Improved tumor infiltrating lymphocyte in-vitro amplification method

Abstract

The invention provides an improved in-vitro amplification method of tumor infiltrating lymphocytes. Specifically, the method for preparing the tumor infiltrating lymphocyte (TIL) comprises the following steps: (S1) providing a tumor tissue; (S2) carrying out fragmentation treatment on the tumor tissue to obtain tumor fragments; (S3) pre-culturing the tumor fragments by using a starting culture medium to obtain a first culture mixture; (S4) determining the first culture mixture so as to screen out the tumor reactive TIL; (S5) performing multiplication culture on the tumor reactive TIL by using an amplification culture medium to obtain a second culture mixture; and (S6) isolating or recovering tumor infiltrating lymphocytes (TIL) from the second culture mixture. The invention also provides the high-tumor-reactivity TIL prepared by the method and application of the high-tumor-reactivity TIL.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology; more specifically, the present invention relates to an improved method for expanding tumor infiltrating lymphocytes (TIL) in vitro. Background Art

[0002] Tumor infiltrating lymphocytes (TIL), as the main component of the tumor immune microenvironment, include T cells and natural killer cells (NK cells), and play a key role in the host's tumor immune response. Adoptive cell transfer (ACT) based on TIL refers to an immunotherapy in which TIL is separated from the resected patient's tumor tissue, and TIL is amplified to a certain number in vitro and then infused back into the patient's body to kill tumor cells and induce the host's anti-tumor immune response. The in vitro expansion culture system of TIL is mainly divided into two stages. In the first stage, the tumor tissue obtained from the patient is cut into pieces or prepared into a cell suspension, and then supplemented with exogenous high-dose IL-2 in vitro for expansion and culture for 2-4 weeks. This stage is called pre-REP. The TIL harvested in pre-REP enters the REP stage. TIL is rapidly expanded under the conditions of anti-CD3 antibodies, IL-2, and irradiated allogeneic PBMC to obtain enough TIL to be infused back into the patient's body to achieve the effect of killing tumors. During the 14-day culture period, TIL will expand to 1000-2000 times. By this method, up to 2 × 10 11 TILs are produced for infusion in patients. In recent years, with the continuous deepening and development of TIL tumor suppression mechanisms and expansion systems, the application of TIL therapy in clinical practice has become a research hotspot. According to statistics from the clinical trial website, there are 278 TIL-related clinical trials underway, including 110 phase I clinical trials, 150 phase II clinical trials, 10 phase III clinical trials, and 8 phase IV clinical trials. The subjects of these clinical trials include patients with solid tumors such as malignant melanoma, lung cancer, liver cancer, and refractory gynecological tumors. Among them, TIL adoptive treatment of patients with metastatic melanoma has the best effect. The results of multiple phase I / II clinical trials show that the objective response rate (ORR) of TIL adoptive treatment of melanoma patients is about 50%, and about 20% of the treated patients can observe complete remission (CR). Most patients have no obvious disease progression after 7 years of treatment.

[0003] However, the current conventional TIL expansion culture system has many shortcomings, such as: First, due to the differences in tumor immune infiltration and tumor heterogeneity among patients, the immune cell overflow rate and cell activity of tumor tissues of different patients vary greatly, making it difficult to separate TIL. The first stage of expansion of traditional TIL therapy only provides mitotic signals for T cell expansion by supplementing high-dose recombinant IL-2, which cannot solve problems such as low TIL overflow rate and poor activity. Second, during the process of TIL induced expansion by IL-2 in vitro, there is an aging process of cell differentiation and exhaustion. Exhausted T cells highly express inhibitory receptors such as programmed cell death-1 (PD-1), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and lymphocyte activation gene 3 protein (LAG-3), thereby blocking signaling pathways such as P13K / Akt and Ras / MEK / Erk, affecting cell survival and proliferation ability. These TILs cannot survive for a long time after being infused back into the patient's body, thus greatly reducing the anti-tumor effect in clinical practice.

[0004] Therefore, it is necessary to develop an improved in vitro expansion method of tumor-infiltrating lymphocytes (TILs) to optimize the quality and efficacy of TILs and provide technical guidance for clinical applications. Summary of the invention

[0005] The present invention intends to further improve the current conventional TIL expansion culture system and provide an optimized TIL in vitro expansion culture scheme to increase the yield of functional effector memory CD8+T cells.

[0006] The object of the present invention is to provide an improved method for in vitro expansion of tumor infiltrating lymphocytes (TIL).

[0007] The first aspect of the present invention provides a method for preparing tumor infiltrating lymphocytes (TIL), comprising the following steps:

[0008] (S1) providing tumor tissue;

[0009] (S2) fragmenting the tumor tissue to obtain tumor fragments;

[0010] (S3) pre-culturing the tumor fragments with a starting culture medium to obtain a first culture mixture; (S4) measuring the IFN-γ secretion level of TIL in the first culture mixture to screen out tumor-reactive TIL;

[0011] (S5) performing expansion culture on the tumor-reactive TIL using an expansion medium to obtain a second culture mixture; and

[0012] (S6) Isolating or recovering tumor infiltrating lymphocytes (TIL) from the second culture mixture.

[0013] In another preferred embodiment, the tumor tissue is fresh and not frozen.

[0014] In another preferred embodiment, the tumor tissue is from an animal model.

[0015] In another preferred embodiment, the tumor tissue is from a mammal.

[0016] In another preferred embodiment, the tumor tissue is from human.

[0017] In another preferred embodiment, the tumor tissue comes from a non-human mammal.

[0018] In another preferred embodiment, the tumor tissue is from a primate.

[0019] In another preferred embodiment, the tumor tissue is from a rodent.

[0020] In another preferred embodiment, the tumor tissue is from: human, mouse, rat, rabbit, monkey, or a combination thereof.

[0021] In another preferred embodiment, the TIL cells are from an animal model.

[0022] In another preferred embodiment, the TIL cells are from mammals.

[0023] In another preferred embodiment, the TIL cells are from humans.

[0024] In another preferred embodiment, the TIL cells are from non-human mammals.

[0025] In another preferred embodiment, the TIL cells are from primates.

[0026] In another preferred embodiment, the TIL cells are from rodents.

[0027] In another preferred embodiment, the TIL cells are from: humans, mice, rats, rabbits, monkeys, or a combination thereof.

[0028] In another preferred embodiment, the CD3 is derived from an animal model.

[0029] In another preferred embodiment, the CD3 is from a mammal.

[0030] In another preferred embodiment, the CD3 is from human.

[0031] In another preferred embodiment, the CD3 is from a non-human mammal.

[0032] In another preferred embodiment, the CD3 is from primates.

[0033] In another preferred embodiment, the CD3 is from a rodent.

[0034] In another preferred embodiment, the CD3 is from: human, mouse, rat, rabbit, monkey, or a combination thereof.

[0035] In another preferred example, the starting culture medium includes RPMI 1640 culture medium.

[0036] In another preferred embodiment, the priming medium further comprises a component selected from the following group: HEPES, L-glutamine, serum, 2-mercaptoethanol, or a combination thereof.

[0037] In another preferred embodiment, the priming culture medium further comprises a component selected from the following group: IL-2, anti-PD-1 antibody, CTLA-4 blocker, IL-15, IL-21, IL-10, or a combination thereof.

[0038] In another preferred embodiment, the PD-1 is derived from an animal model.

[0039] In another preferred embodiment, the PD-1 is from a mammal.

[0040] In another preferred embodiment, the PD-1 is from human.

[0041] In another preferred embodiment, the PD-1 is from a non-human mammal.

[0042] In another preferred embodiment, the PD-1 is from primates.

[0043] In another preferred embodiment, the PD-1 is from rodents.

[0044] In another preferred embodiment, the PD-1 is derived from: human, mouse, rat, rabbit, monkey, or a combination thereof.

[0045] In another preferred embodiment, the IL (interleukin) is derived from an animal model.

[0046] In another preferred embodiment, the IL (interleukin) is from a mammal.

[0047] In another preferred embodiment, the IL (interleukin) is from human.

[0048] In another preferred embodiment, the IL (interleukin) is from a non-human mammal.

[0049] In another preferred embodiment, the IL (interleukin) is from primates.

[0050] In another preferred embodiment, the IL (interleukin) is from rodents.

[0051] In another preferred embodiment, the IL (interleukin) is from: human, mouse, rat, rabbit, monkey, or a combination thereof.

[0052] In another preferred embodiment, the IL-2 concentration is 2000 IU / ml to 10000 IU / ml, preferably 4000 IU / ml to 8000 IU / ml, more preferably 6000 IU / ml.

[0053] In another preferred embodiment, the anti-PD-1 antibody concentration is 1-10 ug / ml.

[0054] In another preferred embodiment, the concentration of the CTLA-4 inhibitor is 1-10 ug / ml.

[0055] In another preferred embodiment, the IL-15 concentration is 500-2000 IU / ml.

[0056] In another preferred embodiment, the IL-21 concentration is 500-3000 IU / ml.

[0057] In another preferred embodiment, the IL-10 concentration is 50-500 IU / ml.

[0058] In another preferred embodiment, the final concentration of the anti-PD-1 antibody is 3 to 7 ug / ml, preferably 5 ug / ml.

[0059] In another preferred embodiment, the CTLA-4 inhibitor is 3 to 7 ug / ml, preferably 5 ug / ml.

[0060] In another preferred embodiment, the final concentration of IL-15 is 10-18 IU / ml, preferably 1500 IU / ml.

[0061] In another preferred embodiment, the final concentration of IL-21 is 1500-2800 IU / ml, preferably 2500 IU / ml.

[0062] In another preferred example, the expansion medium includes RPMI 1640 medium.

[0063] In another preferred embodiment, the expansion medium further comprises a component selected from the following group: HEPES, L-glutamine, serum, 2-mercaptoethanol, or a combination thereof.

[0064] In another preferred embodiment, the serum is from an animal model.

[0065] In another preferred embodiment, the serum is from a mammal.

[0066] In another preferred embodiment, the serum is from human.

[0067] In another preferred embodiment, the serum is from a non-human mammal.

[0068] In another preferred embodiment, the serum is from primates.

[0069] In another preferred embodiment, the serum is from rodents.

[0070] In another preferred embodiment, the serum is from: human, mouse, rat, rabbit, monkey, or a combination thereof.

[0071] In another preferred embodiment, the HEPES concentration is 15-35 mM, preferably 20-30 mM, more preferably 22-28 mM, most preferably 25 mM.

[0072] In another preferred embodiment, the L-glutamine concentration is 1-3 mM, preferably 1.5-2.5 mM, more preferably 1.8-2.2 mM, most preferably 2 mM.

[0073] In another preferred embodiment, the serum concentration is 3% to 17%, preferably 5% to 15% mM, more preferably 8% to 12%, and most preferably 10%.

[0074] In another preferred embodiment, the concentration of 2-mercaptoethanol is 5.5×10 -4 M~5.5×10 -6 M, preferably 1.5×10 -5 M~9.5×10 -5 M, more preferably 3×10 -5 M~8×10 -5 M, most preferably 5.5×10 -5 M.

[0075] In another preferred embodiment, the expansion medium further comprises a component selected from the following group: irradiated allogeneic PBMC, 30 ng / ml anti-CD3 antibody (OKT3), 3000 IU / ml IL-2, anti-CD137 antibody (100-1000 ng / ml), 0.05-1 ng / ml TGF-β1, or a combination thereof.

[0076] In another preferred embodiment, the PBMCs are from an animal model.

[0077] In another preferred embodiment, the PBMCs are from mammals.

[0078] In another preferred embodiment, the PBMCs are from humans.

[0079] In another preferred embodiment, the PBMCs are from non-human mammals.

[0080] In another preferred embodiment, the PBMCs are from primates.

[0081] In another preferred embodiment, the PBMCs are from rodents.

[0082] In another preferred embodiment, the PBMCs are from: humans, mice, rats, rabbits, monkeys, or a combination thereof.

[0083] In another preferred embodiment, the PBMCs are allogeneic PBMCs.

[0084] In another preferred embodiment, the PBMCs are irradiated PBMCs.

[0085] In another preferred embodiment, the PBMCs are irradiated allogeneic PBMCs.

[0086] In another preferred embodiment, the PBMC is 10 6 ~10 10 , preferably 10 7 ~10 9 , the most preferred is 0.5x10 9 indivual.

[0087] In another preferred embodiment, the concentration of the anti-CD3 antibody (OKT3) is 10-50 ng / ml, preferably 20-40 ng / ml, most preferably 30 ng / ml.

[0088] In another preferred embodiment, the IL-2 concentration is 100-10000 IU / ml, preferably 1000-8000 IU / ml, more preferably 1500-5000 IU / ml, most preferably 3000 IU / ml.

[0089] In another preferred embodiment, the concentration of the anti-CD137 antibody is 100-1000 ng / ml.

[0090] In another preferred embodiment, the concentration of TGF-β1 is 0.05-1 ng / ml.

[0091] In another preferred embodiment, the final concentration of the anti-CD137 antibody is 200-800 ng / ml, most preferably 500 ng / ml.

[0092] In another preferred embodiment, the final concentration of TGF-β1 is 0.07-0.7 ng / ml, most preferably 0.1 ng / ml.

[0093] The second aspect of the present invention provides a TIL cell, wherein the TIL cell is prepared by the method described in the first aspect of the present invention.

[0094] The third aspect of the present invention provides a use of the TIL cells according to the second aspect of the present invention for preparing a pharmaceutical composition or preparation, wherein the pharmaceutical composition or preparation is used for:

[0095] (a) treating tumors; and / or

[0096] (b) Modulation of immune response.

[0097] In another preferred embodiment, the tumor is a tumor allogeneic to the TIL cells.

[0098] In another preferred embodiment, the tumor is a solid tumor.

[0099] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, head and neck cancer, pancreatic cancer, or a combination thereof.

[0100] The fourth aspect of the present invention provides a cell preparation or a pharmaceutical composition, wherein the cell preparation or the pharmaceutical composition contains the TIL cell population described in the second aspect of the present invention.

[0101] In another preferred embodiment, the cell preparation or pharmaceutical composition comprises the TIL cell population and a pharmaceutically acceptable carrier.

[0102] The fifth aspect of the present invention provides a method for treating a disease, characterized in that a safe and effective amount of the cell preparation or pharmaceutical composition described in the fourth aspect of the present invention is administered to a subject in need, thereby treating the disease of the subject.

[0103] In another preferred embodiment, the disease is a tumor.

[0104] In another preferred embodiment, the tumor is a tumor allogeneic to the TIL cells.

[0105] In another preferred embodiment, the subject is a human.

[0106] In another preferred embodiment, the subject is a non-human mammal.

[0107] In another preferred embodiment, the subject is a rodent.

[0108] In another preferred embodiment, the subject is a primate.

[0109] In another preferred embodiment, the subject is selected from: mice, rats, monkeys, rabbits, or a combination thereof.

[0110] In another preferred embodiment, the administration dose of the cell preparation or pharmaceutical composition is 10 4 ~10 6 Cells / body, or 10 per person 6 ~10 10 cells / 60kg.

[0111] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1 In one embodiment of the present invention, the present invention provides an in vitro expansion scheme for TILs of cancer patients.

[0113] Figure 2 This is the result of the first stage amplification of TIL from a renal cancer patient in Example 1.

[0114] Figure 3 This is a flow cytometry analysis diagram of TIL from a renal cancer patient in Example 1.

[0115] Figure 4 The results show that after co-culture of human renal cancer TIL and tumor cells, TIL has the ability to kill tumor target cells. DETAILED DESCRIPTION

[0116] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly discovered for the first time an improved method for in vitro expansion of tumor-infiltrating lymphocytes (TILs). The method of the present invention uses an optimized priming medium to pre-culture tumor fragments, and screens out TILs with high tumor reactivity by measuring cytokines, and uses an optimized expansion medium to expand and culture tumor-reactive TILs, thereby preparing a large number of tumor-infiltrating lymphocytes (TILs) with high tumor reactivity and high lethality to tumor cells. The present invention was completed on this basis.

[0117] the term

[0118] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0120] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may occur but does not have to occur, may exist but does not have to exist, and may be 1, 2, or 3.

[0121] The terms "administer" or "administration" refer to introducing the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0122] In vitro expansion method of tumor infiltrating lymphocytes (TIL)

[0123] The present invention provides an improved in vitro amplification method of tumor infiltrating lymphocytes (TIL).

[0124] Typically, the method of the present invention comprises the following steps: obtaining tumor tissue, first-stage expansion of TIL, screening of tumor-reactive TIL, TIL rapid expansion stage, and cell collection.

[0125] In the first stage amplification (pre-amplification), it is preferred to use a modified version of the priming medium, which is based on RPMI 1640 medium and contains the following concentration components: 25 mM HEPES, 2 mM L-glutamine, 10% human serum, 5.5×10 -5M 2-mercaptoethanol, 6000IU / ml human recombinant IL-2, 1-10ug / ml anti-PD-1 antibody, 1-10ug / ml CTLA-4 blocker, 500-2000IU / ml IL-15, 500-3000IU / mlIL-21, 50-500IU / ml IL-10.

[0126] In the TIL rapid expansion stage, it is preferred to use a modified expansion medium or culture system, which includes: a certain amount (e.g., 0.1×10 6 -5×10 8 / ml) irradiated allogeneic PBMCs, 10-50ng / ml (such as about 30ng / ml) anti-CD3 antibody (OKT3), 1500-6000IU / ml (such as about 3000IU / ml) IL-2, 100-1000ng / ml anti-CD137 antibody, 0.05-1ng / ml TGF-β1 and TIL complete culture medium.

[0127] Experiments show that the use of the improved TIL rapid expansion culture medium of the present invention can increase the proportion of CD8+T cells in TIL and enhance the ability of TIL products to kill tumor cells.

[0128] In a preferred embodiment of the present invention, the tumor TIL in vitro expansion and culture method of the present invention comprises the following steps:

[0129] (1) Obtaining tumor tissue

[0130] The starting material for TIL expansion is the resected patient tumor. The tumor is removed from as much surrounding tissue as possible, taking care to remove residual skin to avoid microbial contamination.

[0131] (2) The first stage of TIL expansion

[0132] Prepare TIL complete culture medium, which consists of RPMI 1640 medium, 25 mM HEPES, 2 mM L-glutamine, 10% human serum, 5.5 × 10 5 M 2-mercaptoethanol composition.

[0133] The present invention improves the culture medium used for the first stage expansion of TIL, maintains the expansion ability of TIL by adding cytokines required for T cell expansion, adds immunomodulators to improve the immune microenvironment in tumor tissue, optimizes the culture conditions of TIL, and increases the overflow rate of immune cells in tumor tissue.

[0134] Furthermore, based on the complete TIL medium, a modified TIL priming medium was prepared, which contained the following components: RPMI 1640 medium, 25 mM HEPES, 2 mM L-glutamine, 2-10% human AB serum, 5.5 × 10 -5 M 2-mercaptoethanol, 6000IU / ml IL-2, anti-PD-1 antibody (0-500ng / ml), 1-10ug / ml CTLA-4 blocker, 500-2000IU / ml IL-21, 500-3000IU / mlIL-15, 50-500IU / ml IL-10.

[0135] Furthermore, the culture method of step (2) is specifically to cut the tumor harvested in stage 1) into pieces of about 1-3 mm 3 Small fragments of each were placed in a 24-well plate well pre-filled with TIL modified start-up medium. The plate was placed in a cell culture incubator and cultured at 37°C, 5% CO2. During the culture process, TILs covered at least 50% of the bottom of the well. After gently resuspending the medium several times with a 1ml pipette, half (or less) of the medium was aspirated and transferred to one or more new wells, and then fresh complete medium supplemented with 6000IU / ml IL-2 was added to each split well.

[0136] (3) Screening of tumor-reactive TILs

[0137] TILs were harvested 14 days after culture in the first stage, and the IFN-γ secretion level in each well was tested by ELISA kit to evaluate the killing ability of TILs. If the IFN-γ release was greater than 200 pg / mL, the TILs were defined as having specific reactivity, and tumor-reactive TILs were then used for rapid expansion in the next stage.

[0138] (4) TIL rapid expansion stage

[0139] The present invention further improves the rapid expansion system of TIL by adding co-stimulatory molecules to enhance the expression of CD8+ in TIL, thereby improving the ability of TIL to recognize tumor targets and effectively preventing the exhaustion and apoptosis of T cells during rapid expansion.

[0140] Furthermore, a modified TIL rapid expansion system was prepared, comprising the following components: irradiated allogeneic PBMC, 30 ng / ml anti-CD3 antibody (OKT3), 3000 IU / ml IL-2, 100-1000 ng / ml anti-CD137 antibody, 0.05-1 ng / ml TGF-β1, and TIL complete culture medium.

[0141] More specifically, the culture method of stage 4 is to collect the tumor-reactive TILs harvested in stage 3 and transfer them to a G-Rex 100 flask, add a complete cell culture medium containing 3000 IU / ml IL-2, a certain amount (e.g., 0.1×10 7 -5x10 8 / ml) irradiated allogeneic PBMC, 30ng / ml OKT3 antibody, on day 0 of TIL rapid expansion, 100-1000ng / ml exogenous anti-CD137 antibody and 0.05-1ng / ml TGF-β1 were added to the culture flask, and the flask was cultured in a cell culture incubator.

[0142] Further, on the 5th day of culture, new IL-2 was added to the flask to a final concentration of 3000 IU / ml (assuming that all IL-2 had been consumed).

[0143] On day 7, refresh the medium by carefully removing half of the TIL complete medium from the flask without disturbing the cells at the bottom and adding fresh TIL complete cell medium containing 6000 IU / ml IL-2 (final concentration 3000 IU / ml).

[0144] On day 9, new IL-2 was added to the flask to a final concentration of 3000 IU / ml.

[0145] TIL collection is optional starting from day 9.

[0146] On day 12, new IL-2 was added to the flask to a final concentration of 3000 IU / ml.

[0147] 5) Harvest cells and freeze them for direct transfusion into the patient.

[0148] Typically, TILs are harvested and cryopreserved or returned to the patient by day 14 at the latest.

[0149] In some embodiments, the methods of the present invention include the steps of:

[0150] 1) Under sterile conditions, tumor tissue was obtained from the patient, rinsed several times with sterile PBS, and the tumor was removed from as much surrounding tissue as possible, taking care to remove the remaining skin to avoid microbial contamination;

[0151] 2) Prepare a modified version of the priming medium, cut the tumor into small fragments, place each fragment into a 24-well plate pre-filled with culture medium, place the plate in a cell culture incubator, and culture at 37°C, 5% CO2 for 14 days, changing half of the medium every 2-3 days;

[0152] 3) Testing the IFN-γ secretion level of TIL in each well by ELISA kit to screen tumor-reactive TIL;

[0153] 4) preparing a modified expansion culture system in which tumor-reactive TILs are cultured for 14 days, with the medium replaced every 3-5 days;

[0154] 5) Harvest cells and freeze or return them to the patient.

[0155] In some embodiments, the complete TIL culture medium is based on RPMI 1640 medium and contains the following components: 25 mM HEPES, 2 mM L-glutamine, 10% human serum, 5.5×10 -5 M 2-Mercaptoethanol.

[0156] In some embodiments, the improved priming medium consists of TIL complete medium, 6000 IU / ml IL-2, 1-10 ug / ml anti-PD-1 antibody, 1-10 ug / ml CTLA-4 blocker, 500-2000 IU / ml IL-15, 500-3000 IU / ml IL-21, and 50-500 IU / ml IL-10.

[0157] In some embodiments, the final concentration of anti-PD-1 antibody is 5ug / ml, the final concentration of CTLA-4 blocker is 5ug / ml, the final concentration of IL-15 is 1500IU / ml, and the final concentration of IL-21 is 2500IU / ml.

[0158] In some embodiments, the improved expansion culture system consists of irradiated allogeneic PBMCs, 30 ng / ml anti-CD3 antibody (OKT3), 3000 IU / ml IL-2, anti-CD137 antibody (100-1000 ng / ml), 0.05-1 ng / ml TGF-β1, and TIL complete medium.

[0159] In some embodiments, the final concentration of anti-CD137 antibody is 500 ng / ml, and the final concentration of TGF-β1 is 0.1 ng / ml.

[0160] TIL cells

[0161] Tumor infiltrating lymphocytes (TIL) can efficiently identify and kill tumor cells, achieving the effect of cancer treatment.

[0162] TIL is an infiltrating immune cell that is isolated from tumor tissue and responds to tumor cells. These immune cells have the ability to recognize and attack tumor cells, and TIL cells are currently used to treat tumor patients. Compared with other immunotherapies, such as CAR-T cell therapy, TCR-T cell therapy, and PD-1 / PD-L1 immune checkpoint inhibitors, TIL therapy has the advantages of stronger specificity, higher safety, and low recurrence rate after treatment. However, the number of TIL cells in tumor tissue is small and their activity is usually suppressed by the immune microenvironment. In clinical treatment, a very large number of TIL cells must be prepared to obtain the ideal anti-tumor effect. Therefore, TIL therapy needs to be further optimized.

[0163] TGF-β

[0164] Transforming growth factor-β (TGF-β) is a protein superfamily composed of structurally related multifunctional cytokines, including TGF-β1 / 2 / 3 subtypes, activins, bone morphogenetic proteins, and growth differentiation factors. TGF-β superfamily ligands regulate cell functions through seven type I TGF-β superfamily receptors (activin-like kinases-1 to 7) and five type II TGF-β superfamily receptors (TβRII, ActRII, ActRIIB, AMHRII, and BMPRII), playing an important role in embryonic development, tissue repair, and homeostasis of skeletal muscle, cardiovascular, nervous, endocrine, and immune systems.

[0165] TGF-β is a tumor-promoting factor that can induce epithelial-mesenchymal transition (EMT) of tumor cells and promote tumor cell invasion and metastasis. In addition, TGF-β is a key regulator of T cell responses and plays an important role in regulating immune responses. It can regulate the functions of almost every innate and adaptive immune cell, including dendritic cells, B cells, NK cells, innate lymphocytes, and granulocytes. In the late stage of tumors, most tumor cells can secrete TGF-β. Once the concentration of TGF-β increases, it can block the differentiation of immature T cells into Th1 cells and promote their transformation into Treg subsets. At the same time, it inhibits the antigen presentation function of dendritic cells, thereby leading to the immune escape of tumor cells.

[0166] In the present invention, representative methods of inhibiting the TGFβ pathway include, but are not limited to: overexpressing a dominant inhibitory mutant of the TGFβ receptor (dnTGFβRII) or an antibody to TGFβ, knocking down the TGFβ receptor by RNAi, and knocking out the TGFβ receptor by gene knockout (the TGFβ receptor includes TGFβRI and TGFβRII). A representative method of RNAi is to use shRNA targeting the endogenous TGFβRII transcript.

[0167] It should be understood that in the present invention, dnTGFβRII can be replaced by a TGFβR inhibitor (membrane-bound / secreted), an inhibitor of related proteins in the TGFβR signaling pathway / a means of interfering with the expression of related genes, knocking out or knocking down the TGFβ receptor TGFβRII in immune cells, or overexpressing its dominant inhibitory mutant dnTGFβRII, or overexpressing a membrane-bound TGFβ antibody.

[0168] preparation

[0169] The present invention provides a preparation, which contains the TIL cells described in the second aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the immune cells (such as TIL cells) in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0170] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0171] Therapeutic applications

[0172] The present invention provides a method for preventing or treating mammalian tumors, comprising the following steps: administering the TIL cells of the present invention to the mammal.

[0173] Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. Cancer types treated with the TIL cells of the present invention include, but are not limited to, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer, etc.

[0174] Generally, cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method for treating cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of CAR-T cells or enhanced TIL cells of the present invention.

[0175] The TIL cells of the present invention can be used alone, or as a pharmaceutical composition and diluent and / or in combination with other components such as IL-2, IL-17, IL-7, IL-15, IL-21 or other cytokines or cell groups. In short, the pharmaceutical composition of the present invention can be combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0176] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or can be determined by clinical trials.

[0177] When "therapeutically effective amount", "anti-tumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The pharmaceutical composition comprising the TIL cells described herein can be administered in an amount of 10 4 Up to 10 11 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 8 The TIL cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs, and the treatment is adjusted accordingly.

[0178] The administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The composition described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranode, intraspinal, intramuscularly, by intravenous injection or intraperitoneal administration. In one embodiment, the T cell or enhanced TIL cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the TIL cell composition of the present invention is preferably administered by intravenous injection. The composition of TIL cells can be directly injected into a tumor, a lymph node or an infection site.

[0179] In certain embodiments of the present invention, cells activated and expanded using methods described herein or other methods known in the art to expand TIL cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, at the same time, or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the TIL cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell composition of the present invention is administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, at the same time, or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0180] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 5 to 1×10 12 The TIL cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0181] Main advantages of the present invention

[0182] The method of the present invention is simple and can efficiently amplify TIL cells with high killing activity against tumor cells.

[0183] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0184] Example 1

[0185] - Experimental Materials

[0186] RPMI 1640 medium was purchased from GibCo; IL-2 was purchased from Biolegend; β-mercaptoethanol was purchased from Sigma; HEPES and L-glutamine were purchased from Bio-Time; anti-PD-1 antibody, anti-CD3 antibody, and anti-CD137 antibody were purchased from Biolegend;

[0187] 2. Experimental Methods

[0188] Experimental methods see Figure 1 .

[0189] The first stage of TIL expansion:

[0190] Cut the patient's tumor tissue under sterile conditions and cut the tissue into 1-3mm pieces 3 The tumor fragments were then placed in 24-well plates, each well was supplemented with 1.5 ml of TIL complete medium containing 6000 IU / ml recombinant IL-2 and TIL modified start-up medium, the cells were cultured at 37°C and 5% CO2, half of the medium was changed every 2-3 days, and TIL was collected and counted after 2 weeks.

[0191] Screening for tumor-reactive TILs:

[0192] TILs were collected from each well, and TILs and tumor target cells were cultured in a round-bottom 96-well plate at a ratio of 10:1. The supernatant was collected 24 hours later, and the expression of IFN-γ was measured using an ELISA kit. Tumor-reactive TILs were screened for the next stage of expansion based on the secretion level of IFN-γ.

[0193] Rapid expansion culture of TIL (REP):

[0194] In order to further rapidly expand TILs, 5 × 10 6 TIL in a G-REX 100 bottle, add 5.0×10 8 PBMC treated with mitomycin C, 30 ng / ml anti-CD3 antibody, 3000 IU / ml IL-2, supplemented with TIL conditioned medium to 1000 ml, supplemented or not with exogenous anti-CD137 antibody (100-1000 ng / ml).

[0195] On the 5th day of culture, new IL-2 was added to the flask to a final concentration of 3000 IU / ml.

[0196] On day 7, refresh the medium by carefully pipetting half of the TIL complete medium out of the flask without disturbing the cells at the bottom and add fresh TIL complete cell medium containing 6000 IU / ml IL-2.

[0197] On day 9, new IL-2 was added to the flask to a final concentration of 3000 IU / ml.

[0198] TIL collection is optional starting from day 9.

[0199] On day 12, new IL-2 was added to the flask to a final concentration of 3000 IU / ml.

[0200] On day 14, TILs were harvested.

[0201] LDH measures TIL killing ability:

[0202] Prepare 1% conditioned medium (RPMI 1640; calf serum), transfer the TIL cell suspension to a centrifuge tube, centrifuge at 1500 rpm for 5 min, resuspend the cell pellet in conditioned medium containing 1% serum, count and adjust the cell concentration to 2.5×10 6 / ml; discard the culture medium of tumor cell T25 flask, wash twice with PBS, add trypsin to digest for 5min, add 1ml TIL conditioned medium to resuspend, centrifuge at 1500r.pm for 5min, resuspend the cell pellet with conditioned medium containing 1% serum, count and adjust the cell concentration to 2.5×10 5 Pieces / ml;

[0203] The cell suspension and conditioned medium containing 1% serum were added to the 96-well plate, and the edge wells were supplemented with PBS. The 96-well plate was placed in an incubator and cultured for 24 hours.

[0204] After 23 h of incubation in the 96-well plate, 20 μl of LDH release reagent was added to the tumor cell wells.

[0205] After culturing the LDH 96-well plate for 24 hours, centrifuge at 400g for 5 minutes, take 120ul of supernatant from each well into a new 96-well plate, then add 60ul of LDH working solution to each well, and incubate on a shaker for 30 minutes at room temperature in the dark.

[0206] The OD value of each well was measured at 490 nm, and the cell killing rate (%) was calculated according to the formula: ((experimental group release - effector cell spontaneous release - target cell spontaneous release) / (target cell maximum release - target cell spontaneous release)) × 100%

[0208] result

[0209] The results are as follows Figure 2-4 As shown. Among them, after the first stage of TIL pre-amplification, tumor-reactive TIL was screened. After the second stage of amplification, about 92.91% of the large number of tumor-infiltrating lymphocytes prepared were CD4 +T cells.

[0210] In vitro killing experiments show that after 4 hours or 24 hours of co-culturing the TIL prepared by the present invention with tumor cells, the TIL exhibits a high killing ability against tumor target cells.

[0211] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing tumor infiltrating lymphocytes (TIL), characterized in that: The following steps are involved: (S1) providing tumor tissue; (S2) fragmenting the tumor tissue to obtain tumor fragments; (S3) pre-culturing the tumor fragments with a starting culture medium to obtain a first culture mixture; (S4) measuring the IFN-γ secretion level of TIL in the first culture mixture to screen out tumor-reactive TIL; (S5) performing expansion culture on the tumor-reactive TIL using an expansion medium to obtain a second culture mixture; and (S6) Isolating or recovering tumor infiltrating lymphocytes (TIL) from the second culture mixture.

2. The method according to claim 1, characterized in that The priming medium includes RPMI 1640 medium.

3. The method according to claim 2, characterized in that The priming medium further comprises a component selected from the group consisting of HEPES, L-glutamine, serum, 2-mercaptoethanol, or a combination thereof.

4. A TIL cell, characterized in that: The TIL cells are prepared by the method of claim 1.

5. A use of the TIL cell according to claim 4, characterized in that: For preparing a pharmaceutical composition or preparation, the pharmaceutical composition or preparation is used for: (a) treating tumors; and / or (b) Modulation of immune response.

6. The use according to claim 5, characterized in that The tumor is a tumor allogeneic to the TIL cells.

7. The use according to claim 5, characterized in that The tumor is a solid tumor.

8. The use according to claim 7, characterized in that The solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, head and neck cancer, pancreatic cancer, or a combination thereof.

9. A cell preparation or pharmaceutical composition, characterized in that: The cell preparation or pharmaceutical composition contains the TIL cell population according to claim 4.

10. The cell preparation or pharmaceutical composition according to claim 9, characterized in that: The cell preparation or pharmaceutical composition comprises the TIL cell population and a pharmaceutically acceptable carrier.

Images