WO2024260318A1 - Method for enriching tumor-specific cells and use of tumor-specific cells - Google Patents

Abstract

Provided is a method for enriching tumor-specific cells. The present invention specifically relates to obtaining tumor-specific cells targeting a specific type of tumor by means of enriching a specific marker. Further provided is the use of the tumor-specific cells. The sorted immune cells have a strong ability to release specific cytokines targeting lung tumors and cervical tumors, a strong ability to specifically kill advanced tumors, or a high expression level of tumor specific recognition markers within the corresponding time window after tumor antigen stimulation. The step of sorting 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

The present invention relates to the field of biomedicine, and in particular to a method for enriching tumor-specific cells and an application thereof.

Background Art

Currently, immunotherapy is an effective method for treating patients with poor prognosis. However, the immune cells used in immunotherapy have weak anti-tumor ability, which is mainly reflected in the low proportion of immune cells with tumor-specific killing ability in cell products. Therefore, enriching and separating specific types of tumor-specific immune cells through magnetic bead separation or cell sorting of tumor-specific cell surface markers will have important prospects for the widespread clinical application of cell therapy.

However, since 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, immunosuppression mechanisms and immune cell states in 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 M.L. et al. mentioned in the abstract (Celine M.L. 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 the 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.

Therefore, there is an urgent need in the art for a tumor-specific surface marker for 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.

Summary of the invention

The present invention provides a method for enriching tumor-specific cells and application thereof. The method 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 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.

The content of the present invention is based in part on the following discovery: In specific tumor tissue samples, such as advanced ovarian cancer tissue samples, it was found that the enriched CD137 positive immune cells obtained by sorting with CD137 as a surface marker did not have an improvement in specific killing or specific cytokine release for advanced ovarian cancer cells. However, in lung cancer and cervical cancer tissue samples, the present invention unexpectedly found that the enriched CD137 positive immune cells obtained by sorting with CD137 as a surface marker had specific killing and cytokine production and/or release for lung cancer and cervical cancer cells, respectively.

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, quantity and/or proportion of CD137 positive cells in a tumor sample of a lung cancer and/or cervical cancer patient.

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.

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.

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.

For example, after obtaining the above cells, a cell culture expansion step can be performed to obtain the required amount of cells for treatment. For example, after obtaining the above cells, a cell culture expansion step and/or a cell reinfusion step can be performed to prevent and/or treat lung tumors and/or cervical tumors.

In one aspect, the present invention provides 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.

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

In another aspect, the present invention provides a pharmaceutical composition comprising the cell of the present invention, and optionally a pharmaceutically acceptable carrier.

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

In another aspect, the present invention provides use of the cell of the present invention and/or the pharmaceutical composition of the present invention in the preparation of a drug for preventing and/or treating diseases and/or symptoms including lung tumors and/or cervical tumors.

Those skilled in the art can easily discern other aspects and advantages of the present invention from the detailed description below. In the detailed description below, only exemplary embodiments of the present invention are shown and described. As will be appreciated by those skilled in the art, the content of the present invention enables those skilled in the art to make changes to the disclosed specific 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 merely exemplary and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are briefly described as follows:

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

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

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

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

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

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.

FIG. 7 shows the results of tumor-specific IFN-γ secretion by CD137-positive cells sorted from lung cancer tumor tissues.

FIG. 8 shows the results of tumor-specific IFN-γ secretion of CD137-positive cells sorted from cervical cancer tumor tissues.

FIG. 9 shows the results of tumor-specific IFN-γ secretion by CD137-positive cells sorted from ovarian cancer tumor tissues.

Figure 10 shows the results of tumor-specific IFN-γ secretion of sorted CD137-positive cells in preREP TILs derived from cervical cancer tumor tissue.

Figure 11 shows the results of tumor-specific IFN-γ secretion of sorted CD137-positive cells in preREP TILs derived from lung cancer tumor tissue.

DETAILED DESCRIPTION

The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

Definition of terms

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, In some embodiments, T cells include 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 αβT cells and/or γδT 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), T cell receptor (TCR) modified immune effector cells (e.g., TCR-T cells).

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 causing 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/light chains, and/or single-chain antibodies.

In the present invention, the term "T cell receptor (TCR)" generally refers to an engineered antigen receptor. For example, TCR may include TCR alpha and/or TCR beta chains that have been isolated and cloned from a T cell population that recognizes a specific target antigen. For example, TCR alpha and/or TCR beta genes (i.e., TRAC and TRBC) may 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 resulting in activation and proliferation of TCR engineered cells.

In the present invention, the term "encoding" generally refers to the ability to directly or indirectly infer the structure or composition information of another type of molecule related to it from the structure or composition information of one molecule according to essentially determined rules. For example, the nucleotide sequence can be inferred from the sequence of amino acids, such as the property of deoxyribonucleic acid to transcribe complementary nucleic acids, including nucleic acids that can be translated into polypeptides. For example, deoxyribonucleic acid can encode RNA transcribed from deoxyribonucleic acid. Deoxyribonucleic acid can similarly encode polypeptides translated from RNA transcribed from deoxyribonucleic acid.

In the present invention, the term "NK cell" is also called "natural killer cell", which generally refers to a cell with large granules in the cytoplasm. NK cells develop from bone marrow lymphoid stem cells and can differentiate and develop depending on the bone marrow or thymus microenvironment. In the present invention, the proportion of NK cells in TIL cells can be changed by the method of the present invention.

In the present invention, "CD4 ⁺ cells" generally refer to cells that are CD4 positive, such as T cells. The terms "CD4 ⁺ cells" and "CD4 positive cells" can be used synonymously. 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.

In the present invention, "CD8 ⁺ cells" generally refer to cells that are positive for CD8, such as T cells. The terms "CD8 ⁺ cells" and "CD8-positive cells" can be used synonymously. 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.

In the present invention, the term "tumor infiltrating lymphocytes" or "TIL" generally refers to a cell population originally obtained as white blood cells, and the cells of the present invention have left the subject's bloodstream and migrated into the 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. TIL may include primary TIL and secondary TIL. "Primary TIL" may be those TIL cells obtained from a subject's tissue sample, and "secondary TIL" may 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 separated and purified, or may be mutually infiltrated with tumor cells. For example, the TIL of the present invention May refer to a TIL population.

In the present invention, the term "stage" in "a stage of in vitro expansion", "a single stage of in vitro expansion", or "a first stage of in vitro expansion" generally refers to a period of expansion process that TIL passes through 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, it can be considered that the TIL cells have entered the next stage of in vitro expansion. In one embodiment, when the TIL cells are centrifuged and/or washed, 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 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.

In the present invention, the term "T cell activator" generally refers to a substance that binds to a corresponding binding receptor on a T cell and mediates a T cell co-stimulatory response. A T cell activator may be a substance other than an antigen receptor required for a T cell to produce an effective immune response. A T cell activator may refer to a T cell co-stimulatory molecule. For example, the T cell activator of the present invention may include any substance comprising a variant, homolog, or functionally active fragment thereof. A T cell activator may include Including but not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal lymphocyte activation molecules (SLAM proteins), NK cell activation receptors, BTLA (the gene encoding it may be 151888), Toll ligand receptors, OX40 (the gene encoding it may be 7293), CD2 (the gene encoding it may be 914), CD7 (the gene encoding it may be 924), CD27 (the gene encoding it may be 939), CD28 (the gene encoding it may be 940), CD30 (the gene encoding it may be 943), CD40 (the gene encoding it may be 958), CDS, ICAM-1 (the gene encoding it may be 3383), LFA -1 (CD11a/CD18) (the gene encoding it may be 3689), 4-1BB (CD137) (the gene encoding it may be 3604), B7-H3 (the gene encoding it may 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 57823), D can be 51348), NKp44 (the gene encoding it can be 9436), NKp30 (the gene encoding it can be 259197), NKp46 (the gene encoding it can be 9437), CD19 (the gene encoding it can be 930), CD4 (the gene encoding it can be 920), CD8α (the gene encoding it can be 925), CD8β (the gene encoding it can be 926), IL-2Rβ, IL-2Rγ, IL7Rα (the gene encoding it can be), ITGA4 (the gene encoding it can be 3676), VLA1 (the gene encoding it can be 3672), CD49a (the gene encoding it can be 3672 ）、IA4 (the gene encoding it may be 3732 in GeneID), CD49D (the gene encoding it may be 3676 in GeneID), ITGA6 (the gene encoding it may be 3655 in GeneID), VLA-6 (the gene encoding it may be 3655 in GeneID), CD49f (the gene encoding it may be 3655 in GeneID), ITGAD (the gene encoding it may be 3681 in GeneID), CD11d (the gene encoding it may be 3681 in GeneID), ITGAE (the gene encoding it may be 3682 in GeneID), CD103 (the gene encoding it may be 3682 in GeneID), ITGAL (the gene encoding it may be 3683 in GeneID), CD11a (the gene encoding it may be 3683 in GeneID), LFA-1 (the gene encoding it may be The following are the gene IDs of the genes: 3683), ITGAM (the gene encoding it may be 3684), CD11b (the gene encoding it may be 3684), ITGAX (the gene encoding it may be 3687), CD11c (the gene encoding it may be 3687), ITGB1 (the gene encoding it may be 3688), CD29 (the gene encoding it may be 3688), ITGB2 (the gene encoding it may be 3689), CD18 (the gene encoding it may be 3689), LFA-1 (the gene encoding it may be 3689), ITGB7 (the gene encoding it may be 3695) 、NKG2D (the gene encoding it may be 22914 in GeneID), NKG2C (the gene encoding it may be 3822 in GeneID), TNFR2 (the gene encoding it may be 7133 in GeneID), TRANCE/RANKL (the gene encoding it may be 8600 in GeneID), DNAM1 (CD226) (the gene encoding it may be 10666 in GeneID), SLAMF4 (CD244, 2B4) (the gene encoding it may be 51744 in GeneID), CD84 (the gene encoding it may be 8832 in GeneID), CD96 (Tactile) (the gene encoding it may be 10225 in GeneID), CEACAM1 (the gene encoding it may be 634 in GeneID), 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. A co-stimulatory intracellular signaling domain may refer to an intracellular portion of a T cell activator. An intracellular signaling domain may comprise a complete intracellular portion of a molecule derived therefrom or a complete native intracellular signaling domain or its function Sexual episode.

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 any substance including its variants, homologues or functionally active fragments thereof. 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 in GeneID), IL-4 (the gene encoding it may be 3565 in GeneID), IL-6 (the gene encoding it may be 3569 in GeneID), IL-7 (the gene encoding it may be 3574 in GeneID), IL-10 (the gene encoding it may be 3586 in GeneID), IL-12 (the gene encoding it may be 3592 or 3593 in GeneID), IL-15 (the gene encoding it may be 3600 in GeneID), IL-21 (the gene encoding it may be 59067 in GeneID), TNF-α (the gene encoding it may be 100137091 in GeneID), interferon-γ (the gene encoding it may be 3458 in GeneID), GZMB (the gene encoding it may be 3002 in GeneID), CD107a (the gene encoding it may be 6499 in GeneID), etc.

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, or can be in vitro amplification. The first stage in vitro amplification of the present invention can also be referred to as the preREP (pre-rapid expansion) stage. For example, TILs derived from tumor tissues and not amplified in vitro can be referred to as the first TIL group. For example, the TILs obtained through the first stage in vitro amplification in the culture method of the present invention divided into two steps can be referred to as the second TIL group.

In the present invention, the term "second stage in vitro expansion" generally refers to the stage of further expansion after the tissue is taken out of the subject and expanded. In one embodiment, compared with the TIL expanded in vitro in the first stage, the number of TIL cells expanded in vitro in the second stage of the present invention is increased, 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. In one embodiment, the second stage of in vitro amplification may be different from the culture conditions of the first stage of in vitro amplification, for example, the culture substances added may be different. For example, in the culture method of the present invention divided by the two-step method, the second stage of in vitro amplification may also be referred to as the REP (rapid expansion) stage. For example, in the culture method of the present invention divided by the two-step method, the TILs obtained by the second stage of in vitro amplification may be referred to as the third TIL group.

In the present invention, the term "tumor-specific cells" generally refers to cells that can specifically resist tumor growth. Tumor-specific cells may have specific tumor-specific killing ability or tumor-specific cytokine release ability, for example, by co-culturing with a specific tumor, by detecting cytokine expression, production and/or release, and/or tumor cell apoptosis, to identify tumor-specific cells. Tumor-specific cells may have a more specific ability to resist tumor growth than ordinary cells.

In the present invention, the term "isolated" generally refers to a change or departure from a 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 may exist in a substantially purified form, or may exist in a non-natural environment, such as a delivery vector.

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

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

In the present invention, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the active ingredients. For example, a pharmaceutically acceptable carrier may not interfere with Interfere with the biological activity of the active ingredient; for example, the 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 materials suitable for administration to humans. Other envisioned carriers, excipients, and/or additives that can be used in the preparations 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 counterions (such as sodium), etc. These and other known drug carriers, excipients and/or additives suitable for use in the preparations described herein are known in the art. In the present invention, "pharmaceutically acceptable carrier" can be understood as a vector that does not contain a nucleic acid form used in genetic engineering.

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 of a non-solid tumor in the subject.

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 variation contained in the term "about" or "approximately" may depend on the specific system under study, and can be easily understood by those of ordinary skill in the art.

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

DETAILED DESCRIPTION OF THE INVENTION

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

The present invention is based in part on the discovery that in certain tumor tissue samples, for example For example, in tissue samples of advanced ovarian cancer, it was found that the enriched CD137 positive immune cells obtained by sorting with CD137 as a surface marker did not have any improvement in specific killing or specific cytokine release for advanced ovarian cancer cells. However, in tissue samples of lung cancer and cervical cancer, the present invention unexpectedly found that the enriched CD137 positive and/or CD69 positive immune cells obtained by sorting with CD137 and/or CD69 as surface markers had specific killing and cytokine production and/or release for lung cancer and cervical cancer cells, respectively.

For example, the present invention provides the use of CD137 and/or CD69 as a marker to identify and separate naturally occurring tumor-specific tumor infiltrating lymphocytes (TIL). In one embodiment, CD137 and/or CD69 are a marker 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 separation and cultivation of CD137 positive and/or CD69 positive cell colonies from lung cancer or cervical cancer tissue samples. In such an embodiment, CD137 positive and/or CD69 positive cell colonies can include tumor-specific T cells.

For example, the present invention provides a method 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.

In one embodiment, the CD137-positive and/or CD69-positive cells produce IFN-γ after contacting HLA-matched lung or cervical cancer tumor cells. For example, compared to non-tumor specific cells or original cell populations that have not been sorted for CD137 and/or CD69, the tumor specific cells, after contact with tumor cells from the same subject, produce and/or release IFN-γ in an amount that is about 100,000-fold to about 1%, such as about 100,000-fold, about 10,000-fold, about 1000-fold, about 100-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%.

In one embodiment, CD137-positive and/or CD69-positive cells can express more tumor-specific recognition markers after contacting 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 contacting tumor cells 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 1000 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 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.

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 with the prior art method of not enriching 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.

For example, the present invention includes a method for treating a patient tumor, comprising administering an effective amount of a CD137-positive and/or CD69-positive cell population to a patient in need thereof. In an embodiment of the present invention, CD137-positive and/or CD69-positive cells can be obtained from 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 separation and culture. In an embodiment of the present invention, CD137 positive and/or CD69 positive cells can be separated and cultured from late, metastatic and/or recurrent tumor tissue samples. In an embodiment of the present invention, tumor tissue includes but is not limited to tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and/or peritoneal effusion.

For example, CD137 positive and/or CD69 positive cells may include immune cells.For example, CD137 positive and/or CD69 positive cells may 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 may include αβT cells and or γδT cells.For example, CD137 positive and/or CD69 positive cells may include CAR cells and/or TCR cells.The methods described herein may 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 autologous to the patient.In this case, the cells transferred into the patient include autologous cells of the patient to prevent rejection or adverse immune responses to the administered cells.

On the other hand, CD137 positive and/or CD69 positive cell populations are separated from liquid tissues containing immune cells, such as bone marrow or ascites. Immune cells such as lymphocytes (e.g., TIL, CTL, NK cells and LAK cells) can be separated using a variety of methods known in the art. For example, in a method of separating CTL, allogeneic restricted CTL is produced by stimulating natural splenocytes in vitro with a suitable antigen. For example, a blood sample containing cell precursors taken from a mammal can be used, PBL (peripheral blood lymphocyte) can be obtained after purification, and incubated with stimulating cells of specific antigenic peptides. Human primary NK cells can be expanded in the presence of a bone marrow cell line, which 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 separated 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 obtained, such as lymphocytes (e.g., TIL, CTL, NK cells or LAK cells), include colonies with clonal capacity. In other cases, the cell population obtained may not have clonal capacity or may not have unlimited clonal capacity.

In one embodiment, tumor-specific cells are separated from lung tumors and/or cervical tumor tissues containing CD137 positive and/or CD69 positive cell groups. In most cases, tumor tissues contain a heterogeneous mixture of cells and cell types, including cancer cells and CD137 positive and/or CD69 positive cell groups containing tumor-specific cells. Tumor tissues can also include tumor antigens. In one embodiment, tumor antigens have been exposed to tumor-specific cells, and the tumor-specific cells have been stimulated. Before selectively separating CD137 positive and/or CD69 positive cells, tumor tissue is first 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 further enrich tumor-specific cells. For example, by further separating cells with other biomarkers, CD137 positive and/or CD69 positive cells can further enrich tumor-specific T cells.

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 amplification, for example by flow cytometry, negative or positive selection or other methods. Another embodiment includes obtaining cells from tumor tissue after in vitro amplification. Although the prior art shows that after in vitro amplification in an artificial environment, immune cell surface markers may undergo substantial changes, and the original surface markers cannot be used to sort tumor-specific cells. However, the present invention unexpectedly discovered that after the preREP stage treatment in the field of TIL, 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 a culture medium containing IL-2 at a concentration of 300-9000 IU/mL for about 3-14 days.

On the other hand, the method of culturing tumor infiltrating lymphocytes (TIL) of the present invention comprises: (A) contacting a first TIL group derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis 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 group is obtained by step (A); (B) contacting the second TIL group with a T cell activator and/or a T cell growth factor, and optionally 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). 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 may be enriched for CD137 and/or CD69.

On the other hand, the method of culturing tumor infiltrating lymphocytes (TIL) 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 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 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 days to about 14 days; step (B) is performed for about 0 days to about 8 days; step (C) is performed for about 5 days to about 14 days.

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 through step (A); (B) enriching the second TIL population for CD137 and/or CD69; After collection, contact with a T cell activator and/or a T cell growth factor, and optionally gene editing can be performed, wherein a third TIL population is obtained through step (B); (C) the third TIL population is co-cultured with feeder cells, wherein a fourth TIL population is obtained through step (C). Step (A) is performed for about 7 days to about 14 days; step (B) is performed for about 0 days to about 8 days; and step (C) is performed for about 5 days to about 14 days.

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 a T cell growth factor, and optionally gene editing, wherein a third TIL population is obtained by step (B); (C) enriching the third TIL population for CD137 and/or CD69, and co-culturing with feeder cells, wherein a fourth TIL population is obtained by step (C). Step (A) is performed for about 7 days to about 14 days; step (B) is performed for about 0 days to about 8 days; step (C) is performed for about 5 days to about 14 days.

For example, the method of culturing tumor infiltrating lymphocytes (TIL) of the present invention comprises: the method of obtaining TIL cells from a subject tissue sample can be a patient surgery to obtain an in situ tumor sample or a metastatic tumor sample, the weight can be at least about 1g, or multiple tissues can be combined. Tumor tissue, tumor-related lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and/or peritoneal effusion are transported in a sample transport fluid, such as a commercially commonly used tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid at about 2-8°C and processed within 48 hours. The tissue block can be mechanically broken to a size of about 1-27 cubic millimeters per block, transferred into a breathable culture bag or Grex, and a cell serum-free culture medium and IL-2 at a concentration of 300-9000IU/mL (e.g., 1000-9000IU/mL, such as 6000IU/mL) are added and cultured for about 3-14 days. The cells in the culture medium are collected and transferred into a breathable culture bag, or a Grex, or a Xuri device. The serum-free culture medium of the cells can be supplemented with the CD28 antibody, CD3 antibody and CD28 antibody of the present invention, magnetic beads (such as Dynabeads) containing CD3 antibody and CD28 antibody and/or nanomatrix (such as transACT) containing CD3 antibody and CD28 antibody, at a concentration of 300-9000. IU/mL (e.g., 1000-9000IU/mL, e.g., 6000IU/mL) of IL-2 and optionally editing the expression of the target gene in the cell population (e.g., by using a ribonucleoprotein complex (RNP) formed by carrying gRNA and Cas protein, or LNP containing gRNA and Cas protein, or LNP containing nucleic acid encoding gRNA and Cas protein for transduction gene editing), after activating the TIL of the present invention for a certain period of time, adding irradiated PBMC (TIL and PBMC according to a ratio of about 1:40-about 1:400), and amplifying and culturing for about 3-14 days. Cells in the culture medium can be collected using a cell processing system, washed and frozen, and detected. The final product CD3 ratio can be greater than 80%, the cell viability can be greater than 50%, and cells greater than 80% can be memory effector cells and effector cells. IFN-γ can be secreted after stimulation, and/or can have the characteristics of an increased proportion of activated cells. For example, enrichment of CD137 and/or CD69 can be performed between the various steps in the above method.

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 attached to a physical carrier, such as a magnetic bead, a magnetic particle, a magnetic nanomaterial, a microbead, a column, an adsorption column, and an adsorption membrane. It is well known in the art that antibodies are conjugated to physical supports.

For example, CD137 positive and/or CD69 positive cells can be separated from HLA matched tumor tissue. For example, CD137 positive and/or CD69 positive cells can be selectively separated 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 derived 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 being exposed to HLA matched tumor cell lines (e.g., autologous tumor cells).

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 additive for cell growth known to the skilled artisan.

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.

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.

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 a cell therapy product.

On the other hand, the present invention provides a method for reducing tumor-specific killer cells, 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.

For example, after obtaining the above cells, a cell culture expansion step can be performed to obtain the required amount of cells for treatment. For example, after obtaining the above cells, a cell culture expansion step and/or a cell reinfusion step can be performed to prevent and/or treat lung tumors and/or cervical tumors.

In one aspect, the present invention provides a cell, and the cell of the present invention 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 or a batch of cells cultured by the culture method of the present invention. In one embodiment, the cell provided by the present invention can include multiple or multiple batches of cells cultured by the culture method of the present invention and combined in any proportion.

In some embodiments, 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 The administration may be by intravenous or intravenous infusion, which may last for about 30 to 60 minutes. Other suitable routes of administration may include intraperitoneal, intrathecal, and intralymphatic administration.

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 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 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 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 6 times, about 7 times, about 8 times, about 10 times, about 10 times, about 15 times, about 15 times, about 10 ... 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, 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 cells not enriched based on CD137 and/or CD69, in the enriched cell population of the present invention, the proportion of CD137-positive and/or CD69-positive cells 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%.

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 of CD137 and/or CD69, in the enriched cell population of 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 1 ... 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, 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 enriched cell population of the present invention, after contacting a specific matching tumor cell, the level of tumor cell apoptosis 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 15 times, about 20 times, about 25 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 25 times, about 26 times, about 27 times, about 28 times, about 30 ... 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, 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 receptor of the enriched cells is determined. For example, the TCR derived from the enriched cells can be used to develop engineered TCR cells. For example, relative to a cell population that is not enriched based on CD137 and/or CD69, or screened out for CD137 and/or CD69, the TCR obtained from the enriched cell population of the present invention is used to engineer cells, and the tumor specificity 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 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 detected by the level of tumor-specific cytokines such as IFN-γ produced and/or released after contact with specific matched tumor cells, and/or the number of tumor cells and/or the level of apoptosis after contact with specific matched tumor cells.

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 cells may be administered. In some embodiments, about 1×10 ⁹ to about 12×10 ¹⁰ cells may be administered. In some embodiments, about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ cells may be administered. In some embodiments, about 3×10 ¹⁰ to about 12×10 ¹⁰ cells may be administered. In some embodiments, about 4×10 ¹⁰ to about 10×10 ¹⁰ cells may be administered. In some embodiments, about 5×10 ¹⁰ to about 8×10 ¹⁰ cells may be administered. In some embodiments, about 6×10 ¹⁰ to about 8×10 ¹⁰ cells may be administered. In some embodiments, about 7×10 ¹⁰ to about 8×10 ¹⁰ cells may be administered. In some embodiments, the therapeutically effective dose may 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 cells. In some embodiments, the therapeutically effective dose may be about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ cells. In some embodiments, the therapeutically effective dose may be about 3×10 ¹⁰ to about 12×10 ¹⁰ cells. In some embodiments, the therapeutically effective dose may be about 4×10 ¹⁰ to about 10×10 ¹⁰ cells. In some embodiments, the therapeutically effective dose may be about 5×10 ¹⁰ to about 8×10 ¹⁰ cells. In some embodiments, the therapeutically effective dose may be about 6×10 ¹⁰ to about 8×10 ¹⁰ cells. In some embodiments, the therapeutically effective dose may be about 7×10 ¹⁰ to about 8×10 ¹⁰ cells.

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 2 days. In some embodiments, the administration of the cells can be continuous administration.

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

In one aspect, the present invention provides a kit, which may include a cell activator, a cell growth factor and/or a feeder cell of the cell culture method of the present invention and an instruction manual recording the steps of the cell culture method of the present invention. In one aspect, the present invention provides a kit, which may include a cell of the present invention and/or a pharmaceutical composition of the present invention.

In one aspect, the present invention provides a method of affecting the growth of cells, such as tumor cells, which may include administering cells of the present invention and/or pharmaceutical compositions of the present invention to a subject. In some embodiments, affecting tumor growth may include reducing the volume of the tumor to about 99-0.1% 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%.

On the one hand, the present invention provides the use of the cell of the present invention and/or the pharmaceutical composition of the present invention in the preparation of a drug, and the drug of the present invention 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 kidney cancer. For example, the tumor of the present invention can be an advanced solid tumor.

In one aspect, the present invention provides a method for preventing and/or treating a disease and/or symptom, which may include 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 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 may be selected from the following groups: One or more of: 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 can be an advanced solid tumor.

On the one hand, 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.

Without intending to be bound by any theory, the following embodiments 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.

Example

Example 1 Sorting and amplification of tumor infiltrating lymphocytes

1.1 Tumor tissue reception and processing

1.1.1 Organization reception

Receive tumor tissue from the donor, check and record sample information, and print corresponding sample labels.

1.1.2 Tissue processing, digestion and pre-culture

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 culture medium or other commercial T cell culture medium, such as T cell culture medium of brands such as Stem Cell, Lonza, Thermo, Miltenyi Biotec, 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. Use sterile ophthalmic forceps to remove the tumor tissue from the sample tube into a 10 cm culture dish, wash the tissue and replace the culture dish. Use ophthalmic scissors and ophthalmic forceps to perform preliminary shearing to remove adipose tissue and necrotic tissue, and use a disposable scalpel to continue to mince each tissue block to a size of about 1-27 cubic millimeters (preferably 1-3 cubic millimeters). According to 1g of tissue/tube, take non-suspended tumor tissue blocks and transfer them to a pre-set 4ml complete culture medium. The single cell digestion tube (RWD or Miltenyi) of the base was placed and the corresponding dose of digestive enzyme (Miltenyi) was added. The single cell digestion tube was placed on the single cell suspension preparation instrument (RWD or Miltenyi), and the corresponding tissue digestion program was selected for digestion. After digestion, the tissue suspension was collected and filtered with a 70-micron sterile mesh (Miltenyi) to remove the incompletely digested participating tissues. The single cell suspension was collected in a 15 ml centrifuge tube (JET or ThermoFisher) and 10 ml of complete medium was added for washing, 25 ° C, 500-600g, centrifuged for 10 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in complete medium and counted. According to the counting results, the single cells obtained by tumor digestion were cultured in a 6-well culture plate (Thermo Fisher) at a density of 2.0E6/ml, and the culture volume per well was 3 ml. The culture plate was 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 "a subpopulation").

1.1.3 Tissue processing and culture

Take several 10 cm culture dishes and add an appropriate amount of rewarmed complete medium. The complete medium can be any X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell 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. Use sterile ophthalmic forceps to remove the tumor tissue from the sample tube into a 10 cm culture dish, wash the tissue and replace the culture dish. Use ophthalmic scissors and ophthalmic forceps to perform preliminary shearing, remove fat tissue and necrotic tissue, and use a disposable scalpel to continue to mince each tissue block to about 1-27 cubic millimeters. Use a pipette to transfer the non-suspended tumor tissue block to a 6-well culture plate pre-filled with 3 ml of complete medium at about 0.1-0.2 g/well. Place the culture plate in a carbon dioxide incubator, and replenish or half-change the medium 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: add serum-free culture medium and IL-2 at a concentration of 300-9000 IU/mL (e.g., 1000-9000 IU/mL, such as 6000 IU/mL) to the cell population and culture for about 3-14 days. Until prePRP is harvested, a preREP TILs population is obtained.

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

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

Single cells obtained from tumor digestion were pre-cultured for 22-24 hours and harvested and counted, washed with pre-cooled cell sorting solution (PBS + 0.5% BSA + 2mM EDTA), and centrifuged at 4°C, 500g for 10 minutes. Seventy percent of the pre-cultured cell population (subpopulation a) (as "subpopulation c") was taken and added with the corresponding volume of Anti-CD137 PE (Miltenyi or Biolegend) antibody according to the number of cells, with a volume of 100ul/1.0×10 ⁷ total cells, and incubated at 2-8°C in the dark for 30 minutes. After staining, the cells were washed with pre-cooled cell sorting solution, centrifuged at 4°C, 300g for 10 minutes. Single cells were resuspended in a pre-cooled cell sorting solution at a ratio of 80ul/1.0×10 ⁷ total cells and Anti-PE magnetic beads (Miltenyi) were added at a ratio of 20ul/1.0×10 ⁷ total cells, mixed thoroughly, and incubated at 2-8°C in the dark for 15 minutes. After incubation, wash with pre-cooled cell sorting solution, centrifuge at 4°C, 300g for 10 minutes, and resuspend the cells with pre-cooled cell sorting solution at a ratio of 500ul/1.0× ¹⁰⁷ total cells.

1.2.2 Step (A) Magnetic bead sorting of CD137 positive and negative cells from pre-cultured cells

According to the number of cells, a magnetic separation column (Miltenyi Biotec) is selected and placed in the slot of the corresponding magnet (Miltenyi Biotec), and the separation column is washed with a corresponding volume of cell sorting solution. After washing, a single cell suspension containing magnetic bead-labeled cells is added to the separation column, and a certain volume of sorting solution is added for washing after it is completely dripped. The single cell suspension that flows through the separation column and drips is marked as CD137-negative cells, and the separated CD137-negative cells (as "d subpopulation") are centrifuged and frozen at 1-2×10 ⁶ /tube. The separation column is removed from the magnet slot and placed in a suitable 15ml centrifuge tube (JET or ThermoFisher), and the corresponding volume of separation solution is added and the magnetic bead-bound cells in the separation column are flushed out with the help of the matching piston and marked as CD137-positive cells (as "e subpopulation"), and they are temporarily stored at 2-8°C after counting.

1.2.3 Step (A) Magnetic bead labeling of CD3 positive cells

Take 25% of the pre-cultured cell population (subpopulation a) in step 1.2.1 (A) (as "subpopulation f") and centrifuge and discard the supernatant. Add Anti-CD3 magnetic beads (Miltenyi Biotec) and cell sorting solution according to the number of cells, mix thoroughly and incubate at 2-8℃ in the dark for 15 minutes. After incubation, wash with pre-cooled cell sorting solution and centrifuge at 4℃, 300g for 10 minutes. Resuspend the cells with pre-cooled cell sorting solution according to the ratio of 500ul/1.0×10 ⁷ total cells.

1.2.4 Step (A) Magnetic bead sorting of CD3 positive and negative cells

Select a magnetic separation column (Miltenyi Biotec) according to the number of cells and place the separation column in the slot of the corresponding magnet (Miltenyi Biotec). 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 and frozen at 1-2×10 ⁶ /tube. Remove the separation column from the magnet slot and place it in a suitable 15ml centrifuge tube, add the corresponding volume of separation solution 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 subpopulation"), count them and store them temporarily at 2-8°C.

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

Complete medium is used. The complete medium can be arbitrarily selected from X-vivo 15 medium or other commercial T cell medium, such as T cell medium of brands such as Stem Cell, Lonza, Thermo, Miltenyi Biotech, 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) is added. According to the counting results, the CD137-positive cells and CD3-positive cells obtained by sorting are distributed to G-rex 24-well culture plates (Wilson Wolf) at a density of 1-5.0×10 ⁵ /well for separate culture, and feeder cells (irradiated healthy donor PBMC T cells) are added to each sorted cell culture well at a ratio of 4-5×10 ⁶ /well. And according to the cell status, the fluid is replenished or half-replaced until harvest. After 14-21 days of expansion, the cells that have completed in vitro expansion in step (A) are collected and centrifuged, the culture medium is discarded, and the cells are washed once with PBS or normal saline to obtain CD137-positive sorted cells (as "i subpopulation") and CD3-positive sorted cells (as "j subpopulation") after in vitro expansion in step (A), respectively. Samples are counted to retain about 5×10 ⁶ to 1×10 ⁷ cells for subsequent functional testing, and all the remaining cells are added to the freezing solution, and the cell density is adjusted to 1-5×10 ⁷ cells/mL for freezing.

1.3 Step (B) Sorting and expansion of CD137 positive cells in preREP TILs

1.3.1 Step (B) Magnetic bead labeling of CD137-positive cells in preREP TILs

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 ⁷ cells (as "subpopulation k") were retained and washed with pre-cooled cell sorting solution (PBS + 0.5% BSA + 2mM EDTA), and centrifuged at 4°C, 500g for 10 minutes. According to the number of cells, the corresponding volume of Anti-CD137 PE (Miltenyi or Biolegend) antibody was added, and the chromosome volume was 100ul/1.0×10 ⁷ total cells, and incubated at 2-8°C in the dark for 30 minutes. After staining, the cells were washed with pre-cooled cell sorting solution, and centrifuged at 4°C, 300g for 10 minutes. Resuspend single cells in pre-cooled cell sorting buffer at a ratio of 80ul/1.0×10 ⁷ total cells and add Anti-PE magnetic beads (Miltenyi Biotec) at a ratio of 20ul/1.0×10 ⁷ total cells. Mix thoroughly and incubate at 2-8°C in the dark for 15 minutes. After incubation, wash with pre-cooled cell sorting buffer and centrifuge at 4°C, 300g, for 10 minutes. Resuspend cells in pre-cooled cell sorting buffer at a ratio of 500ul/1.0×10 ⁷ total cells.

1.3.2 Step (B) Sorting of CD137-positive and -negative cells in preREP TILs

According to the number of cells, a magnetic separation column (Miltenyi Biotec) is selected and placed in the slot of the corresponding magnet (Miltenyi Biotec). The separation column is washed with a corresponding volume of cell sorting solution. After washing, a single cell suspension containing a magnetic bead-labeled k subpopulation is added to the separation column. After it is completely dripped, a certain volume of sorting solution is added 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 frozen at 1-2×10 ⁶ /tube. The separation column is removed from the magnet slot and placed in a suitable 15ml centrifuge tube. The corresponding volume of separation solution is added and the magnetic bead-bound cells in the separation column are flushed out with the help of the matching piston and marked as CD137-positive cells (as "m subpopulation"), and they are temporarily stored at 2-8°C after counting.

1.3.3 Step (B) Amplification and harvesting of CD137-positive and unsorted preREP TILs obtained by magnetic bead sorting

Complete culture medium is used. The complete culture medium can be any X-vivo 15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 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 Select preREP TILs (subpopulation b) and distribute them to G-rex 24-well culture plates (Wilson Wolf) at a density of 1-5.0×10 ⁵ /well and culture them separately. Feeder cells (irradiated healthy donor PBMC T cells) are added to each sorted cell culture well at a ratio of 4-5×10 ⁶ /well, and the medium is replenished or half-replaced according to the cell status until harvest. After 14 days of expansion, the cells that have completed in vitro expansion in step (B) are collected, centrifuged, the culture medium is discarded, and the cells are washed once with PBS or saline to obtain CD137 positive sorted cells (as "subpopulation n") and unsorted TILs (as "subpopulation o") after in vitro expansion in step (B), and samples are counted to retain about 5×10 ⁶ to 1×10 ⁷ cells for subsequent functional testing. All other cells are added to the freezing solution and the cell density is adjusted to 1-5×10 ⁷ cells/mL for freezing.

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

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

Collect 5% of the pre-cultured cell population (subpopulation a) (as "subpopulation p") or (subpopulation b) preREP TILs 1-5×10 ⁵ (as "subpopulation q") after harvesting, wash with pre-cooled PBS, centrifuge at 4°C, 500g for 10 minutes. Flow cytometry was used to detect the expression of CD137 on the surface of TIL cells before sorting to provide data support for magnetic bead sorting of CD137-positive cells. Sources of main reagents and materials for flow cytometry: V-bottom 96-well plate, manufacturer Corning, item number 3894; flow tube, manufacturer Corning, item number 352052; flow antibodies purchased from BD or Biolegend. Add 1-5×10 ⁵ cell samples from each group to flow tubes or V-bottom 96-well plates. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, 1mL/tube of flow tubes and 200μL/well of 96-well plates, 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 active detection dye 1:10000. 100μL/tube for flow tubes, 50μL/well for 96-well plates, incubate at 2-8℃ in the dark for 30 minutes. After surface staining, wash the cells once with PBS (200μL/time for 96-well plates, 1mL/time for flow tubes), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant after centrifugation. Resuspend the cells with 100-500μL PBS for flow cytometry detection.

FIG. 2 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.

Figure 3 shows that there is clear CD137 expression in preREP CD3+CD4+ or CD3+CD8+ TILs after tissue expansion from lung cancer and cervical cancer.

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

The CD137-positive sorted cells (i subgroup) and CD3-positive sorted cells (j subgroup) after sorting and expansion of the pre-cultured cell group (a subgroup), or the CD137-positive sorted cells (n subgroup) and unsorted TILs (o subgroup) after sorting and expansion of the preREP TILs group (b subgroup) were collected, and each group of TIL cells was co-cultured with the target cells, i.e., the recovered autologous CD3-negative cells (g subgroup) in a round-bottom 96-well culture plate at an effector-target ratio (T cells: target cells, E:T=5:1-10:1), and the target cells should be pretreated with IFN-γ (20ng/ml) for 12-18 hours. 100 μL 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 concentration of transACT working solution 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 it at -20 or -80°C for testing, and collect the remaining cells in the well for tumor-specific recognition markers (4-1BB/CD69). Sources of the main reagents and materials for flow cytometry: V-bottom 96-well plate, manufacturer Corning, catalog number 3894; flow tube, manufacturer Corning, catalog number 352052; flow antibodies purchased from BD or Biolegend. Add 1-5×10 ⁵ cell samples from each group to a flow tube or V-bottom 96-well plate. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, add 1mL/tube to flow tubes and 200μL/well to 96-well plates, 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, and contains active detection dye 1:10000. Add 100μL/tube to flow tubes and 50μL/well to 96-well plates, 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 plates and 1mL/time for flow tubes), centrifuge at 600g for 3 minutes at room temperature, and discard the supernatant after centrifugation. Resuspend the cells with 100-500μL PBS and perform flow cytometry detection.

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.

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.

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 is significantly higher than that of unsorted preREP TILs. The CD3-positive sorted amplified TILs group is a test group that only sorted T cell markers (CD3) but not CD137.

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

The supernatant collected after the above co-incubation step is completed can be used for cytokine (IFN-γ) detection. The cytokine secretion detection method can refer to the instructions of the cytokine detection kit (BD), and the human Th1/Th2/Th17 cytokine standard lyophilized powder (BD) is reconstituted with 2mL Assay Diluent diluent (BD) (the concentration of each cytokine in the standard stock solution is 5000pg/mL) and diluted in the order of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, and marked as "standard tube". Take 1 tube containing only Assay Diluent (test diluent) as a reference. Add each Capture Beads (BD) at 2 μL/Beads/well, then add PE Detection Reagent (PE Detection Reagent) (BD) at 10 μL/well and mix to prepare a mixture, add 22 μL/well to a V-bottom 96-well plate, then add the supernatant of each standard and experimental group at 10 μL/well and mix, incubate at room temperature in the dark for 3 hours. At the end of incubation, add 200 μL Wash Buffer (BD) to each well and centrifuge at 500g for 3 minutes. At the end of centrifugation, add 100 μL Wash Buffer (BD) to each well, resuspend, and perform flow analysis.

FIG. 7 shows that the tumor-specific IFN-γ secretion of CD137-positive cells sorted from lung cancer tumor tissues is clearly higher than that of CD3-positive cells.

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

Figure 9 shows the tumor specificity of CD137 positive cells sorted from ovarian cancer tumor tissues IFN-γ secretion was not significantly higher than that of CD3 positive cells.

Figure 10 shows that the tumor-specific IFN-γ secretion of sorted CD137-positive cells in preREP TILs derived from cervical cancer tumor tissue is higher than that of unsorted preREP TILs. Among them, the CD3-positive sorted amplified TILs group is a test group that only sorted T cell markers (CD3) but not CD137.

Figure 11 shows that the tumor-specific IFN-γ secretion of sorted CD137-positive cells in preREP TILs derived from lung cancer tumor tissue is significantly higher than that of unsorted preREP TILs. Among them, the CD3-positive sorted amplified TILs group is a test group that only sorted T cell markers (CD3) but not CD137.

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 are obvious to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims (19)

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. The method of claim 1, wherein the CD137-positive cells comprise immune cells. The method according to any one of claims 1 to 2, wherein the CD137-positive cells comprise T cells, natural killer (NK) cells, and/or natural killer-like T (NKT) cells. According to the method according to any one of claims 1 to 3, the CD137-positive cells comprise αβT cells and/or γδT cells. According to the method according to any one of claims 1-4, the CD137-positive cells comprise tumor infiltrating lymphocytes (TIL). According to the method according to any one of claims 1 to 5, 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. According to the method according to any one of claims 1 to 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. The method according to any one of claims 1 to 7, further comprising, before isolating the CD137-positive cells, performing in vitro amplification 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 lung tumor and/or cervical tumor subject. According to the method of claim 8, 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 subject with the lung tumor and/or cervical tumor in a culture environment with an IL-2 concentration of 300 to 9000 IU/mL. The method according to any one of claims 1 to 9, further comprising expanding the CD137-positive cells in vitro after isolating the CD137-positive cells. According to the method of claim 10, the in vitro expansion comprises culturing the CD137-positive cells in a culture environment with a concentration of IL-2 of 300 to 9000 IU/mL, and anti-CD3 antibodies and/or anti-CD28 antibodies. A cell obtained by the method according to any one of claims 1 to 11. A pharmaceutical composition comprising the cell according to claim 12, and optionally a pharmaceutically acceptable carrier. 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. 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. A medicine 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. 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. The cell according to claim 12 and/or the pharmaceutical composition according to claim 13 is used for preventing and/or treating lung tumors and/or cervical tumors. 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.