Culture method for and use of immune cells

Abstract

A culture method for and a use of immune cells, specifically relating to contact between isolated immune cells and organoids. The cultured immune cells exhibit a strong cytokine release capacity or specific killing ability. The culture step improves the anti-tumor activity of cells in cell therapy.

Description

Immune cell culture method and application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for culturing immune cells and applications thereof. 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 within the cell product. Therefore, providing a culture method to enhance the specific cytokine release or killing capacity of immune cells against tumor cells holds great promise for the widespread clinical application of cell therapy.

[0003] However, because immune cell culture methods typically use interleukin-2, CD3 antibodies, and / or PBMCs from healthy donors, the anti-tumor ability of the resulting immune cells still needs to be further improved. Therefore, there is an urgent need in the art for a method of culturing immune cells to enhance the anti-tumor ability of in vitro cultured immune cells for widespread application in cell therapy. Summary of the Invention

[0004] The present invention provides a method for culturing immune cells and its application. The method of the present invention has one or more of the following advantages: the immune cells obtained by culture have a strong ability to release specific cytokines for tumors, the immune cells obtained by culture have a strong ability to specifically kill tumors, or the immune cells obtained by culture have a high expression level of tumor-specific recognition and activation markers, 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.

[0005] In one aspect, the present invention provides a method for culturing immune cells, comprising contacting isolated immune cells with three-dimensional cell aggregates and / or cells derived from three-dimensional cell aggregates. For example, the three-dimensional cell aggregates comprise tumor organoids (e.g., tumor-derived organoids).

[0006] In another aspect, the present invention provides a method for enhancing tumor-specific killing, comprising contacting isolated immune cells with three-dimensional cell aggregates and / or cells derived from the three-dimensional cell aggregates. For example, after obtaining the cells, the cells may be cultured and expanded to obtain the required amount of cells for treatment. For example, after obtaining the cells, the cells may be cultured and expanded and / or reinfused to prevent and / or treat tumors.

[0007] In another aspect, the present invention provides a cell (population) obtained by the culture method of the present invention.

[0008] In another aspect, the present invention provides a pharmaceutical composition comprising the cells (population) obtained by the culture method of the present invention, and optionally a pharmaceutically acceptable carrier.

[0009] In another aspect, the present invention provides a method for influencing tumor cell growth, comprising administering the cells (population) obtained by the culture method of the present invention and / or the pharmaceutical composition of the present invention.

[0010] In another aspect, the present invention provides use of the cell (population) obtained by the culture method of the present invention and / or the pharmaceutical composition of the present invention in preparing a drug for preventing and / or treating tumors.

[0011] In another aspect, the present invention provides a medicament for preventing and / or treating tumors, comprising the cell (population) obtained by the culture method of the present invention and / or the pharmaceutical composition of the present invention as an active ingredient.

[0012] In another aspect, the present invention provides a method for preventing and / or treating tumors, comprising administering the cells (population) obtained by the culture method of the present invention and / or the pharmaceutical composition of the present invention to a subject in need thereof.

[0013] In another aspect, the present invention provides cells (populations) obtained by the culture method of the present invention and / or the pharmaceutical composition of the present invention, which are used for preventing and / or treating tumors.

[0014] 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

[0015] The accompanying drawings are briefly described as follows:

[0016] FIG1 shows an exemplary culture flow chart of the present invention.

[0017] 2A-2B show the effects of CD107a expression and IFN-γ secretion under different stimulation conditions.

[0018] Figures 3A-3B show the activation marker expression and tumor cell killing effect of different PDO and TILs co-incubation effector-target ratios.

[0019] 4A-4F show the anti-tumor activity of immune cells, optionally sorted for tumor-specific markers, in different tumor types.

[0020] 5A-5B show the tumor killing ability of immune cells optionally sorted for tumor-specific markers. DETAILED DESCRIPTION

[0021] 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.

[0022] Definition of terms

[0023] As used herein, the term "organoid" generally refers to a three-dimensional aggregate of one or more cell types. For example, the organoid forms in vitro and exhibits a three-dimensional organ structure. For example, the organoid can mimic the surface appearance or the actual structure or function of a tissue or organ.

[0024] 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).

[0025] 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 an antibody. In some embodiments, the extracellular domain of CAR may include an antigen-binding fragment derived from an antibody. The antigen-binding domains of the present invention may include, for example, scFv, antibodies, antigen-binding regions of antibodies, variable regions of heavy chains / light chains, and / or single-chain antibodies.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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. TIL can include primary TIL and secondary TIL. "Primary TIL" can be those TIL cells obtained from a subject's tissue sample, and "secondary TIL" 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 infiltrated with tumor cells. For example, the TIL of the present invention may refer to a TIL population.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The terms "above," "below," "at most," and "at least" are inclusive.

[0042] Detailed Description of the Invention

[0043] The present invention is based in part on the discovery that, while immune cell cultures typically utilize PMBCs from healthy donors co-cultured with isolated immune cells, the resulting immune cells' cytokine release and specific killing abilities need to be improved. Furthermore, co-culturing various cells expressing or presenting tumor antigens with immune cells has not significantly improved their cytokine release and specific killing abilities.

[0044] The present invention unexpectedly discovered that contacting isolated immune cells with three-dimensional cell aggregates and / or cells derived from three-dimensional cell aggregates can improve the immune cells' cytokine release capacity and / or specific killing capacity. In one aspect, the present invention provides a method for culturing immune cells, comprising contacting isolated immune cells with three-dimensional cell aggregates and / or cells derived from three-dimensional cell aggregates.

[0045] For example, the three-dimensional cell aggregates include tumor organoids (e.g., tumor-derived organoids). For example, the cells derived from the three-dimensional cell aggregates include cells dissociated from the three-dimensional cell aggregates. For example, the extracellular matrix of the three-dimensional cell aggregates can be destroyed by a commercially available kit (cell digestion solution or collagenase hydrolysis solution) to dissociate the cells of the three-dimensional cell aggregates.

[0046] For example, the culture method of the present invention can include co-culturing tumor organoids with immune cells. The method includes the step of mixing the organoids described in the present invention with immune cells in an in vitro culture. The mixing can include sequentially layering the T cells and organoids into the same wells in a multi-well plate, or can include sequentially pipetting the T cells and organoids into a gel. For example, cells can be dissociated from the organoids by digestion and co-cultured with the immune cells.

[0047] For example, a method for preparing an organoid comprises culturing tumor-derived cells in an organoid culture medium to prepare the at least one organoid. For example, before mixing the at least one organoid with the immune cells, the organoid culture medium (optionally including any extracellular matrix) is removed from the at least one organoid. The extracellular matrix can be destroyed using a commercially available kit (cell digestion solution or collagenase hydrolysis solution). For example, an alternative matrix can be used to replace the removed matrix.

[0048] For example, the method of the present invention further comprises expanding the isolated immune cells in vitro prior to contacting the isolated immune cells with the tumor organoids and / or cells derived from the tumor organoids. The co-culture of immune cells with tumor organoid-derived material can be ex vivo and / or in vitro.

[0049] For example, the organoids of the present invention may comprise autologous cells (i.e., cells obtained from the same patient) or be composed of allogeneic cells (i.e., cells obtained from different patients). For example, organoids can be obtained by culturing tumor cells. For example, the organoids of the present invention comprise organoids in a culture medium containing interleukins (such as IL-2, IL-7, IL-15, or IL-21). For example, the at least one organoid comprises or is composed of mammalian cells. For example, before mixing with immune cells, the organoids are separated into groups that share one or more genotypes, phenotypes, and / or epigenetic markers. For example, organoids that have a partial matching with the immune cells of the present invention (wherein partial matching means that the immune cells and the organoids may have at least one HLA typing in common) can be used in the culture method of the present invention.

[0050] For example, organoid culture medium can be used to prepare organoids for co-cultivation, for example, by promoting growth, division (amplification), structural organization or other development to produce organoids suitable for co-cultivation. For example, suitable organoid culture medium for different tissues is known in the art. Preferred organoid culture medium includes Wnt agonists, mitogenic growth factors (e.g., selected from EGF, FGF, HGF and BDNF) and / or BMP inhibitors. For example, organoid culture medium also includes TGF-β inhibitors.

[0051] For example, organoid culture media can comprise one or more basal media (e.g., DMEM / F12 medium, Gibco), Wnt ligands, Wnt agonists, BMP inhibitors, EGF, and TGF-β inhibitors, and optionally further comprise one or more (or all) of the following: p38 MAPK inhibitors, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27, and / or antimicrobial agents (e.g., primocin).

[0052] For example, the co-culture medium of immune cells and tumor organoid-derived cells comprises a portion of immune cell culture medium (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) and a portion of organoid cell culture medium (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). For example, in a preferred embodiment, the co-culture medium comprises about 50% immune cell culture medium (e.g., T cell culture medium, e.g., RPMI1640 (Gibco)) and about 50% organoid culture medium.

[0053] For example, by providing a biomaterial or synthetic material that interacts with cell membrane proteins, it is possible to imitate cell niches and cultivate cells for forming organoids. For example, the extracellular matrix (ECM) for cultivating organoids comprises any biomaterial or synthetic material or a combination thereof. For example, a type of ECM is secreted by epithelial cells, endothelial cells, luminal endoderm-like cells and connective tissue cells. This ECM comprises a variety of polysaccharides, water, elastin and glycoprotein, wherein glycoprotein comprises collagen, nestin (entactin / nidogen), fibronectin and laminin. For example, in some embodiments, collagen is used as ECM. Different types of ECM are known, including different compositions containing different types of glycoproteins and / or different glycoprotein combinations.

[0054] For example, examples of commercially available extracellular matrices include: extracellular matrix proteins (Invitrogen) and basement membrane preparations from EHS mouse sarcoma cells (e.g., Cultrex basement membrane extract (Trevigen) or Matrigel (BD Biosciences). For example, the ECM is Matrigel (BD Biosciences), which contains laminin, entactin, and type IV collagen. In some embodiments, the ECM contains laminin, entactin, type IV collagen, and heparin sulfate proteoglycans. For example, the ECM can be a synthetic ECM. For example, a synthetic ECM (such as ProNectin) can be used. In another example, the ECM can be a plastic (such as a polyester) or a hydrogel. In some embodiments, the synthetic ECM can be coated with a biomaterial, such as one or more glycoproteins, such as collagen or laminin.

[0055] For example, the method comprises contacting the isolated immune cells with the three-dimensional cell aggregates and / or cells derived from the three-dimensional cell aggregates at a cell ratio of about 2: 1 to 50: 1. For example, the method comprises contacting the isolated immune cells with cells of the dissociated three-dimensional cell aggregates at a cell ratio of about 1: 100 to 100: 1. For example, the method comprises contacting the isolated immune cells with cells of the dissociated three-dimensional cell aggregates at a cell ratio of about 1: 100, about 1:50, about 1:20, about 1:10, about 1:5, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 50:1, or about 100:1.

[0056] For example, the method comprises contacting the isolated immune cells with the three-dimensional cell aggregates and / or cells derived from the three-dimensional cell aggregates for about 2 hours or more. For example, the method comprises contacting the isolated immune cells with cells of the dissociated three-dimensional cell aggregates for about 2 hours to 7 days, for example, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

[0057] For example, the method further comprises expanding the immune cells in vitro before the contacting. For example, the method comprises expanding the immune cells in vitro for about 2 hours to 28 days, such as about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, or about 28 days, before the isolated immune cells are contacted with the three-dimensional cell aggregates and / or cells derived from the three-dimensional cell aggregates.

[0058] For example, the in vitro expansion comprises culturing tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and / or ascites derived from a tumor subject in a culture environment with an IL-2 concentration of 300 to 9000 IU / mL.

[0059] For example, the method further comprises culturing the immune 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 before and / or after the contacting.

[0060] For example, the method 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 ascites and not expanded in vitro with one or more T cell growth factors (such as IL-2), wherein a second TIL population is obtained through step (A); (B) contacting the second TIL population with a T cell activator (such as an anti-CD3 antibody and / or an anti-CD28 antibody) and / or a T cell growth factor, and optionally performing gene editing, wherein a third TIL population is obtained through step (B); (C) co-culturing the third TIL population with feeder cells, wherein a fourth TIL population is obtained through step (C).

[0061] 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), the isolated immune cells can be contacted with the three-dimensional aggregates of cells and / or cells derived from the three-dimensional aggregates of cells. For example, for the first TIL group obtained, the second TIL group obtained, the third TIL group obtained, and / or the fourth TIL group obtained, the isolated immune cells can be contacted with the three-dimensional aggregates of cells and / or cells derived from the three-dimensional aggregates of cells. For example, step (A) is cultured for about 3-14 days. For example, step (B) is cultured for about 3-14 days. For example, step (C) is cultured for about 3-14 days.

[0062] For example, the method 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 (such as IL-2), wherein a second TIL population is obtained after about 10 to 14 days after step (A); (B) contacting the second TIL population with a T cell activator (such as an anti-CD3 antibody and / or an anti-CD28 antibody) and / or a T cell growth factor, and optionally performing gene editing, wherein a third TIL population is obtained after about 6 hours to 8 days after step (B); (C) co-culturing the third TIL population with feeder cells, wherein a fourth TIL population is obtained after about 10 to 14 days after step (C); and (D) contacting the isolated immune cells with three-dimensional cell aggregates and / or cells derived from the three-dimensional cell aggregates, wherein a fifth TIL population is obtained after about 6 hours to 8 days after step (D). Optionally, the fifth TIL population obtained can be further sorted to obtain immune cells positive for tumor-specific recognition markers (such as CD39, CD103, CD137, OX40, ICOS, CD25, CD69, or PD-1, etc.).

[0063] For example, compared to the original cell population that has not been in contact with the three-dimensional cell aggregates or cells derived from the three-dimensional cell aggregates, the contacted immune cells can produce and / or release more cytokines (such as GZMB, IFN-γ, CD107a, TNF-α), and / or can express more tumor-specific recognition markers (such as CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, CD107) after contact with tumor cells derived from the same subject.

[0064] In one embodiment, IFN-γ is produced after contact with a three-dimensional cell aggregate or a cell derived from a three-dimensional cell aggregate. For example, compared to the original cell population that has not been in contact with a three-dimensional cell aggregate or a cell derived from a three-dimensional cell aggregate, the immune cell produces and / or releases IFN-γ at an amount that is about 100,000 times to about 1% after contact with tumor cells from the same subject, for example, an increase of 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, cytokine production and / or release amount can be detected by cytokine detection kit and / or cell flow cytometry.

[0065] In one embodiment, after contact with a three-dimensional cell aggregate or a cell derived from a three-dimensional cell aggregate, more tumor-specific recognition markers can be expressed. For example, compared to the original cell population that has not been in contact with a three-dimensional cell aggregate or a cell derived from a three-dimensional cell aggregate, the immune cells, after contact with tumor cells from the same subject, express tumor-specific recognition markers selected from CD39, CD103, CD137, OX40, ICOS, CD25, CD69, or PD-1. The proportion of cells in the total cells is increased by about 10,000 times to about 1%, for example, by about 10,000 times, about 1,000 times, about 100 times, about 50 times, about 40 times, or about 30 times. %, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, 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 expression ratio of the cell marker can be detected by flow cytometry.

[0066] For example, the immune cells comprise tumor infiltrating lymphocytes 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, or kidney tumor tissue sample. In an embodiment of the present invention, the immune cells comprise cells isolated and cultured from a late-stage, metastatic, and / or recurrent tumor tissue sample. 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.

[0067] For example, immune 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, immune cells can include αβT cells and or γδT cells. For example, immune 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, the immune cells are autologous to the patient. In this case, the cells transferred into the patient include the patient's own cells to prevent rejection or adverse immune reactions to the administered cells.

[0068] On the other hand, immune 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 stimulator 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 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 such as lymphocytes (e.g., TIL, CTL, NK cells or LAK cells) obtained include colonies with clonal capacity. In other cases, the cell population obtained may not have clonal capacity or may not have unlimited clonal capacity.

[0069] In one embodiment, CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 positive cells are cultured in an in vitro cell culture environment. The enriched CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 positive cells can be further used as enriched tumor-specific cells. For example, by further isolating cells having CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 cell markers, the cells can be used as enriched tumor-specific T cells.

[0070] Any cell separation method can be used to isolate the cells of the present invention from a biological sample. For example, antibodies that bind CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 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.

[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 cell 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 nucleic acid encoding gRNA and Cas protein). After activating the TIL of the present invention for a certain period of time, cell three-dimensional aggregates or cells derived from cell three-dimensional aggregates are added (TIL and cells derived from cell three-dimensional aggregates are at a ratio of about 2: 1 to about 50: 1), and the culture is expanded for about 3-14 days. Cells in culture can be collected 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 effector cells and effector cells. Upon stimulation, they can secrete IFN-γ and / or exhibit an increased proportion of activated cells. For example, enrichment for CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 can be performed between the various steps in the above method.

[0072] 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.

[0073] In another aspect, the present invention provides a cell (population) obtained by the method of the present invention. In one embodiment, the cells provided by the present invention may comprise one or a batch of cells cultured using the culture method of the present invention. In one embodiment, the cells provided by the present invention may comprise multiple or multiple batches of cells cultured using the culture method of the present invention and combined in any proportion.

[0074] On the other hand, the present invention provides a kind of pharmaceutical composition, it comprises the cell (group) of the present invention, and optional pharmaceutically acceptable carrier.In some embodiments, pharmaceutical composition can be the suspension of cell in sterile buffer.Use the cell of amplification of the present invention to be administered by any suitable route known in the art.In some embodiments, cell can be administered with single intra-arterial or intravenous infusion, and infusion can last about 30 to 60 minutes.Other suitable administration routes can include intraperitoneal, intrathecal and intralymphatic administration.

[0075] For example, after culture, the cell product of the present invention increases the proportion of CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 positive cells. For example, compared to cells not in contact with three-dimensional cell aggregates or cells derived from three-dimensional cell aggregates, the proportion of CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 positive cells in the cell population after culture of the present invention increases by about 10,000 times to about 0.01%, for example, by 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 7 times, about 8 times, about 9 times, about 10 ... 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%, about 1%, about 0.5%, about 0.1%, about 0.05%, or about 0.01%.

[0076] For example, after enrichment, the cell product of the present invention has an increased proportion of CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 positive cells. For example, relative to cells not in contact with three-dimensional cell aggregates or cells derived from three-dimensional cell aggregates, the cell population after culture of the present invention has a CD39, CD103, CD137, OX40, ICOS, CD25, CD69, and / or PD-1 positive cell proportion of about 99.99% to 0.1%, for example, about 99.99%, about 99.9%, about 99%, about 95%, about 90%, about 80%, about 100%, about 15 ... 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%.

[0077] For example, the cell products of the present invention have enhanced tumor-specific cytokine release capacity after contact with three-dimensional cell aggregates or cells derived from three-dimensional cell aggregates. For example, compared with a cell population that has not been contacted with three-dimensional cell aggregates or cells derived from three-dimensional cell aggregates, the production and / or release level of tumor-specific cytokines such as IFN-γ in the cell population cultured according to the present invention after contact with specifically matched tumor 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, or about 10 times. 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%.

[0078] For example, the cell products of the present invention have enhanced tumor-specific killing ability after contact with three-dimensional cell aggregates or cells derived from three-dimensional cell aggregates. For example, compared with a cell population that has not been in contact with three-dimensional cell aggregates or cells derived from three-dimensional cell aggregates, the level of tumor cell apoptosis in the cell population cultured according to the present invention is increased by about 10,000 times to about 1% after contact with specifically matched tumor cells, for example, by 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%.

[0079] For example, the cell product of the present invention can be used for the discovery of specific TCRs after contacting with cell three-dimensional aggregates or cells derived from cell three-dimensional aggregates. For example, the type or sequence of the antigen-binding receptors of the cultured cells is determined. For example, TCRs derived from cultured cells can be used to develop engineered TCR cells. For example, relative to a cell population that has not been in contact with cell three-dimensional aggregates or cells derived from cell three-dimensional aggregates, the tumor specificity of the TCR obtained from the cultured cell population of the present invention is increased by about 10,000 times to about 1%, for example, by 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, For example, 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%, 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, expression of specific activation molecule markers, and / or the number and / or level of apoptosis of tumor cells after contact with specifically matched tumor cells.

[0080] In another aspect, the present invention provides a method for influencing tumor cell growth, comprising administering the cells (populations) of the present invention and / or the pharmaceutical composition of the present invention. For example, after obtaining the cells, the cells may be cultured and expanded to obtain the required number of cells for treatment. For example, after obtaining the cells, the cells may be cultured and expanded and / or reinfused to prevent and / or treat tumors.

[0081] In another aspect, the present invention provides use of the cell (population) of the present invention and / or the pharmaceutical composition of the present invention in the preparation of a medicament for preventing and / or treating tumors.

[0082] In another aspect, the present invention provides a medicament for preventing and / or treating tumors, comprising the cell (population) of the present invention and / or the pharmaceutical composition of the present invention as an active ingredient.

[0083] In another aspect, the present invention provides a method for preventing and / or treating tumors, comprising administering the cell (population) of the present invention and / or the pharmaceutical composition of the present invention to a subject in need thereof.

[0084] In another aspect, the present invention provides the cell (population) of the present invention and / or the pharmaceutical composition of the present invention, for use in preventing and / or treating tumors.

[0085] 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 1×10 9to about 13.7×10 10 cells, preferably 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.

[0086] 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.

[0087] 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%.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] Example

[0093] Example 1 Sorting and expansion of tumor-infiltrating lymphocytes

[0094] 1.1 Tumor tissue receipt and processing

[0095] 1.1.1 Organization reception

[0096] Receive tumor tissue from the donor, verify and record the sample information, and print the corresponding sample label.

[0097] 1.1.2 Tissue processing, TILs culture and expansion

[0098] Take several 10 cm culture dishes and add an appropriate amount of rewarmed complete culture medium. Complete culture medium can be X-vivo 15 medium or other commercially available T cell culture medium, such as those from Stem Cell, Lonza, Thermo, or Miltenyi Biopharmaceuticals. Essential amino acids and antibiotics can be supplemented, along with IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, or 6000 IU / mL). Use sterile ophthalmic forceps to remove tumor tissue from the sample tube and place it in a 10 cm culture dish. Wash the tissue and replace the dish. Perform a preliminary shearing operation using ophthalmic scissors and forceps to remove adipose and necrotic tissue. Use a disposable scalpel to mince each tissue block to approximately 1-27 cubic millimeters. Use a pipette to transfer 90% of the non-suspended tumor tissue at a rate of approximately 0.1-0.2 g / well to a 6-well culture plate containing 3 ml of complete culture medium. The culture plate was placed in a carbon dioxide incubator, and the medium was replenished or half-volume medium was replaced according to the cell status until preREP was harvested. The culture was cultured for about 10 to 14 days to obtain a preREP TILs population (as shown in Figure 1, subpopulation a).

[0099] After preREP TILs were harvested and counted, the cells were resuspended in complete culture medium and the cell concentration was adjusted to 2 × 10 6 / ml and add OKT3 (30ng / ml) for stimulation for 24-48 hours, then add feeder cells at a ratio of 1:50-200, place the culture plate in a carbon dioxide incubator, and replenish the fluid or replace half of the fluid according to the cell status until REP is harvested. Culture for about 10 to 14 days to obtain a REP TILs population (as shown in Figure 1, subpopulation b).

[0100] 1.1.3 PDO model cultivation

[0101] The remaining 10% of non-suspended tumor tissue from the TILs culture in step 1.1.2 was further minced with a scalpel and added to a dedicated tissue digestion solution (BioGenous, Organoid Dissociation Solution) and digested at 37°C for 2-5 minutes. After digestion, 5 volumes of culture medium containing 10% FBS were added to terminate the digestion. The tissue suspension was filtered through a 100 μm filter and washed, then placed on ice and thoroughly mixed with Matrigel. The mixture was then applied to the bottom of the cell culture plate and placed in a 37°C incubator. After the Matrigel was fully solidified, complete culture medium was carefully added for incubation.

[0102] When organoids grow to approximately 300 microns in diameter, they are passaged or cryopreserved to create a PDO (Patient-Derived Organoid) model. Continued culture and passages yield PDOs (as shown in Figure 1, subpopulation c).

[0103] 1.2 Co-incubation and amplification with PDO

[0104] 1.2.1 Co-incubation

[0105] The PDO (c subpopulation) amplified in step 1.1.2 was digested and counted, and about 2.5×10 5 to 2×10 6 PDO cells were resuspended in 1 ml T cell culture medium. The REP TILs population (subpopulation b) obtained in step 1.1.2 was harvested and counted; or the preREP TILs population (subpopulation a) obtained in step 1.1.2 was harvested and counted, and about 1×10 7 to 2×10 7 cells and resuspend in 9 ml T cell culture medium.

[0106] 1 ml of PDO single cell suspension was mixed with 9 ml of TILs cell suspension (preREP TILs group, or REP TILs group) and added to 1-2 wells of a 6-well culture plate for co-incubation. The ratio of TILs to PDO was approximately 2:1 to 60:1. The culture plate was placed in a carbon dioxide incubator and incubated for approximately 6 to 48 hours to obtain unsorted REP TILs (as shown in Figure 1, e subpopulation). Positive controls (transACT stimulation) and negative controls (no stimulation) were also set up.

[0107] 1.2.2 Optional Sorting of Tumor-Specific Marker-Positive and -Negative Cells

[0108] After co-incubation, cells were harvested and counted. 85% of the total cell volume (as shown in Figure 1, subpopulation d) was washed with pre-cooled cell sorting buffer (PBS + 0.5% BSA + 2mM EDTA) and centrifuged at 4°C, 500g for 10 minutes. According to the number of cells, a corresponding volume of tumor-specific marker staining antibody, such as Anti-CD137 PE (Miltenyi Biotec or Biolegend) antibody, was added, 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 proportion of total cells was 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.

[0109] Select a magnetic separation column (Miltenyi Biotec) according to the cell number 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 magnetic beads to the separation column. After it is completely dripped, add a certain volume of separation solution for washing.

[0110] The single cell suspension that flows through the separation column and drips off is separated to obtain tumor-specific marker-negative cells, such as CD137-negative cells (as shown in FIG1 , h subpopulation), which are counted and temporarily stored at 2-8° C.

[0111] Remove the separation column from the magnet slot and place it in a suitable 15 ml centrifuge tube. Add the corresponding volume of separation solution and use the matching piston to flush out the magnetic bead-bound cells in the separation column and mark them as tumor-specific marker-positive cells, such as CD137-positive cells (as shown in Figure 1, subpopulation g). After counting, store them at 2-8°C.

[0112] 1.2.3 Expansion and Harvest

[0113] Complete culture medium can be 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 Biopharm. Essential amino acids and antibiotics can be added, and IL-2 can be added at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL).

[0114] According to the counting results, 14% of the total cells after incubation in the above 1.2.1 process were unsorted REP TILs (e subpopulation) according to the 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. 6Feeder cells (irradiated healthy donor PBMC T cells) were added to each sorted cell culture well at a ratio of 1:1 / well. The medium was replenished or replaced halfway according to the cell status until harvest. After 14 days of expansion, the cells that had completed in vitro expansion were collected, centrifuged and the culture medium was discarded. The cells were washed once with PBS or saline and cultured for about 0 days (i.e., they can be harvested directly) to 14 days to obtain the unsorted REP TIL population after expansion (as shown in Figure 1, k subpopulation). A sample of about 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.

[0115] Similarly, according to the counting results, the tumor-specific markers, such as CD137-positive cells (g subpopulation) or the tumor-specific markers, such as CD137-negative cells (h subpopulation), obtained by the optional sorting step are cultured for about 10 to 14 days with reference to the above-mentioned expansion step to obtain in vitro amplified tumor-specific marker-positive sorted cells (as shown in Figure 1, i subpopulation) or tumor-specific marker-negative sorted cells (as shown in Figure 1, j subpopulation), respectively.

[0116] FIG1 shows an exemplary culture flow chart of the present invention.

[0117] Example 2 Comparison of tumor-specific responses of immune cells expanded under different stimulation conditions

[0118] Preparation of APCs (antigen presenting cells, including DCs and EBV-B cells) and their mediation of antigen-specific T cells

[0119] CD14+ monocytes and CD19+ B lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) using CD14- and CD19-specific magnetic beads (Miltenyi Biotec). These were then used to prepare monocyte-derived dendritic cells (DCs) or immortalized EBV-B cells derived from B lymphocytes.

[0120] After the preparation, the harvested DCs were incubated with EBV-B peptide fragment antigens as reported in the art (for example, refer to the method described in Levin N et al. Clinical Cancer Research, 2021, 27(18): 5084-5095), loaded, and expressed. They were then co-incubated with autologous PBMCs for 14-21 days and harvested. 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.

[0121] In the control invention, the prepared APCs (antigen presenting cells, including DC cells and EBV-B cells) were used instead of PDO cells to stimulate TIL cells.

[0122] Comparison of CD107a expression and IFN-γ secretion ability of antigen-specific T cells under different stimulation conditions

[0123] Unsorted TILs (k subset) stimulated and expanded by the PDO of the present invention were collected separately, and T cells stimulated by DCs or EBV-B cells loaded with peptide fragment antigens of the control invention were collected. Each group of T cells was co-cultured with the corresponding target cells PDO (l subset), DCs or EBV-B cells carrying the antigen at an effector-target ratio of 4:1-10:1 in a round-bottom 96-well culture plate.

[0124] PDO cells should be pretreated with IFN-γ (20 ng / ml) for 12-18 hours. Target cells and T cells should be plated in duplicate, with 100 μL each. A transACT-stimulated group should serve as a positive control, adding transACT (approximately 100 to 500 nm in diameter, Miltenyi) to a transACT working solution concentration of 1:200 (v / v). An unstimulated group should serve as a negative control, adding an equal volume of cytokine-free medium. Add the following volumetric ratios: Golgistop 0.7:1000, Golgiplug 1:1000, and CD107a antibody 1:500 (2 μL / mL). Incubate at 37°C for 6-18 hours. After incubation, wash once with 200 μL / well PBS, centrifuge at 600g for 3 minutes, and discard the supernatant. Prepare a mixed antibody working solution for cell surface staining of CD3 / CD4 / CD8 at an antibody concentration of 1:100 and a cell viability assay dye concentration of 1:10,000. Add 50 μL / well of a 96-well plate and 100 μL / tube to a flow cytometry tube and incubate at 2-8°C in the dark for 30 minutes. Wash cells once with PBS (200 μL / well for a 96-well plate and 1 mL / well for a flow cytometry tube), centrifuge at 600 g for 3 minutes at room temperature, and discard the supernatant. Add 100 μL of Fixation / Permeabilization buffer (BD, Fixation / Permeabilization) to each well and incubate at 2-8°C in the dark for 20-40 minutes. After fixation and permeabilization, wash twice with 1× Perm / Wash Buffer (200 μL / well for a 96-well plate and 1 mL / well for a flow cytometry tube), centrifuge at 600 g for 3 minutes, and discard the supernatant. Prepare cytokine detection antibodies (e.g., GZMB, TNF-α, IFN-γ) in 1× Perm / Wash Buffer and resuspend TIL cells (50 μL / well for 96-well plates, 100 μL / tube for flow cytometry staining). Incubate at 2-8°C in the dark for 30 minutes. After cytokine staining, wash 1-2 times with 1× Perm / Wash Buffer (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes). Centrifuge at 600 g for 3 minutes and discard the supernatant. Resuspend cells in 100-500 μL PBS for flow cytometry analysis.

[0125] Figures 2A-2B show the expression of CD107a and IFN-γ secretion capacity under different stimulation conditions.

[0126] The results showed that the antigen-specific T cells obtained by PDO stimulation and expansion in the present invention had stronger killing and cytokine secretion abilities than the specific T cells expanded by APCs (DC cells or EBV-B cells) stimulation in the control invention.

[0127] Example 3 Tumor-specific response of TILs after stimulation by co-incubation of PDO and TILs at different effector-target ratios

[0128] REP TILs (subpopulation b) and PDO (subpopulation c) were collected and co-cultured in round-bottom 96-well plates at effector-target ratios of 2:1, 4:1, and 50:1, respectively. PDO cells were pretreated with IFN-γ (20 ng / ml) for 12-18 hours. Target cells and T cells were added in 100 μL each, with two replicates set up for each group. A non-stimulated group was set up as a negative control, with only the same volume of cell culture medium added. Repeated groups were set up on different 96-well plates with the same cell number and effector-target ratio.

[0129] The first group of cells was incubated at 37°C for 6-18 hours. After incubation, the remaining cells in the wells were collected and assayed for the surface activation marker CD137. The main reagents and materials for flow cytometry assays were: V-bottom 96-well plates, Corning, catalog number 3894; flow cytometry tubes, Corning, catalog number 352052; and 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] For the second group of cells, add the following volume ratios: Golgistop 0.7:1000, Golgiplug 1:1000, and CD107a antibody 1:500 (2 μL / mL). Incubate at 37°C for 6-18 hours. After incubation, wash once with 200 μL / well PBS, centrifuge at 600 g for 3 minutes, and discard the supernatant. Prepare a mixed antibody working solution for cell surface staining of CD3 / CD4 / CD8 at an antibody concentration of 1:100 and a cell viability assay dye concentration of 1:10,000. 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube were added for staining. Incubate at 2-8°C in the dark for 30 minutes. Wash the cells once with PBS (200 μL / well for a 96-well plate and 1 mL / well for a flow cytometry tube), centrifuge at 600 g for 3 minutes at room temperature, and discard the supernatant. Add 100 μL of Fixation / Permeabilization buffer (BD, Fixation / Permeabilization) to each well and incubate at 2-8°C in the dark for 20-40 minutes. After fixation and permeabilization, wash twice with 1× Perm / Wash Buffer (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes), centrifuge at 600 g for 3 minutes, and discard the supernatant. Prepare cytokine detection antibodies (e.g., GZMB, TNF-α, IFN-γ) in 1× Perm / Wash Buffer and resuspend TIL cells (50 μL / well for 96-well plates, 100 μL / tube for flow cytometry staining). Incubate at 2-8°C in the dark for 30 minutes. After cytokine staining, wash once or twice with 1× Perm / Wash Buffer (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes), centrifuge at 600 g for 3 minutes, and discard the supernatant. Resuspend the cells in 100-500 μL PBS and perform flow cytometry analysis.

[0131] Figures 3A-3B show the activation marker expression and tumor cell killing effect of different PDO and TILs co-incubation effector-target ratios.

[0132] The results showed that the secretion capacity of immune cell activation cytokines and the killing function at each co-incubation ratio were higher than those of the negative control group without stimulation.

[0133] Example 4 Effect of Optional Tumor-Specific Marker Sorting

[0134] After expansion, tumor-specific marker-positive cells, such as CD137-positive sorted cells (subpopulation i), tumor-specific marker-negative cells, such as CD137-negative sorted cells (subpopulation j), and unsorted TILs (subpopulation k) were collected. Each group of TILs was co-cultured with digested PDO cells (subpopulation l) or single tumor cells (subpopulation m) at an effector-to-target ratio of 4:1 (T cell:target cell, E:T) in round-bottom 96-well culture plates. PDO cells should be pretreated with IFN-γ (20 ng / ml) for 12-18 hours. Target cells and T cells were each diluted in 100 μL, with duplicate wells set up for each group. A non-stimulated group was also established as a negative control, with an equal volume of cytokine-free culture medium added.

[0135] Add the following volume ratios: Golgistop 0.7:1000, Golgiplug 1:1000, and CD107a antibody 1:500 (2 μL / mL). Incubate at 37°C for 6-18 hours. After incubation, wash once with 200 μL / well PBS, centrifuge at 600 g for 3 minutes, and discard the supernatant. Prepare a mixed antibody working solution for cell surface staining of CD3 / CD4 / CD8 at an antibody concentration of 1:100 and a cell viability assay dye concentration of 1:10,000. Add 50 μL / well of a 96-well plate and 100 μL / tube of a flow cytometry tube. Incubate at 2-8°C in the dark for 30 minutes. Wash cells once with PBS (200 μL / well for a 96-well plate and 1 mL / well for a flow cytometry tube), centrifuge at 600 g for 3 minutes at room temperature, and discard the supernatant. Add 100 μL of Fixation / Permeabilization buffer (BD, Fixation / Permeabilization) to each well and incubate at 2-8°C in the dark for 20-40 minutes. After fixation and permeabilization, wash twice with 1× Perm / Wash Buffer (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes), centrifuge at 600 g for 3 minutes, and discard the supernatant. Prepare cytokine detection antibodies (e.g., GZMB, TNF-α, IFN-γ) in 1× Perm / Wash Buffer and resuspend TIL cells (50 μL / well for 96-well plates, 100 μL / tube for flow cytometry staining). Incubate at 2-8°C in the dark for 30 minutes. After cytokine staining, wash once or twice with 1× Perm / Wash Buffer (200 μL / well for 96-well plates, 1 mL / well for flow cytometry tubes), centrifuge at 600 g for 3 minutes, and discard the supernatant. Resuspend the cells in 100-500 μL PBS and perform flow cytometry analysis.

[0136] 4A-4F show the anti-tumor activity of immune cells, optionally sorted for tumor-specific markers, in different tumor types.

[0137] Figure 4A shows the expression level of the killing marker (CD107a) of each sorted subpopulation of REP TILs derived from cervical cancer (neuroendocrine type). Figure 4B shows the expression ratio of tumor-specific cytokine secretion (IFN-γ or TNF-α) and killing marker (IFN-γ+CD107a) associated with cytokine secretion of each sorted subpopulation of REP TILs derived from cervical cancer (neuroendocrine type). Figures 4C-4D show the expression ratio of tumor-specific cytokine secretion (IFN-γ) and killing marker (CD107a) of each sorted subpopulation of REP TILs derived from lung cancer. Figures 4E-4F show the expression ratio of tumor-specific cytokine secretion (IFN-γ) and killing marker (CD107a) of each sorted subpopulation of REP TILs derived from cervical cancer.

[0138] The results showed that the anti-tumor activity of immune cells before and after sorting was higher than that of the control group. Optional sorting for tumor-specific markers can further enhance the anti-tumor activity of immune cells.

[0139] After expansion, cells positive for tumor-specific markers, such as CD137-positive sorted cells (subpopulation i), cells negative for tumor-specific markers, such as CD137-negative sorted cells (subpopulation j), and unsorted TILs (subpopulation k) were collected and co-incubated with digested PDO cells (subpopulation e) at an effector:target ratio of 1:1 or 1:3 (T cell:target cell, E:T). 100 μL of target cells and 100 μL of T cells were added to each well, with triplicate wells set up for each group. A control group containing only target cells was also set up. According to the instructions for the apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL of the apoptosis detection reagent was added per well, and 25 μL of culture medium was added per well to dilute the Caspase 3 / 7 Green Dye. The activity of Caspase 3 / 7 was recorded using an Incucyte recorder (Sartorius) to analyze the killing ability of TIL cells on target cells. The activity was recorded every 1.5 hours, and the total recording time was approximately 1 day.

[0140] 5A-5B show the tumor killing ability of immune cells optionally sorted for tumor-specific markers.

[0141] The results showed that all sorted subpopulations of REP TILs derived from cervical cancer (neuroendocrine type) exhibited tumor-specific killing ability after co-incubation with PDO at different effector-target ratios (1:1 or 1:3). Preferably, optional tumor-specific marker sorting can further enhance the tumor-killing ability of immune cells.

[0142] 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

A method for culturing immune cells, comprising contacting isolated immune cells with three-dimensional cell aggregates and / or cells derived from three-dimensional cell aggregates.

2. The method of claim 1, wherein the three-dimensional cell aggregates comprise tumor organoids (eg, tumor-derived organoids).

3. The method according to any one of claims 1-2, wherein the cells derived from the three-dimensional cell aggregates comprise cells dissociated from the three-dimensional cell aggregates.

4. The method according to any one of claims 1 to 3, comprising contacting the isolated immune cells with three-dimensional cell aggregates and / or cells derived from three-dimensional cell aggregates at a cell ratio of about 2:1 to 50:

1.

5. The method according to any one of claims 1 to 4, further comprising expanding the immune cells in vitro before the contacting.

6. The method according to claim 5, wherein the in vitro expansion comprises culturing immune cells derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and / or ascites of a tumor subject in a culture environment with an IL-2 concentration of 300 to 9000 IU / mL.

7. The method according to any one of claims 1 to 6, further comprising culturing the immune 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 before and / or after the contacting.

8. The method according to any one of claims 1 to 7, further comprising isolating cells positive for tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107).

9. The method according to any one of claims 1 to 8, wherein the immune cells comprise T cells, natural killer cells, and / or natural killer-like T cells.

10. The method according to any one of claims 1 to 9, wherein the immune cells comprise αβT cells and / or γδT cells.

11. The method of any one of claims 1-10, wherein the immune cells comprise tumor infiltrating lymphocytes.

12. The method according to any one of claims 1 to 4, wherein the immune cells comprise cells derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, paracancerous tissue, pleural effusion and / or peritoneal effusion of a tumor subject.

13. The method according to any one of claims 1 to 12, wherein the contacted immune cells are capable of producing and / or releasing more cytokines (e.g., GZMB, CD107a, TNF-α, or IFN-γ) and / or expressing more tumor-specific recognition markers (e.g., CD39, CD103, CD137, OX40, ICOS, CD25, CD69, PD-1, or CD107) after contact with tumor cells derived from the same subject, compared to the original cell population that has not been in contact with the three-dimensional cell aggregates or cells derived from the three-dimensional cell aggregates.

14. A cell (population), wherein the cells are obtained by the method according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising the cell (population) according to claim 14, and optionally a pharmaceutically acceptable carrier.

16. A method for influencing tumor cell growth, comprising administering the cell (population) according to claim 14 and / or the pharmaceutical composition according to claim 15.

17. Use of the cell (population) according to claim 14 and / or the pharmaceutical composition according to claim 15 in the preparation of a medicament for preventing and / or treating tumors.

18. A medicament for preventing and / or treating tumors, comprising the cell (population) according to claim 14 and / or the pharmaceutical composition according to claim 15 as an active ingredient.

19. A method for preventing and / or treating tumors, comprising administering the cell (population) according to claim 14 and / or the pharmaceutical composition according to claim 15 to a subject in need thereof.

20. The cell (population) according to claim 14 and / or the pharmaceutical composition according to claim 15, for use in preventing and / or treating tumors.

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