumor-specific cytokine secretion in each subpopulation after amplification

[0134] The supernatant collected after the above co-incubation step is used for cytokine (IFN-γ) detection. Cytokine secretion detection methods can be referred to the instructions of the Cytokine Assay Kit (BD). Human Th1 / Th2 / Th17 cytokine standard lyophilized powder (BD) is reconstituted with 2 mL of Assay Diluent (BD) (the concentration of each cytokine in the standard stock solution is 5000 pg / mL) and serially diluted in the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, and labeled "Standard Tube." One tube containing only Assay Diluent (test diluent) is used as a reference. Add 2 μL / Beads / well of each Capture Bead (BD), followed by 10 μL / well of PE Detection Reagent (BD) and mix to prepare a mixture. Add 22 μL / well to a V-bottom 96-well plate. Then, add 10 μL / well of the supernatant from each standard and experimental group and mix. Incubate at room temperature for 3 hours in the dark. At the end of the incubation, add 200 μL of Wash Buffer (BD) to each well and centrifuge at 500 g for 3 minutes. After centrifugation, add 100 μL of Wash Buffer (BD) to each well and resuspend the cells for flow cytometry analysis.

[0135] FIG7 shows that the tumor-specific IFN-γ secretion of CD137-positive cells sorted from lung cancer tumor tissues is significantly higher than that of CD3-positive cells.

[0136] FIG8 shows that the tumor-specific IFN-γ secretion of CD137-positive cells sorted from cervical cancer tumor tissues is significantly higher than that of CD3-positive cells.

[0137] FIG9 shows that the tumor-specific IFN-γ secretion of CD137-positive cells sorted from ovarian cancer tumor tissues was not significantly higher than that of CD3-positive cells.

[0138] Figure 10 shows that the secretion of tumor-specific IFN-γ by sorted CD137-positive cells in preREP TILs derived from cervical cancer tumor tissue was higher than that of unsorted preREP TILs. The CD3-positive sorted and expanded TILs group was a test group that was only sorted for T cell markers (CD3) but not CD137.

[0139] Figure 11 shows that the secretion of tumor-specific IFN-γ by CD137-positive cells in preREP TILs derived from lung cancer tissue was significantly higher than that of unsorted preREP TILs. The CD3-positive sorted and expanded TILs group was a test group that was only sorted for T cell markers (CD3) but not CD137.

[0140] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A method for enriching tumor-specific cells, the method comprising isolating CD137-positive cells derived from a subject with lung tumor and / or cervical tumor.

2. The method of claim 1, wherein the CD137-positive cells comprise immune cells.

3. The method of any one of claims 1-2, wherein the CD137-positive cells comprise T cells, natural killer (NK) cells, and / or natural killer-like T (NKT) cells. 4 . The method according to claim 1 , wherein the CD137-positive cells comprise αβ T cells and / or γδ T cells.

5. The method according to any one of claims 1 to 4, wherein the CD137-positive cells comprise tumor infiltrating lymphocytes (TIL).

6. The method according to any one of claims 1 to 5, wherein the CD137-positive cells comprise cells derived from the subject's tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and / or peritoneal effusion.

7. According to the method according to any one of claims 1-6, the CD137-positive cells comprise tumor-specific cells, and compared to the original cell population of unseparated CD137-positive cells, the tumor-specific cells can produce and / or release more IFN-γ and / or can express more tumor-specific recognition markers selected from 4-1BB or CD69 after contact with tumor cells derived from the same subject.

8. The method according to any one of claims 1 to 7, further comprising, before isolating the CD137-positive cells, performing in vitro expansion on tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and / or peritoneal effusion derived from the subject with the lung tumor and / or cervical tumor.

9. The method according to claim 8, wherein the in vitro expansion comprises culturing the tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and / or peritoneal effusion derived from the lung tumor and / or cervical tumor subject in a culture environment with an IL-2 concentration of 300 to 9000 IU / mL.

10. The method according to any one of claims 1 to 9, further comprising, after isolating the CD137-positive cells, expanding the CD137-positive cells in vitro. 11 . The method according to claim 10 , wherein the in vitro expansion comprises culturing the CD137-positive cells in a culture environment containing IL-2 at a concentration of 300 to 9000 IU / mL and anti-CD3 antibodies and / or anti-CD28 antibodies.

12. A cell obtained by the method according to any one of claims 1 to 11.

13. A pharmaceutical composition comprising the cell according to claim 12, and optionally a pharmaceutically acceptable carrier.

14. A method for influencing the growth of lung tumor and / or cervical tumor cells, comprising administering the cells of claim 12 and / or the pharmaceutical composition of claim 13.

15. Use of the cell according to claim 12 and / or the pharmaceutical composition according to claim 13 in the preparation of a drug for preventing and / or treating lung tumors and / or cervical tumors.

16. A medicament for preventing and / or treating lung tumors and / or cervical tumors, comprising the cell according to claim 12 and / or the pharmaceutical composition according to claim 13 as an active ingredient.

17. A method for preventing and / or treating lung tumors and / or cervical tumors, comprising administering the cell of claim 12 and / or the pharmaceutical composition of claim 13 to a subject in need thereof.

18. The cell according to claim 12 and / or the pharmaceutical composition according to claim 13, for use in preventing and / or treating lung tumors and / or cervical tumors.

19. A method for identifying an antigen binding receptor, comprising sequencing the cell of claim 12 to identify at least one antigen binding receptor, wherein the antigen binding receptor comprises a T cell receptor.

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