Method for culturing TIL from hydrothorax and ascites

Abstract

The invention relates to a culture method of TIL from pleural effusion and / or ascites. Specifically, the invention provides the in-vitro culture method of the pleural effusion and / or ascites derived TIL, and the method comprises the following steps: 1) obtaining TIL seed cells from pleural effusion and / or ascites, and 2) sorting the TIL seed cells to obtain intermediate cells which are CD45RA-cells, CD39 + cells, or CD45RA-CD39 + cells.

Description

[0001] This invention claims priority to Chinese application No. 202411501545.0, filed on October 25, 2024, entitled "A method for culturing TILs derived from pleural and peritoneal fluid". Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to a method for culturing TILs derived from pleural and peritoneal fluid. Background Technology

[0003] Since the first report of tumor-infiltrating lymphocytes (TILs) in clinical oncology treatment in 1988, TILs have demonstrated an increasingly important role in the treatment of solid tumors. In recent years, it has been found that TILs have a very significant therapeutic effect on large, difficult-to-treat tumors, especially solid tumors, for adoptive metastasis therapy (ACT). Iovance BioTherapeutics in the United States currently has one approved TIL drug, and several other TIL drugs have entered Phase II or pivotal Phase III clinical trials, with indications including melanoma, cervical cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, colon cancer, pancreatic cancer, and sarcoma, among others.

[0004] Generally, obtaining tumor cells (TILs) in vitro usually requires first obtaining solid tumor tissue from the patient through surgery or biopsy. Surgery is relatively difficult and invasive, and not feasible for some patients whose physical condition cannot tolerate surgery. Biopsy usually yields a smaller amount of tumor tissue, and the tumor tissue itself has greater heterogeneity. The T cell diversity in TIL cell populations obtained through biopsy tissue culture is usually lower than that of TILs derived from surgical tissue.

[0005] In some patients with highly malignant tumors, significant amounts of pleural effusion or ascites are common, and tumor-specific intraepithelial lymph nodes (TILs) are frequently present in these fluids. A high proportion of these patients are not candidates for surgery. Therefore, their own pleural or ascites fluid may be another important source of TILs for these patients. New culture methods for culturing tumor-specific TILs derived from pleural or ascites fluid are urgently needed. Summary of the Invention

[0006] The first aspect of this invention provides a method for culturing TILs derived from pleural effusion and / or ascites, comprising the steps of:

[0007] 1) Obtain TIL seed cells from pleural effusion and / or ascites;

[0008] 2) The TIL seed cells were sorted to obtain intermediate cells, which were CD45RA cells. -Cells, CD39 + Cells, or CD45RA - CD39 + cell;

[0009] Optional 3) culture the intermediate cells described in 2) to obtain TIL.

[0010] In one or more embodiments, pleural effusion and / or ascites are pleural effusion (pleural fluid) and / or ascites (ascites).

[0011] In one or more embodiments, the method includes: obtaining TIL seed cells from pleural effusion and / or ascites, sorting to obtain CD45RA-TIL cells, and expanding and culturing the CD45RA-TIL cells.

[0012] In one or more embodiments, the method further includes: after obtaining the CD45RA-TIL cells, sorting to obtain CD45RA-CD39+TIL cells, and expanding and culturing the CD45RA-CD39+TIL cells. Preferably, the CD45RA-CD39+TIL cells are obtained by sorting using a CD39 antibody.

[0013] In one or more embodiments, obtaining TIL seed cells includes the following steps: centrifuging pleural fluid and / or ascites fluid to precipitate, washing with physiological saline, centrifuging again to precipitate, and obtaining the seed cells. In one or more embodiments, the centrifugation rate is 200-500 g, for example 200-400 g or 300-500 g, and the centrifugation time is 5-10 minutes, for example 5-8 minutes. In one or more embodiments, obtaining TIL seed cells further includes the step of removing erythrocytes from the seed cells. Preferably, a erythrolytic agent is added to the seed cells to remove erythrocytes.

[0014] In one or more embodiments, the step of culturing the TIL seed cells may optionally include culturing the TIL seed cells in a system for culturing the TIL seed cells for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, or at least 12 days.

[0015] In one or more embodiments, the system for culturing the TIL seed cells includes a culture medium suitable for TIL cells.

[0016] Preferably, the culture medium suitable for TIL cells includes AIM-V, X-VIVO, DMEM, RPMI 1640, and OpTmizer. TMBasic culture media such as FUJIFILM Irvin MHM-C, and serum-free cell culture dishes (e.g., GIBCO). ® AIM-V ® CTS TM Serum-free cell culture medium), DMEM medium, RPMI 1640 medium.

[0017] In one or more embodiments, the system for culturing the TIL seed cells further includes antibodies and cytokines.

[0018] In one or more embodiments, the cytokines include one or more selected from IL-2, IL-7, IL-15, IL-21, thymosin α1, αCD3, αCD28, TWS119, vitamin C, IFN-γ, TNF-α, and GM-CSF.

[0019] In one or more embodiments, the antibody comprises an immune checkpoint antibody or an antigen-binding fragment thereof. In one or more embodiments, the immune checkpoint antibody comprises any one or more selected from PD-1 antibody, CTLA-4 antibody, LAG3 antibody, TIM3 antibody, TIGIT antibody, and BTLA antibody.

[0020] In one or more embodiments, the antibody includes any one or more selected from CD3 antibody, CD28 antibody, CD137 antibody, OX40 antibody, GITR antibody, ICOS antibody, CD206 antibody, and CD40 antibody.

[0021] In one or more embodiments, the system for culturing the seed cells comprises IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α, and serum.

[0022] In one or more of the above embodiments, the concentration of IL-2 in the system for culturing the seed cells is 200-6000 IU / mL, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 IU / mL, or a range between any two of the above values. Preferably, the concentration of IL-2 is 500-5000, 1000-4000, or 1500-3500 ng / mL.

[0023] In one or more of the above embodiments, the concentration of IL-7 in the system for culturing the seed cells is 5-100 ng / mL, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL, or a range between any two of the above values. Preferably, the concentration of IL-7 is 5-65, 5-55, 5-45, 5-35, or 5-25 ng / mL.

[0024] In one or more of the above embodiments, the concentration of IL-15 in the system for culturing the seed cells is 5-100 ng / mL, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL, or a range between any two of the above values. Preferably, the concentration of IL-15 in the system for culturing the seed cells is 5-65, 5-55, 5-45, 5-35, or 5-25 ng / mL.

[0025] In one or more of the above embodiments, the concentration of GM-CSF in the system for culturing the seed cells is 200-5000 U / mL, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 U / mL, or a range between any two of the above values. Preferably, the concentration of GM-CSF is 200-1500, 200-1200, 200-950, 200-850, 200-750, 200-650, 200-550, or 200-500 U / mL.

[0026] In one or more of the above embodiments, the concentration of IFN-γ in the system for culturing the seed cells is 100-3000 U / mL, for example, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, or 3000 U / mL, or a range between any two of the above values. Preferably, the concentration of IFN-γ is 200-2000, 300-1800, 500-1500, or 800-1200 U / mL.

[0027] In one or more of the above embodiments, the concentration of TNF-α in the system for culturing the seed cells is 1-100 ng / mL, for example, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ng / mL, or a range between any two of the above values. Preferably, the concentration of TNF-α is 5-45, 5-35, 5-25, or 5-15 ng / mL.

[0028] In one or more of the above embodiments, the concentration of CD137 antibody in the system for culturing the seed cells is 1-100 μg / mL, for example, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL, or a range between any two of the above values. Preferably, the concentration of CD137 antibody is 1-95, 1-85, 1-75, 1-65, 1-55, 1-45, 1-35, 1-25, 1-15, 1-9, 1.5-8, 2-7, 2.5-6, 2.5-5, or 3-5 μg / mL.

[0029] In one or more of the above embodiments, the concentration of CD28 antibody in the system for culturing the seed cells is 1-100 μg / mL, for example, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL, or a range between any two of the above values. Preferably, the concentration of CD28 antibody is 1-95, 1-85, 1-75, 1-65, 1-55, 1-45, 1-35, 1-25, 1-15, 1-9, 1.5-8, 2-7, 2.5-6, 2.5-5, or 3-5 μg / mL.

[0030] In one or more of the above embodiments, the concentration of PD-1 antibody in the system for culturing the seed cells is 1-100 µg / mL, for example, 1, 2, 3, 5, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µg / mL, or a range between any two of the above values. Preferably, the concentration of PD-1 antibody is 1-65, 1-55, 1-45, 1-35, 1-25, 1-15, 1-8, 1-6, 1-4, 1-3 µg / mL.

[0031] In one or more embodiments, each antibody is a monoclonal antibody (mAb).

[0032] In one or more embodiments, the serum is selected from human AB serum, the subject's own serum, or animal-derived serum.

[0033] In one or more embodiments, the serum concentration is 1-10%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the serum concentration is 1.5-9.5%, 2.5-8.5%, 3.5-7.5%, or 4.5-6.5%.

[0034] In one or more embodiments, the system for culturing the seed cells further includes an antibiotic. Preferably, the antibiotic is selected from one or more of penicillin and streptomycin. In one or more embodiments, the concentration of penicillin in the culture medium is 10-1000 IU / mL, 20-800 IU / mL, or 50-100 IU / mL.

[0035] In one or more embodiments, the sorting includes: sorting techniques based on cell culture characteristics, sorting techniques based on cell physical properties, and sorting techniques based on affinity, preferably sorting based on cell membrane surface molecules (antibody affinity-dependent sorting).

[0036] In one or more embodiments, the sorting method is selected from density gradient centrifugation, affinity chromatography, flow cytometry, and magnetic bead separation and purification.

[0037] In one or more embodiments, the pleural effusion and / or ascites is pleural effusion and / or ascites from a patient suffering from one or more cancers selected from the following: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer (including non-small cell lung cancer), melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, colon cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the pleural effusion and / or ascites is pleural effusion and / or ascites from a patient suffering from one or more cancers selected from the following: melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and colon cancer.

[0038] The present invention also provides TIL cells cultured as described in any embodiment of the first aspect herein.

[0039] The present invention also provides a cell cryopreservation formulation comprising TIL cells and cryopreservation solution as described in any embodiment herein.

[0040] Preferably, the cell cryopreservation preparation further contains one or more of sugars, serum albumin, glycine, vitamin E, and electrolytes, wherein the sugars are monosaccharides selected from glucose, fructose, and dextrose, and / or disaccharides selected from lactose and trehalose.

[0041] In one or more embodiments, the cryopreservation solution is a cell cryopreservation solution or a tissue cryopreservation solution.

[0042] In one or more embodiments, the cryopreservation solution is a serum-free cryopreservation solution, such as BioLife Solutions' CryoStor CS10 cryopreservation solution.

[0043] The present invention also provides a method for treating solid tumors, comprising:

[0044] (1) Obtain pleural effusion and / or ascites from patients with solid tumors, and prepare TIL cells using the method described in any of the embodiments described herein;

[0045] (2) TIL cells from (1) were reinfused into patients with solid tumors.

[0046] In one or more embodiments, the solid tumor includes gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma.

[0047] The present invention also provides a pharmaceutical composition comprising TIL cells as described herein, and pharmaceutically acceptable excipients.

[0048] In one or more embodiments, the pharmaceutical composition further includes a chemotherapy drug.

[0049] The present invention also provides a kit containing the TIL cells described herein.

[0050] The present invention also provides a method for improving the target cell killing rate of TIL cells, including sorting TIL seed cells to obtain CD45RA- cells, CD39+ cells, or CD45RA-CD39+ cells.

[0051] In one or more embodiments, the method includes: negatively sorting seed cells to remove CD45RA+ cells, and / or performing positive sorting with CD39 antibody to obtain the TIL cells.

[0052] In one or more embodiments, the TIL seed cells are obtained from pleural effusion and / or ascites of cancer patients.

[0053] In one or more embodiments, the pleural effusion and / or ascites is pleural effusion and / or ascites from a patient suffering from one or more cancers selected from the following: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer (including non-small cell lung cancer), melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, colon cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the pleural effusion and / or ascites is pleural effusion and / or ascites from a patient suffering from one or more cancers selected from the following: melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and colon cancer.

[0054] In some implementations, the target cells are tumor cells from patients with TILs.

[0055] The present invention also provides the use of TIL cells as described in any embodiment herein in the preparation of a medicament. In one or more embodiments, the medicament is used to treat a patient with cancer.

[0056] In one or more embodiments, the TIL seed cells are obtained from the patient's pleural effusion and / or ascites.

[0057] In one or more embodiments, the cancer is selected from: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the cancer is selected from: melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and colon cancer.

[0058] The present invention also provides the use of reagents that specifically bind CD45RA and CD39 in the preparation of drugs or kits, wherein the drugs comprise TILs derived from pleural effusion and / or ascites with enhanced target cell killing rates, and the kits are used to prepare TILs derived from pleural effusion and / or ascites with enhanced target cell killing rates.

[0059] In one or more embodiments, the TIL is prepared by the method of any embodiment of the first aspect of this document.

[0060] In one or more embodiments, the reagent that specifically binds to CD45RA is an antibody. In one or more embodiments, the antibody is conjugated to a solid support (e.g., magnetic beads).

[0061] In one or more embodiments, the reagent that specifically binds to CD39 is an antibody. In one or more embodiments, the antibody is conjugated to a solid support (e.g., magnetic beads).

[0062] In one or more embodiments, the drug is used to treat cancer.

[0063] In one or more embodiments, the cancer is selected from: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma.

[0064] The present invention also provides a kit containing reagents that specifically bind to CD45RA and CD39. The kit is used to prepare TILs with enhanced target cell killing rate.

[0065] In one or more embodiments, the reagent that specifically binds to CD45RA is an antibody. In one or more embodiments, the antibody is conjugated to a solid support (e.g., magnetic beads).

[0066] In one or more embodiments, the reagent that specifically binds to CD39 is an antibody. In one or more embodiments, the antibody is conjugated to a solid support (e.g., magnetic beads).

[0067] In one or more embodiments, the kit further contains reagents for obtaining TIL seed cells from pleural effusion and / or ascites.

[0068] In one or more embodiments, the kit further contains reagents for culturing TIL seed cells.

[0069] In one or more embodiments, the kit further contains reagents for amplifying TIL cells.

[0070] In one or more embodiments, the kit further contains reagents for activating TIL cells. Detailed Implementation

[0071] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0072] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0073] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0074] In this document, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0075] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0076] In this article, the sum of the percentages of all components in the composition is 100%.

[0077] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0078] In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), biantibodies, and single-chain molecules, as well as antibody fragments. An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. Antibody fragments are preferably antigen-binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multi-specific antibodies formed from antibody fragments; and any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes.

[0079] In this article, pleural effusion or ascites, also referred to as pleural effusion or peritoneal effusion, refers to the abnormal accumulation of fluid in the pleural or abdominal cavity. Normally, a small amount of fluid exists in the pleural or abdominal cavity to lubricate organs, but when the amount of fluid exceeds the normal range, pleural or peritoneal effusion occurs. The formation of pleural effusion or ascites can be associated with a variety of diseases, including liver disease (such as cirrhosis), heart disease, renal insufficiency, cancer, and infections. In one or more embodiments, the pleural effusion or ascites described herein refers to the pleural effusion or ascites of a cancer patient.

[0080] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0081] The inventors discovered that, for solid tumor tissue, pleural effusion, and / or ascites from the same individual, TILs obtained by expanding culture after CD45RA negative selection and / or CD39 positive selection from solid tumor tissue did not show a significant increase in killing power against homologous tumor target cells compared to TILs obtained without sorting. However, TILs obtained by expanding culture after the above sorting of TIL seed cells from pleural effusion and / or ascites showed a significant increase in killing power against homologous tumor target cells compared to TILs obtained without sorting.

[0082] Therefore, the present invention first provides a method for culturing TILs derived from pleural effusion and / or ascites, comprising the steps of: 1) obtaining TIL seed cells from pleural effusion and / or ascites; 2) sorting the TIL seed cells to obtain intermediate cells, wherein the intermediate cells are CD45RA. - Cells, CD39 + Cells, or CD45RA - CD39 + Cells; optionally 3) culture the intermediate cells described in 2) to obtain TIL. In some embodiments, the pleural effusion and / or ascites are derived from pleural effusion (pleural fluid) and / or ascites (ascites).

[0083] CD45RA is a subtype of the CD45 complex, with restricted expression among different subtypes of lymphoid cells. CD45 (LCA, leukocyte common antigen) is a receptor-type protein tyrosine phosphatase widely expressed in all nucleated hematopoietic cells, accounting for approximately 10% of all lymphocyte surface proteins. CD45 glycoprotein plays a crucial role in lymphocyte development and antigen signal transduction and is an important regulator of SRC family kinases.

[0084] In one or more embodiments, the method includes: obtaining TIL seed cells from pleural fluid and / or ascites, sorting to obtain CD45RA-TIL cells, and expanding and culturing the CD45RA-TIL cells. In one or more embodiments, the method includes: obtaining TIL seed cells from pleural fluid and / or ascites, sorting to obtain CD39+TIL cells, and expanding and culturing the CD39+TIL cells.

[0085] In a preferred embodiment, the method of the present invention involves multiple sorting, namely CD45RA- sorting and CD39+ sorting. The two sorting processes can be performed sequentially or simultaneously, and their order and interval are not limited. For example, CD45RA- sorting can be performed first, followed by CD39+ sorting; or CD39+ sorting can be performed first, followed by CD45RA- sorting; or both can be performed simultaneously.

[0086] In some embodiments, the method includes: obtaining TIL seed cells from pleural fluid and / or ascites, sorting to obtain CD45RA-TIL cells, and expanding and culturing the CD45RA-TIL cells; after obtaining the CD45RA-TIL cells, sorting to obtain CD45RA-CD39+TIL cells, and expanding and culturing the CD45RA-CD39+TIL cells. Negative and positive sorting are relative concepts, depending on the nature of the obtained cells. In one or more embodiments, the step of sorting to obtain CD45RA-TIL cells (which can be obtained from TILs or from CD39+TILs) is a negative sorting step, and the step of sorting to obtain CD39+TIL cells (which can be obtained from TILs or from CD45RA-TILs) is a positive sorting step.

[0087] Methods for sorting immune cells based on cell surface protein markers are known in the art, and these methods are all included within the scope of this invention, such as any method based on the binding reaction of antigens with antibodies or ligands. Exemplary methods include: magnetic bead sorting, flow cytometry sorting, immune density gradient cell sorting, and microfluidic cell sorting. In the methods described herein, forward sorting and / or negative sorting includes sorting techniques based on cell culture characteristics, sorting techniques based on cell physical properties, and sorting techniques based on biochemical affinity, preferably sorting based on cell membrane surface markers (antibody affinity-dependent sorting). Methods for sorting immune cells based on cell surface protein markers are known in the art, and these methods are all included within the scope of this invention, such as any method based on the binding reaction of antigens with antibodies or ligands. Exemplary methods include: density gradient centrifugation, affinity chromatography, magnetic bead separation and purification, flow cytometry sorting, etc., preferably magnetic bead separation and purification or flow cytometry sorting. Typically, antibody incubation is required before sorting.

[0088] In some embodiments, the CD39 forward sorting includes the step of co-incubating a biotin-conjugated CD39 antibody with biotin-coated magnetic beads. In an exemplary embodiment, the magnetic beads are Miltenyi Biotec Cat#: 130-090-485, the antibody is Miltenyi Biotec Cat#: 130-110-649, and the forward sorting step is performed according to the aforementioned product instructions.

[0089] In some implementations, CD45RA negative sorting is performed using a CD45RA negative sorting system. An exemplary implementation uses Miltenyi Biotec's CliniMACS. ®CD45RA Product Line.

[0090] The method described herein obtains seed cells containing tumor-specific tumor-associated endothelial cells (TILs) using commonly used cell isolation methods in the art, which may include centrifugation, washing, and precipitation steps. Specifically, it includes: centrifuging pleural effusion and / or ascites to precipitate the effusion, washing with physiological saline, and obtaining the seed cells. Typically, after obtaining pleural effusion and / or ascites, the effusion from the patient is centrifuged in a sterile environment to remove the supernatant. The centrifugation rate can be 200-500 g, for example, 200-400 g or 300-500 g, and the centrifugation time can be 5 minutes or more, for example, 5-10 minutes or 5-8 minutes. The centrifuged cell pellet is washed multiple times with physiological saline, repeating the centrifugation and washing steps, followed by washing with erythrocyte lysis buffer. After washing, the cell pellet is obtained by centrifugation, which is the seed cells containing TILs. In this method, the physiological saline is freshly prepared and contains 80-120 U / mL penicillin, 80-120 μg / mL streptomycin, and 30-60 μg / mL gentamicin. In an exemplary embodiment, the saline solution contains 100-120 U / mL of penicillin, 100-120 μg / mL of streptomycin, and 50-60 μg / mL of gentamicin.

[0091] In some embodiments, obtaining seed cells containing tumor-specific TILs includes the following steps: centrifuging pleural fluid and / or ascites to precipitate the effusion, washing with physiological saline, centrifuging again to precipitate the effusion, and obtaining the seed cells. In one or more embodiments, the centrifugation rate is 200-500 g, for example 200-400 g or 300-500 g, and the centrifugation time is 5-10 minutes, for example 5-8 minutes.

[0092] The method described herein may further include a step of culturing the TIL seed cells before and / or after any sorting step. The culturing step may involve culturing the TIL seed cells in a system for culturing the TIL seed cells for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, or at least 12 days. In an exemplary embodiment, seed cells are added to a culture medium and amplified at 35–37°C with 5% CO2. At regular intervals (e.g., every 1 day, every 2 days, every 4 days, every 6 days, or every 8 days), half the volume of the old culture medium is removed and half the volume of fresh amplification culture medium is added. The culture system may be a commonly used culture medium for TIL cells in the art, such as AIM-V, X-VIVO, DMEM, RPMI1640, or OpTmizer. TMBasic culture media such as FUJIFILM Irvin MHM-C, and serum-free cell culture dishes (e.g., GIBCO). ® AIM-V ® CTS TM Serum-free cell culture medium, DMEM medium, RPMI 1640 medium. Antibodies and cytokines may also be added to this medium as needed. In one or more embodiments, the system for culturing the TIL seed cells further includes antibodies and cytokines. Cytokines may include one or more of IL-2, IL-7, IL-15, IL-21, thymosin α1, αCD3, αCD28, TWS119, vitamin C, IFN-γ, TNF-α, and GM-CSF. Antibodies may include monoclonal antibodies and bispecific antibodies, such as one or more of anti-CD137 mAb, anti-CD28 mAb, and anti-PD-1 mAb.

[0093] In one or more embodiments, the antibodies in the system for culturing TIL seed cells include immune checkpoint antibodies or antigen-binding fragments thereof. In one or more embodiments, the immune checkpoint antibody includes any one or more selected from PD-1 antibody, CTLA-4 antibody, LAG3 antibody, TIM3 antibody, TIGIT antibody, and BTLA antibody.

[0094] In one or more embodiments, the antibody in the system for culturing TIL seed cells includes any one or more selected from CD3 antibody, CD28 antibody, CD137 antibody, OX40 antibody, GITR antibody, ICOS antibody, CD206 antibody, and CD40 antibody.

[0095] In one or more embodiments, the system for culturing the seed cells comprises IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α, and serum.

[0096] In one or more of the above embodiments, the concentration of IL-2 in the system for culturing the seed cells is 200-6000 IU / mL, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 IU / mL, or a range between any two of the above values. Preferably, the concentration of IL-2 is 500-5000, 1000-4000, or 1500-3500 ng / mL.

[0097] In one or more of the above embodiments, the concentration of IL-7 in the system for culturing the seed cells is 5-100 ng / mL, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL, or a range between any two of the above values. Preferably, the concentration of IL-7 is 5-65, 5-55, 5-45, 5-35, or 5-25 ng / mL.

[0098] In one or more of the above embodiments, the concentration of IL-15 in the system for culturing the seed cells is 5-100 ng / mL, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL, or a range between any two of the above values. Preferably, the concentration of IL-15 in the system for culturing the seed cells is 5-65, 5-55, 5-45, 5-35, or 5-25 ng / mL.

[0099] In one or more of the above embodiments, the concentration of GM-CSF in the system for culturing the seed cells is 200-5000 U / mL, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 U / mL, or a range between any two of the above values. Preferably, the concentration of GM-CSF is 200-1500, 200-1200, 200-950, 200-850, 200-750, 200-650, 200-550, or 200-500 U / mL.

[0100] In one or more of the above embodiments, the concentration of IFN-γ in the system for culturing the seed cells is 100-3000 U / mL, for example, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, or 3000 U / mL, or a range between any two of the above values. Preferably, the concentration of IFN-γ is 200-2000, 300-1800, 500-1500, or 800-1200 U / mL.

[0101] In one or more of the above embodiments, the concentration of TNF-α in the system for culturing the seed cells is 1-100 ng / mL, for example, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ng / mL, or a range between any two of the above values. Preferably, the concentration of TNF-α is 5-45, 5-35, 5-25, or 5-15 ng / mL.

[0102] In one or more of the above embodiments, the concentration of CD137 antibody in the system for culturing the seed cells is 1-100 μg / mL, for example, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL, or a range between any two of the above values. Preferably, the concentration of CD137 antibody is 1-95, 1-85, 1-75, 1-65, 1-55, 1-45, 1-35, 1-25, 1-15, 1-9, 1.5-8, 2-7, 2.5-6, 2.5-5, or 3-5 μg / mL.

[0103] In one or more of the above embodiments, the concentration of CD28 antibody in the system for culturing the seed cells is 1-100 μg / mL, for example, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL, or a range between any two of the above values. Preferably, the concentration of CD28 antibody is 1-95, 1-85, 1-75, 1-65, 1-55, 1-45, 1-35, 1-25, 1-15, 1-9, 1.5-8, 2-7, 2.5-6, 2.5-5, or 3-5 μg / mL.

[0104] In one or more of the above embodiments, the concentration of PD-1 antibody in the system for culturing the seed cells is 1-100 µg / mL, for example, 1, 2, 3, 5, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µg / mL, or a range between any two of the above values. Preferably, the concentration of PD-1 antibody is 1-65, 1-55, 1-45, 1-35, 1-25, 1-15, 1-8, 1-6, 1-4, 1-3 µg / mL.

[0105] In one or more embodiments, each antibody is a monoclonal antibody (mAb).

[0106] In one or more embodiments, the serum is selected from human AB serum, the subject's own serum, or animal-derived serum.

[0107] In one or more embodiments, the serum concentration is 1-10%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the serum concentration is 1.5-9.5%, 2.5-8.5%, 3.5-7.5%, or 4.5-6.5%.

[0108] In one or more embodiments, the system for culturing the seed cells further includes an antibiotic. Preferably, the antibiotic is selected from one or more of penicillin and streptomycin. In one or more embodiments, the concentration of penicillin in the culture medium is 10-1000 IU / mL, 20-800 IU / mL, or 50-100 IU / mL.

[0109] In some embodiments, the culture medium used to culture TIL seed cells contains X-VIVO 15 basal medium containing IL-2, IL-7, IL-15, GM-CSF, IFN-γ, TNF-α, anti-CD137 mAb, anti-CD28 mAb, anti-PD-1 mAb, human AB serum, and 1×PS penicillin antibody. In an exemplary embodiment, an exemplary amplification medium is 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to the final volume.

[0110] Optionally, in the method described herein, after obtaining TIL cells through multiple sorting, the TIL cells are further cultured using an expansion medium to obtain finished TIL cells. Those skilled in the art are familiar with the expansion medium and culture conditions used for TIL cells. Typically, the expansion medium contains IL-2, IL-7, IL-15, serum, and basal medium, and the culture conditions are 37°C and 5% CO2. In an exemplary embodiment, the expansion medium used to expand and culture the TIL seed cells includes 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to the final volume.

[0111] Optionally, the method described herein, after obtaining TIL cells through multiple sorting, further includes a step of activating the TIL seed cells. Those skilled in the art are familiar with the reagents and methods used to activate TIL cells. Typically, TIL cells can be activated by contacting TIL seed cells with any one or more of anti-CD3 antibodies, anti-CD28 antibodies, and anti-CD137 antibodies (e.g., in an amplification medium) for at least one day. The anti-CD3, anti-CD28, and anti-CD137 antibodies can contact the TILs in solution or in a form immobilized on a substrate. The substrate can be magnetic beads, plates, dishes, well plates, or cell culture bags, etc.

[0112] Therefore, the present invention also provides TIL cells cultured as described in any embodiment herein.

[0113] In some embodiments, the present invention also provides a cell cryopreservation formulation comprising a TIL cell population and a cryopreservation solution as described in any embodiment herein. Typically, the cell cryopreservation formulation may also contain one or more of carbohydrates, serum albumin, glycine, vitamin E, and electrolytes, wherein the carbohydrates are monosaccharides selected from glucose, fructose, and dextrose, and / or disaccharides selected from lactose and trehalose. In one or more embodiments, the cryopreservation solution is a cell cryopreservation solution or a tissue cryopreservation solution. In one or more embodiments, the cryopreservation solution is a serum-free cryopreservation solution, such as BioLifeSolutions' CryoStor CS10 cryopreservation solution.

[0114] In some embodiments, the present invention also provides a method for treating solid tumors, comprising: (1) obtaining pleural effusion and / or ascites from a patient with a solid tumor and preparing TIL cells using the method described in any embodiment herein; and (2) reinfusing the TIL cells from (1) into the patient with a solid tumor.

[0115] In one or more embodiments, the solid tumor includes gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma.

[0116] In some embodiments, the present invention also provides a pharmaceutical composition comprising TIL cells as described herein, and pharmaceutically acceptable excipients. In one or more embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent.

[0117] In this article, pharmaceutically acceptable excipients refer to carriers, diluents, and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, and preservatives. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for TIL cell reinfusion (e.g., intravenous infusion).

[0118] The present invention also provides a kit containing the TIL cells described herein.

[0119] This invention also provides a method for improving the target cell killing rate of TIL cells, comprising sorting TIL seed cells to obtain CD45RA- cells, CD39+ cells, or CD45RA-CD39+ cells. In one or more embodiments, the method comprises: negatively sorting the seed cells to remove CD45RA+ cells, and / or performing positive sorting with CD39 antibodies to obtain the TIL cells. In one or more embodiments, the TIL seed cells are obtained from pleural effusion and / or ascites of cancer patients. In one or more embodiments, the pleural effusion and / or ascites is pleural effusion and / or ascites from patients suffering from one or more cancers selected from: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer (including non-small cell lung cancer), melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, colon cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the pleural effusion and / or ascites is pleural effusion and / or ascites from a patient suffering from one or more cancers selected from: melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and colon cancer. In some embodiments, the target cells are tumor cells from a TIL-derived patient.

[0120] This invention also provides the use of TIL cells as described herein in the preparation of medicaments. In one or more embodiments, the medicament is used to treat a patient with cancer. In one or more embodiments, the TIL seed cells are obtained from the patient's pleural effusion and / or ascites. In one or more embodiments, the cancer is selected from: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the cancer is selected from: melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and colon cancer.

[0121] The present invention also provides the use of reagents that specifically bind CD45RA and CD39 in the preparation of drugs or kits, wherein the drugs comprise TILs derived from pleural effusion and / or ascites with enhanced target cell killing rates, and the kits are used to prepare TILs derived from pleural effusion and / or ascites with enhanced target cell killing rates.

[0122] In one or more embodiments, the TIL is prepared by a method according to any embodiment of the first aspect of this document. In one or more embodiments, the reagent that specifically binds to CD45RA is an antibody. In one or more embodiments, the antibody is conjugated to a solid-phase support (e.g., magnetic beads). In one or more embodiments, the reagent that specifically binds to CD39 is an antibody. In one or more embodiments, the antibody is conjugated to a solid-phase support (e.g., magnetic beads). In one or more embodiments, the drug is used to treat cancer. In one or more embodiments, the cancer is selected from: gastric cancer, thyroid tumors, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma.

[0123] The present invention also provides a kit containing reagents that specifically bind to CD45RA and CD39. The kit is used to prepare TILs with enhanced target cell killing rate.

[0124] In one or more embodiments, the reagent that specifically binds to CD45RA is an antibody. In one or more embodiments, the antibody is conjugated to a solid support (e.g., magnetic beads). In one or more embodiments, the reagent that specifically binds to CD39 is an antibody. In one or more embodiments, the antibody is conjugated to a solid support (e.g., magnetic beads). In one or more embodiments, the kit further contains reagents for obtaining TIL seed cells from pleural fluid and / or ascites. In one or more embodiments, the kit further contains reagents for culturing TIL seed cells. In one or more embodiments, the kit further contains reagents for expanding TIL cells. In one or more embodiments, the kit further contains reagents for activating TIL cells. The kit of the present invention may also include instructions describing CD45RA negative selection of TIL cells (to obtain CD45RA-TIL cells) or CD39 positive selection (to obtain CD39+TIL) or CD45RA negative selection and CD39 positive selection (to obtain CD45RA-CD39+TIL).

[0125] In this document, pleural effusion and / or ascites refers to pleural effusion and / or ascites in patients suffering from one or more of the following cancers: gastric cancer, thyroid tumors, gallbladder cancer, bile duct cancer, lung cancer (including non-small cell lung cancer), melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, colon cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the pleural effusion and / or ascites refers to pleural effusion and / or ascites in patients suffering from one or more of the following cancers: melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and colon cancer.

[0126] The present invention has the following beneficial effects:

[0127] The TILs obtained by expanding culture of TIL seed cells derived from pleural effusion and / or ascites after CD45RA negative selection and / or CD39 positive selection have a significantly enhanced killing effect on homologous tumor target cells compared with TILs obtained without sorting.

[0128] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0129] Example 1: Processing of solid tumor tissue and pleural or ascites samples and TIL culture

[0130] The tumor tissue samples and pleural effusion or ascites samples used in the examples are shown in Table 1 below:

[0131] Table 1. Tumor tissue samples and pleural or ascites samples

[0132] Sample No. Cancer Type Sample Type T01T Melanoma Tumor Tissue T01MA Melanoma Ascites T02T Cervical Cancer Tumor Tissue T02MPE Cervical Cancer Pleural Effusion T03T Gastric Cancer Tumor Tissue T03MA Gastric Cancer Ascites T04T Ovarian Cancer Tumor Tissue T04MPE Ovarian Cancer Pleural Effusion T05T Non-Small Cell Lung Cancer Tumor Tissue T05MPE Non-Small Cell Lung Cancer Pleural Effusion T06T Colon Cancer Tumor Tissue T06MA Colon Cancer Ascites

[0133] Note: Samples with the same number in their sample number indicate that they came from the same patient. The letters T, MA, and MPE after the number represent the sample type as tumor tissue, ascites, and pleural effusion, respectively.

[0134] For each solid tumor tissue sample, the following methods were used for processing and TIL culture:

[0135] Method 1:

[0136] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0137] 2) Under aseptic conditions in a biosafety cabinet, the freshly isolated tumor tissue samples from the tumor patients were washed in a 10cm culture dish containing 30 mL of the physiological saline prepared in step 1), and then transferred to a new 10cm culture dish containing 30 mL of the physiological saline prepared in step 1). This washing process was repeated 3 times.

[0138] 3) Using a sterile scalpel blade, remove adipose and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm. 3 Forty-two randomly selected tumor tissue blocks were placed in two G-REX100 culture jars (purchased from Wilsonwolf). Seed cell culture medium was added to each jar, consisting of: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume. Excess tumor tissue blocks were cryopreserved in CryoStor10 (purchased from BioLifeSolutions) cryopreservation solution using a programmed freezing system in liquid nitrogen.

[0139] 4) After adding 1L of the above seed cell culture medium to the G-REX100 culture vessel containing tumor tissue blocks in 3), the tumor tissue blocks were cultured at 37℃ and 5% CO2. Every 4 days, half the volume of old seed cell culture medium was removed and half the volume of fresh seed cell culture medium was added. On the 12th day, the TIL seed cells were harvested by centrifugation and the total number of cells and the viability were counted.

[0140] 5) Take the seed cells harvested in step 4) and resuspend them in expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to a final volume, bringing the solution to 5.0 × 10⁻⁶. 5 / mL was added to cell culture dishes pre-treated with anti-CD3 mAb, anti-CD28 mAb, and anti-CD137 mAb coatings. The cells were activated at 37°C with 5% CO2 for 2 days. The activated cells were then collected by centrifugation and seeded into G-REX500M culture flasks containing pre-warmed expansion medium (the same expansion medium as described above). Each G-REX500M flask contained 5L of expansion medium. The activated seed cells were then cultured at a rate of 2.5 × 10⁻⁶. 5 / cm 2 Inoculate at the desired density and culture at 37°C with 5% CO2. Count cells every 4 days, then remove half the volume of old expansion medium and add half the volume of fresh expansion medium. Continue until the total cell count in each G-REX 500M jar reaches 1.0 × 10⁻⁶ cells / mL. 10 Then, the cells were divided into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to a total volume of 5L before further culturing. After a total of 12 days of culturing in the expansion medium in a G-REX 500M culture vessel, the cells were harvested to obtain the finished TIL product.

[0141] Method 2:

[0142] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0143] 2) Under aseptic conditions in a biosafety cabinet, the freshly isolated tumor tissue samples from the tumor patients were washed in a 10cm culture dish containing 30 mL of the physiological saline prepared in step 1), and then transferred to a new 10cm culture dish containing 30 mL of the physiological saline prepared in step 1). This washing process was repeated 3 times.

[0144] 3) Using a sterile scalpel blade, remove adipose and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm. 3 Forty-two randomly selected tumor tissue blocks were placed in two G-REX100 culture jars (purchased from Wilsonwolf). Seed cell culture medium was added to each jar, consisting of: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume. Excess tumor tissue blocks were cryopreserved in CryoStor10 (purchased from BioLifeSolutions) cryopreservation solution using a programmed freezing system in liquid nitrogen.

[0145] 4) After adding 1L of the above seed cell culture medium to the G-REX100 culture vessel containing tumor tissue blocks in 3), the tumor tissue blocks were cultured at 37℃ and 5% CO2. Every 4 days, half the volume of old seed cell culture medium was removed and half the volume of fresh seed cell culture medium was added. On the 12th day, the TIL seed cells were harvested by centrifugation and the total number of cells and the viability were counted.

[0146] 5) Collect TIL seed cells obtained in step 4) and use the CD45RA negative sorting system (CliniMACS). ® CD45RA Product Line (Miltenyi Biotec) removes the CD45RA+ cell population according to the instructions to obtain CD45RA-TIL seed cells;

[0147] 6) Take the CD45RA-TIL seed cells harvested in step 5) and resuspend them in expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to a final volume, bringing the temperature to 5.0 × 10⁻⁶. 5 / mL was added to cell culture dishes pre-treated with anti-CD3 mAb, anti-CD28 mAb, and anti-CD137 mAb coatings. The cells were activated at 37°C with 5% CO2 for 2 days. The activated cells were then collected by centrifugation and seeded into G-REX500M culture flasks containing pre-warmed expansion medium (the same expansion medium as described above). Each G-REX500M flask contained 5L of expansion medium. The activated seed cells were then cultured at a rate of 2.5 × 10⁻⁶. 5 / cm 2 Inoculate at the desired density and culture at 37°C with 5% CO2. Count cells every 4 days, then remove half the volume of old expansion medium and add half the volume of fresh expansion medium. Continue until the total cell count in each G-REX 500M jar reaches 1.0 × 10⁻⁶ cells / mL. 10 Then, the cells were divided into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to a total volume of 5L before further culturing. After a total of 12 days of culturing in the expansion medium in a G-REX 500M culture vessel, the cells were harvested to obtain the finished TIL product.

[0148] Method 3

[0149] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0150] 2) Under aseptic conditions in a biosafety cabinet, the freshly isolated tumor tissue samples from the tumor patients were washed in a 10cm culture dish containing 30 mL of the physiological saline prepared in step 1), and then transferred to a new 10cm culture dish containing 30 mL of the physiological saline prepared in step 1). This washing process was repeated 3 times.

[0151] 3) Using a sterile scalpel blade, remove adipose and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm.3 Forty-two randomly selected tumor tissue blocks were placed in two G-REX100 culture jars (purchased from Wilsonwolf). Seed cell culture medium was added to each jar, consisting of: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume. Excess tumor tissue blocks were cryopreserved in CryoStor10 (purchased from BioLifeSolutions) cryopreservation solution using a programmed freezing system in liquid nitrogen.

[0152] 4) After adding 1L of the above seed cell culture medium to the G-REX100 culture vessel containing tumor tissue blocks in 3), the tumor tissue blocks were cultured at 37℃ and 5% CO2. Every 4 days, half the volume of old seed cell culture medium was removed and half the volume of fresh seed cell culture medium was added. On the 12th day, the TIL seed cells were harvested by centrifugation and the total number of cells and the viability were counted.

[0153] 5) Collect TIL seed cells obtained in step 4) and use the CD45RA negative sorting system (CliniMACS). ® Following the instructions of the CD45RA Product Line (Miltenyi Biotec), the CD45RA+ cell population was removed to obtain CD45RA-TIL seed cells. Biotin-conjugated CD39 antibody (CD39 Antibody, anti-human, REAfinity™ biotin, Miltenyi Biotec, Cat#: 130-110-649) was co-incubated with biotin-coated magnetic beads (Anti-Biotin MicroBeads, Miltenyi Biotec, Cat#: 130-090-485). The CD45RA-TIL seed cells were then subjected to CD39 positive selection according to the instructions to obtain CD45RA-CD39+ TIL seed cells.

[0154] 6) Take the CD45RA-CD39+ TIL seed cells harvested in step 5) and resuspend them in expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to a final volume, resuspending them to 5.0 × 10⁻⁶. 5 / mL was added to cell culture dishes pre-treated with anti-CD3 mAb, anti-CD28 mAb, and anti-CD137 mAb coatings. The cells were activated at 37°C with 5% CO2 for 2 days. The activated cells were then collected by centrifugation and seeded into G-REX500M culture flasks containing pre-warmed expansion medium (the same expansion medium as described above). Each G-REX500M flask contained 5L of expansion medium. The activated seed cells were then cultured at a rate of 2.5 × 10⁻⁶. 5 / cm 2 Inoculate at the desired density and culture at 37°C with 5% CO2. Count cells every 4 days, then remove half the volume of old expansion medium and add half the volume of fresh expansion medium. Continue until the total cell count in each G-REX 500M jar reaches 1.0 × 10⁻⁶ cells / mL. 10 Then, the cells were divided into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to a total volume of 5L before further culturing. After a total of 12 days of culturing in the expansion medium in a G-REX 500M culture vessel, the cells were harvested to obtain the finished TIL product.

[0155] For each pleural effusion or ascites sample, it was processed and cultured using TIL according to the following methods:

[0156] Method 4:

[0157] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0158] 2) Centrifuge 200-500 mL of pleural or peritoneal fluid from cancer patients at 300 g for 8 minutes. Discard the supernatant in a sterile environment within a biosafety cabinet. Wash the cell pellet three times with the physiological saline prepared in step 1), centrifuging at 300 g for 8 minutes each time. Repeat this process three times. Add erythrocyte lysis buffer (BD Pharm Lyse™ Lysing Buffer, Cat#555899, BD Biosciences). Lyse the red blood cells according to the manufacturer's instructions, then wash three times with the physiological saline prepared in step 1), centrifuge at 300 g for 8 minutes, and obtain the cell pellet.

[0159] 3) Add seed cell culture medium to the G-REX100 culture vessel. The components of the seed culture medium include: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume.

[0160] 4) After adding 1L of the above seed cell culture medium to the G-REX100 culture jar in 3), add the cells from the precipitate obtained in 2) to the seed culture medium and culture at 37℃ with 5% CO2. Every 4 days, remove half the volume of old seed cell culture medium and add half the volume of fresh seed cell culture medium. On the 12th day, centrifuge to harvest TIL seed cells and count the total number of cells and viability.

[0161] 5) Take the seed cells harvested in step 4) and resuspend them in expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to a final volume, bringing the solution to 5.0 × 10⁻⁶. 5 / mL was added to cell culture dishes pre-treated with anti-CD3 mAb, anti-CD28 mAb, and anti-CD137 mAb coatings. The cells were activated at 37°C with 5% CO2 for 2 days. The activated cells were then collected by centrifugation and seeded into G-REX500M culture flasks containing pre-warmed expansion medium (the same expansion medium as described above). Each G-REX500M flask contained 5L of expansion medium. The activated seed cells were then cultured at a rate of 2.5 × 10⁻⁶. 5 / cm 2 Inoculate at the desired density and culture at 37°C with 5% CO2. Count cells every 4 days, then remove half the volume of old expansion medium and add half the volume of fresh expansion medium. If the total cell count in each G-REX 500M jar reaches 1.0 × 10⁻⁶ cells / mL... 10 Then, the cells were divided into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to a total volume of 5L before further culturing. After a total of 12 days of culturing in the expansion medium in a G-REX 500M culture vessel, the cells were harvested to obtain the finished TIL product.

[0162] Method 5:

[0163] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0164] 2) Centrifuge 200-500 mL of pleural or peritoneal fluid from cancer patients at 300 g for 8 minutes. Discard the supernatant in a sterile environment within a biosafety cabinet. Wash the cell pellet three times with the physiological saline prepared in step 1), centrifuging at 300 g for 8 minutes each time. Repeat this process three times. Add erythrocyte lysis buffer (BD Pharm Lyse™ Lysing Buffer, Cat#555899, BD Biosciences). Lyse the red blood cells according to the manufacturer's instructions, then wash three times with the physiological saline prepared in step 1), centrifuge at 300 g for 8 minutes, and obtain the cell pellet.

[0165] 3) Add seed cell culture medium to the G-REX100 culture vessel. The components of the seed culture medium include: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume.

[0166] 4) After adding 1L of the above seed cell culture medium to the G-REX100 culture jar in 3), add the cells from the precipitate obtained in 2) to the seed culture medium and culture at 37℃ with 5% CO2. Every 4 days, remove half the volume of old seed cell culture medium and add half the volume of fresh seed cell culture medium. On the 12th day, centrifuge to harvest TIL seed cells and count the total number of cells and viability.

[0167] 5) Collect TIL seed cells obtained in step 4) and use the CD45RA negative sorting system (CliniMACS). ® CD45RA Product Line (Miltenyi Biotec) removes the CD45RA+ cell population according to the instructions to obtain CD45RA-TIL seed cells;

[0168] 6) Take the CD45RA-TIL seed cells harvested in step 5) and resuspend them in expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to a final volume, bringing the temperature to 5.0 × 10⁻⁶. 5 / mL was added to cell culture dishes pre-treated with anti-CD3 mAb, anti-CD28 mAb, and anti-CD137 mAb coatings. The cells were activated at 37°C with 5% CO2 for 2 days. The activated cells were then collected by centrifugation and seeded into G-REX500M culture flasks containing pre-warmed expansion medium (the same expansion medium as described above). Each G-REX500M flask contained 5L of expansion medium. The activated seed cells were then cultured at a rate of 2.5 × 10⁻⁶. 5 / cm 2 Inoculate at the desired density and culture at 37°C with 5% CO2. Count cells every 4 days, then remove half the volume of old expansion medium and add half the volume of fresh expansion medium. If the total cell count in each G-REX 500M jar reaches 1.0 × 10⁻⁶ cells / mL... 10 Then, the cells were divided into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to a total volume of 5L before further culturing. After a total of 12 days of culturing in the expansion medium in a G-REX 500M culture vessel, the cells were harvested to obtain the finished TIL product.

[0169] Method 6:

[0170] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0171] 2) Centrifuge 200-500 mL of pleural or peritoneal fluid from cancer patients at 300 g for 8 minutes. Discard the supernatant in a sterile environment within a biosafety cabinet. Wash the cell pellet three times with the physiological saline prepared in step 1), centrifuging at 300 g for 8 minutes each time. Repeat this process three times. Add erythrocyte lysis buffer (BD Pharm Lyse™ Lysing Buffer, Cat#555899, BD Biosciences). Lyse the red blood cells according to the manufacturer's instructions, then wash three times with the physiological saline prepared in step 1), centrifuge at 300 g for 8 minutes, and obtain the cell pellet.

[0172] 3) Add seed cell culture medium to the G-REX100 culture vessel. The components of the seed culture medium include: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume.

[0173] 4) After adding 1L of the above seed cell culture medium to the G-REX100 culture jar in 3), add the cells from the precipitate obtained in 2) to the seed culture medium and culture at 37℃ with 5% CO2. Every 4 days, remove half the volume of old seed cell culture medium and add half the volume of fresh seed cell culture medium. On the 12th day, centrifuge to harvest TIL seed cells and count the total number of cells and viability.

[0174] 5) Collect TIL seed cells obtained in step 4) and use the CD45RA negative sorting system (CliniMACS). ® Following the instructions of the CD45RA Product Line (Miltenyi Biotec), the CD45RA+ cell population was removed to obtain CD45RA-TIL seed cells. Biotin-conjugated CD39 antibody (CD39 Antibody, anti-human, REAfinity™ biotin, Miltenyi Biotec, Cat#: 130-110-649) was co-incubated with biotin-coated magnetic beads (Anti-Biotin MicroBeads, Miltenyi Biotec, Cat#: 130-090-485). The CD45RA-TIL seed cells were then subjected to CD39 positive selection according to the instructions to obtain CD45RA-CD39+ TIL seed cells.

[0175] 6) Take the CD45RA-CD39+ TIL seed cells harvested in step 5) and resuspend them in expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS penicillin antibody, and X-VIVO 15 basal medium to a final volume, resuspending them to 5.0 × 10⁻⁶. 5 / mL was added to cell culture dishes pre-treated with anti-CD3 mAb, anti-CD28 mAb, and anti-CD137 mAb coatings. The cells were activated at 37°C with 5% CO2 for 2 days. The activated cells were then collected by centrifugation and seeded into G-REX500M culture flasks containing pre-warmed expansion medium (the same expansion medium as described above). Each G-REX500M flask contained 5L of expansion medium. The activated seed cells were then cultured at a rate of 2.5 × 10⁻⁶. 5 / cm 2 Inoculate at the desired density and culture at 37°C with 5% CO2. Count cells every 4 days, then remove half the volume of old expansion medium and add half the volume of fresh expansion medium. If the total cell count in each G-REX 500M jar reaches 1.0 × 10⁻⁶ cells / mL... 10Then, the cells were divided into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to a total volume of 5L before further culturing. After a total of 12 days of culturing in the expansion medium in a G-REX 500M culture vessel, the cells were harvested to obtain the finished TIL product.

[0176] Solid tumor tissue was taken from each of the T01-T06 samples and cut into pieces with a diameter of 3×3×3 mm. 3 The small pieces were divided into three equal parts, and each sample was cultured according to methods 1-3 above. Pleural or peritoneal fluid from samples T01-T06 were also divided into three equal parts, and each sample was cultured according to methods 4-6 above. The obtained TILs are shown in Table 2 below.

[0177] Table 2 TILs obtained by different culture methods

[0178]

[0179] Example 2: Obtaining primary tumor cells derived from solid tumor tissue

[0180] Fresh T01T-T06T solid tumor tissues from Example 1 were processed according to the method described in the paper published by Chiara M Cattaneo et al. (Tumor organoid-T-cell coculture systems Nat Protoc. 2020 Jan;15(1):15-39., which is incorporated herein by reference in its entirety). Patient-derived organoid (PDO) culture was then performed, and all reagent kits and instruments used for culture were purchased according to the description in that publication. Successfully cultured PDO cells that had been passaged 2-3 times were enzymatically digested, and the resulting primary tumor cell suspension was prepared for subsequent experiments.

[0181] Example 3: In vitro killing effect of different TILs on homologous primary tumor cells

[0182] Primary tumor cells prepared from fresh T01T-T06T solid tumor tissues in Example 2 were selected as target cells. The in vitro killing activity of each TIL prepared in Example 1 was detected using an Agilent Technologies Real-Time Label-Free Cell Function Analyzer (RTCA). The specific steps are as follows:

[0183] (1) Zeroing: Add 50 μL of DMEM or 1640 culture medium to each well, place it in the instrument, select step 1, and zero it;

[0184] (2) Target cell plating: target cells were plated at a density of 10 per well. 450 μL of cells were seeded in a plate containing the detection electrode and left to stand for a few minutes to allow the cells to stabilize before placing them in the instrument to begin step 2, cell culture.

[0185] (3) Adding effector cells: When the target cells are cultured for 24 hours and the cell index is about 1.0, pause step 2 and add 50 μL of TIL as effector cells to each well. Add effector cells at a ratio of 4:1 based on the TIL cell viability. Start step 3 and continue co-culturing for more than 60 hours. Observe the cell proliferation curve and calculate the target cell killing rate. The formula for calculating the target cell killing rate is as follows:

[0186]

[0187] Where A represents the cell index of the group containing only target cells (i.e. tumor cells) without any effector cells, and B represents the cell index of each group with effector cells.

[0188] The results are shown in Table 3. Compared with the TILs obtained from solid tumor tissues cultured by Method 1, the target cell killing rate of the corresponding samples of solid tumor tissue TILs cultured by Method 2 and Method 3 was not significantly improved. In contrast, compared with the TILs obtained from pleural effusion or ascites effusions cultured by Method 4, the target cell killing rate of the corresponding samples of pleural effusion or ascites TILs cultured by Method 5 and Method 6 was significantly improved.

[0189] Table 3 RTCA killing rate of different TIL target cells

[0190]

[0191] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for culturing TILs derived from pleural effusion and / or ascites, comprising the steps of: 1) Obtain TIL seed cells from the pleural effusion and / or ascites; 2) The TIL seed cells were sorted to obtain intermediate cells, which were CD45RA cells. - Cells, CD39 + Cells, or CD45RA - CD39 + cell; Optional 3) culture the intermediate cells described in 2) to obtain TIL; Optionally, the pleural effusion and / or ascites is pleural effusion and / or ascites in a patient suffering from one or more of the following cancers: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, colon cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma.

2. The method as described in claim 1, characterized in that, The method includes: obtaining TIL seed cells from the pleural effusion and / or ascites, sorting to obtain CD45RA-TIL cells, and expanding and culturing the CD45RA-TIL cells. Preferably, the method further includes: after obtaining the CD45RA-TIL cells, sorting to obtain CD45RA-CD39+TIL cells, and expanding and culturing the CD45RA-CD39+TIL cells; preferably, the CD45RA-CD39+TIL cells are obtained by sorting using a CD39 antibody.

3. The method as described in claim 1, characterized in that, The process of obtaining TIL seed cells includes the following steps: centrifuging the pleural fluid and / or ascites to precipitate the effusion, washing with physiological saline, centrifuging again to precipitate the effusion, and obtaining the TIL seed cells.

4. The method as described in claim 1, characterized in that, The method further includes step 1.5) between step 1) and step 2): culturing the TIL seed cells. Preferably, the system for culturing the TIL seed cells includes a culture medium suitable for TIL cells; Preferably, the system for culturing the TIL seed cells further includes antibodies and cytokines. Preferably, the system for culturing the seed cells contains any one or more of IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α, and serum.

5. A population of TIL cells obtained by the method described in any one of claims 1-4.

6. A cell cryopreservation formulation comprising a TIL cell population obtained by the method of any one of claims 1-4 and a cryopreservation solution. Preferably, the cryopreservation solution is a cell cryopreservation solution or a tissue cryopreservation solution; Preferably, the cryopreservation solution is a serum-free cryopreservation solution.

7. A pharmaceutical composition comprising a population of TIL cells obtained by the method of any one of claims 1-4, and pharmaceutically acceptable excipients.

8. A method for improving the target cell killing rate of TIL cells, comprising sorting TIL seed cells to obtain CD45RA- cells, CD39+ cells, or CD45RA-CD39+ cells; Preferably, the method includes: The TIL seed cells are negatively sorted to remove CD45RA+ cells, and / or positively sorted with CD39 antibody to obtain TIL cells with improved target cell killing rate. Preferably, the TIL seed cells are obtained from pleural effusion and / or ascites of tumor patients. Preferably, the pleural effusion and / or ascites is pleural effusion and / or ascites from a patient suffering from one or more of the following cancers: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer (including non-small cell lung cancer), melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, colon cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma.

9. The use of the TIL cell population obtained by the method according to any one of claims 1-4 in the preparation of a medicament for treating cancer in patients. The cancers are selected from: gastric cancer, thyroid tumors, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma; preferably, the cancers are selected from: melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and colon cancer.

10. The use of reagents that specifically bind CD45RA and / or specifically bind CD39 in the preparation of a drug or kit, said drug comprising a TIL derived from pleural effusion and / or ascites with enhanced target cell killing rate, said kit for preparing a TIL derived from pleural effusion and / or ascites with enhanced target cell killing rate. Preferably, the reagent that specifically binds to CD45RA is an antibody; Preferably, the reagent that specifically binds to CD39 is an antibody.

11. A kit comprising a reagent that specifically binds to CD45RA and a reagent that specifically binds to CD39.

Images