Tumor infiltrating lymphocyte multiplication culture process

Abstract

The invention relates to a multiplication culture process of tumor infiltration lymphatic (TIL) cells. Specifically, the invention provides a propagation method of TIL cells, which comprises the step of carrying out rapid propagation culture on preliminarily cultured TIL cells in a propagation culture medium. In the TIL cell expanding culture method, hundreds of times of amplification of the TIL cells can be realized in a short time without depending on an activated antibody CD3, high-dose IL-2 and feeder cells, the requirement of TIL cell therapy on the number of the TIL cells clinically can be met, and the TIL cell expanding culture method has a wide application prospect in the field of TIL cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a process for amplifying and culturing tumor-infiltrating lymphocytes. Background Art

[0002] Tumor infiltrating lymphocytes (TIL) can naturally recognize and target tumor cells. Compared with T cells in peripheral blood, TIL is usually rich in tumor antigen-specific T cell clones. As an effective adoptive immunotherapy, TIL has found good therapeutic effects in different cancer types, especially solid tumors. It has been reported that TIL therapy has a positive effect in the treatment of HPV-related cancers, melanoma, lung cancer, breast cancer, etc.

[0003] TIL therapy is a personalized adoptive cell immunotherapy, and the infusion product must be produced according to the specific situation of the patient, which leads to relatively high cost. In addition, highly specialized good manufacturing practice (GMP) facilities and well-trained personnel are required, which is also expensive and time-consuming. Although in the future, with the development of computerized bioreactors, at least some of the processing work of production and personnel can be replaced, the process cannot be fully automated due to the collection of tumor heterogeneous raw materials such as tumor fragments.

[0004] Secondly, the production time of TIL products is long. The total production time of a TIL treatment may require up to 8 weeks of highly personalized, labor-intensive production, which is not conducive to the control of the condition of some patients with rapidly progressive cancers. The process of producing TILs usually starts with a pre-rapid expansion phase (pre-REP), in which TILs dissociate or migrate from tumor fragments and undergo initial expansion. TILs are then further expanded in a rapid expansion phase (REP) in response to stimuli such as IL-2 or feeder cells. Traditional TIL production procedures are used for specific tumor identification and usually take 6-8 weeks.

[0005] Chinese patent application CN110785486A describes a production method for preparing TIL, which uses a G-Rex500MCS system to convert 2.3×10 10 -1.3×10 11 The production cycle of cells of the same order of magnitude was reduced to 22 days. In the TIL production scheme recorded in Chinese patent application CN114686430A, different culture and expansion systems were used, which could expand to 1-2×10 within 27 days. 11 The Chinese patent application CN114908050A describes that the two-end expansion time of TIL is 27 days. How to reduce the production time and reduce the production cost is a major challenge facing TIL therapy.

[0006] In addition, the existing TIL culture scheme uses CD3 antibodies as activation antibodies, but the use of activation antibodies may lead to over-activation of TIL cells, making TIL cells prone to exhaustion. In addition, existing TIL culture usually relies on high concentrations of IL-2, but high-dose IL-2 is associated with serious adverse events and also places higher requirements on cell washing.

[0007] Therefore, there is an urgent need to develop novel TIL cell culture protocols in this field. Summary of the invention

[0008] The purpose of the present invention is to provide a novel TIL cell culture scheme.

[0009] In a first aspect of the present invention, a method for amplifying and culturing tumor infiltrating lymphocytes (TIL) cells is provided, comprising the steps of:

[0010] A. Provide TIL cells after initial culture;

[0011] B. amplifying and culturing the TIL cells in an expansion culture medium, wherein the expansion culture medium contains IL-2; and the expansion culture medium does not contain or substantially does not contain anti-CD3 antibodies;

[0012] C. Harvest TIL cells after expansion culture.

[0013] In another preferred embodiment, in step A, the TIL cells are derived from tumor tissue.

[0014] In another preferred embodiment, the tumor is a solid tumor or a blood tumor, preferably a solid tumor.

[0015] In another preferred embodiment, the preliminary culture comprises: dissociating or separating TIL cells from tumor tissue, and preliminarily amplifying them to obtain a certain number of TIL cells, for example, greater than 1×10 5 TIL cells, preferably greater than 1×10 6 of TIL cells.

[0016] In another preferred embodiment, the preliminary culture is carried out in a cell basal culture medium containing IL-2, and the amount of IL-2 used is 1000-5000 IU / mL, preferably 2000-4000 IU / mL.

[0017] In another preferred embodiment, in step B, the amount of IL-2 in the expansion medium is 100-600 IU / ml, preferably 200-400 IU / ml, more preferably 300±50 IU / ml.

[0018] In another preferred embodiment, the expansion culture medium further comprises the following components: cell basal culture medium, serum replacement, L-glutamine or its replacement.

[0019] In another preferred embodiment, the cell basal culture medium is a culture medium suitable for mammalian cell culture.

[0020] In another preferred embodiment, the cell basal culture medium is selected from the group consisting of RPMI 1640 culture medium, AIM-V TM medium, SFM medium, X-VIVO medium, or a combination thereof.

[0021] In another preferred embodiment, the cell basal culture medium includes Advanced RPMI 1640 culture medium, CTS TM AIM-V TM Culture medium.

[0022] In another preferred embodiment, the cell basal culture medium is Advanced RPMI 1640 culture medium and CTS TM AIM-V TM culture medium 1:1 mixed with the resulting culture medium.

[0023] In another preferred embodiment, the L-glutamine substitute is Gluta Max TM .

[0024] In another preferred embodiment, the Gluta Max in the expansion medium TM The content is 0.5-2%, preferably 1%.

[0025] In another preferred embodiment, the serum substitute is CTS TM Immune cell serum replacement.

[0026] In another preferred embodiment, the CTS in the expansion culture medium TM The content of immune cell serum replacement is 1%-5%, preferably 2.5%.

[0027] In another preferred embodiment, the expansion medium contains or does not contain anti-CD137 antibodies.

[0028] In another preferred embodiment, feeder cells are added or not added during the expansion culture process.

[0029] In another preferred embodiment, no feeder cells are added during the expansion culture process.

[0030] In another preferred embodiment, the feeder cells include but are not limited to: PBMC, K562.

[0031] In another preferred embodiment, the feeder cells are irradiated feeder cells.

[0032] In another preferred embodiment, the ratio of the added feeder cells to the TIL cells is (5-100):1, preferably (10-100):1.

[0033] In another preferred embodiment, no feeder cells are added during the expansion culture process.

[0034] In another preferred embodiment, the expansion culture is carried out for 5-14 days, preferably 6-12 days, and more preferably 7-10 days.

[0035] In another preferred embodiment, the temperature of the amplification culture is 37°C±2°C, preferably 37°C±1°C.

[0036] In another preferred embodiment, the concentration of CO2 in the expansion culture is 3-8%, preferably 4-6%, and more preferably 5%.

[0037] In another preferred embodiment, the amplification culture is further divided into the steps of:

[0038] B1. Static culture for 2-5 days, preferably 3-4 days;

[0039] B2. Dynamic culture for 3-7 days, preferably 3-6 days.

[0040] In another preferred embodiment, the static culture and the dynamic culture use the same culture medium.

[0041] In another preferred embodiment, the static culture is carried out in the presence or absence of feeder cells.

[0042] In another preferred embodiment, the dynamic culture is performed without adding feeder cells.

[0043] In another preferred embodiment, the static culture is carried out in a container selected from the group consisting of a breathable culture flask, a cell culture bag, a culture dish, and a cell plate.

[0044] In another preferred embodiment, the breathable culture bottle is a bottom breathable culture bottle.

[0045] In another preferred embodiment, the dynamic culture is carried out under conditions selected from the group consisting of: a bioreactor, a cell expansion system, a shaker, an automated cell processing instrument, such as a fully automated cell processing instrument.

[0046] In another preferred embodiment, the dynamic culture is carried out in a bioreactor.

[0047] In another preferred embodiment, the parameters of the bioreactor include: a rocking speed of 2-15 rpm and a rocking angle of 2-10°.

[0048] In another preferred embodiment, during the expansion culture process, the cells are replenished with fluid every 1-3 days.

[0049] In another preferred embodiment, during the expansion culture process, the cell density is 0.1-1×10 6 / ml, more preferably 0.1-0.5×10 6 Pieces / ml.

[0050] In another preferred example, in step C, the harvested TIL cells are expanded ≥200 times, preferably ≥300 times, more preferably ≥400 times, and more preferably ≥500 times compared to the cells in step A.

[0051] In another preferred example, compared with a control group to which CD3 antibodies were added and other conditions were the same, the total number of TIL viable cells harvested by the method was increased by ≥1.4 times, preferably ≥1.5 times, and more preferably ≥1.6 times.

[0052] In another preferred example, compared with a control group to which CD3 antibodies were added and other conditions were the same, the proportion of Tscm in TILs harvested by the method was increased by ≥1.2 times, preferably ≥1.25 times, and more preferably ≥1.3 times.

[0053] In another preferred example, compared with a control group to which CD3 antibodies were added and other conditions were the same, the increase in INF-γ secretion of TILs harvested by the method was ≥1.1 times, preferably ≥1.15 times, and more preferably ≥1.2 times.

[0054] In another preferred embodiment, in step C, the cell viability of the harvested TIL cells is ≥95%.

[0055] In another preferred embodiment, the method further comprises the steps of:

[0056] D. Wash, purify and freeze the harvested TIL cells.

[0057] In another preferred embodiment, in step D, the cell washing solution contains IL-2.

[0058] In another preferred embodiment, the dosage of IL-2 is 100-1000 IU / ml, preferably 200-800 IU / ml, more preferably 600 U / ml.

[0059] In another preferred embodiment, the cell washing solution further comprises the following components: compound electrolyte injection solution and human serum albumin.

[0060] In another preferred embodiment, the content of human serum albumin in the cell washing solution is 0.1-10% (wt), preferably 0.5-5%, and more preferably 1%.

[0061] In another preferred embodiment, the cell washing and purification is performed for 2-5 rounds, more preferably 3 rounds.

[0062] In another preferred embodiment, in step D, the freezing solution used for freezing comprises the following components: cell washing solution, CS10.

[0063] In another preferred embodiment, the freezing solution is a cell washing solution and Cryopreservation solution obtained by mixing 1:1 with CS10.

[0064] In a second aspect of the present invention, a tumor infiltrating lymphocyte (TIL) cell is provided, wherein the TIL cell is obtained by culturing the method described in the first aspect of the present invention.

[0065] In the third aspect of the present invention, a pharmaceutical composition or preparation is provided, comprising: (a) the TIL cells described in the second aspect of the present invention; and (b) a pharmaceutically acceptable carrier, excipient or diluent.

[0066] In a fourth aspect of the present invention, there is provided a use of the TIL cells according to the second aspect of the present invention for preparing a drug for treating a disease.

[0067] In another preferred embodiment, the disease is a tumor.

[0068] In another preferred embodiment, the tumor is a solid tumor or a blood tumor, preferably a solid tumor.

[0069] In another preferred embodiment, the tumor includes: melanoma, breast cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer, or a combination thereof.

[0070] In the fifth aspect of the present invention, a TIL cell expansion medium is provided, wherein the expansion medium contains IL-2; and the expansion medium does not contain or substantially does not contain anti-CD3 antibodies.

[0071] In another preferred embodiment, the amount of IL-2 in the expansion medium is 100-600 IU / ml, preferably 200-400 IU / ml, more preferably 300±50 IU / ml.

[0072] In another preferred embodiment, the expansion culture medium further comprises the following components: cell basal culture medium, serum replacement, L-glutamine or its replacement.

[0073] In another preferred embodiment, the cell basal culture medium is a culture medium suitable for mammalian cell culture.

[0074] In another preferred embodiment, the cell basal culture medium is selected from the group consisting of RPMI 1640 culture medium, AIM-V TM medium, SFM medium, X-VIVO medium, or a combination thereof.

[0075] In another preferred embodiment, the cell basal culture medium includes Advanced RPMI 1640 culture medium, CTS TM AIM-V TM Culture medium.

[0076] In another preferred embodiment, the cell basal culture medium is Advanced RPMI 1640 culture medium and CTS TM AIM-V TM culture medium 1:1 mixed with the resulting culture medium.

[0077] In another preferred embodiment, the L-glutamine substitute is Gluta Max TM .

[0078] In another preferred embodiment, the Gluta Max in the expansion medium TM The content is 0.5-2%, preferably 1%.

[0079] In another preferred embodiment, the serum substitute is CTS TM Immune cell serum replacement.

[0080] In another preferred embodiment, the CTS in the expansion culture medium TM The content of immune cell serum replacement is 1%-5%, preferably 2.5%.

[0081] In another preferred embodiment, the expansion medium contains or does not contain anti-CD137 antibodies.

[0082] 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

[0083] Figure 1 A comparison chart of cell proliferation multiples on day 7 of culture in Example 1 is shown.

[0084] Figure 2 A comparison chart of the total number of cells on the 7th day of expansion culture in Example 1 is shown.

[0085] Figure 3 The INF-γ secretion level of cells at day 7 of culture in Example 1 is shown. DETAILED DESCRIPTION

[0086] After extensive and in-depth research, the inventors have developed a TIL cell expansion method that can achieve a large number of TIL cells in a short period of time. In the TIL cell expansion method of the present invention, the activation antibody CD3 antibody is not added to avoid over-activation of TIL cells; the expansion method of the present invention reduces the amount of IL-2, reduces side effects and safety risks; the expansion method of the present invention does not rely on feeder cells. The TIL cell expansion method of the present invention can achieve hundreds of times of expansion of TIL cells in as short as 7-8 days, which can meet the clinical demand for the number of TIL cells in TIL cell therapy, and the prepared TIL cells have a high level of stemness and INFγ secretion ability. The present invention was completed on this basis.

[0087] definition

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0089] As used herein, the term "tumor infiltrating lymphocytes" or "TIL cells" is a type of immune cell present in the tumor microenvironment. TIL cells have significant advantages in the field of cancer treatment. First, they can be directly extracted from the patient's body and re-injected into the patient's body after in vitro amplification and activation, thereby achieving personalized treatment. Secondly, since TIL cells are derived from the patient himself, there is no risk of allogeneic rejection and the safety is higher. In addition, TIL cell therapy has shown significant therapeutic effects on certain refractory tumors or advanced tumors in clinical trials.

[0090] As used herein, the term "IL-2" or "interleukin 2" is a cytokine that plays an important role in regulating immune responses, promoting cell proliferation and differentiation, etc. IL-2 in the present invention includes all forms of IL-2, such as IL-2 derived from humans or other mammals, and includes IL-2 wild type and mutant types, as long as the mutant type has an activity similar to that of the wild type. In the present invention, IL-2 includes recombinant human IL-2.

[0091] In some embodiments of the present invention, the "anti-CD3 antibody" refers to any form of antibody targeting CD3, such as an anti-CD3 monoclonal antibody, and its source is not limited.

[0092] In some embodiments of the present invention, the "anti-CD137 (4-1BB) antibody" refers to an anti-CD137 monoclonal antibody, the source of which is not limited, for example, it can be self-developed or commercially available. In a preferred embodiment, the anti-CD137 monoclonal antibody is a commercially available antibody.

[0093] As used herein, the term "feeder cell" generally refers to a cultured cell that grows in vitro and secretes at least one factor into the culture medium and can be used to support the growth of another cell of interest. In one embodiment, the feeder cell may include PBMC.

[0094] As used herein, the term "CD3+ cells" refers to CD3-positive cells in TIL cells, which represent mature T cells and thymocytes infiltrating the tumor, including helper T cells, regulatory T cells, and killer T cells.

[0095] As used herein, the term "CD8+ cells" refers to CD8-positive cells in TIL cells, which represent cytotoxic T cells or cytotoxic CD8+ T lymphocytes (CTLs), which are key components of the adaptive immune system and play an important role in the immune system's defense against pathogens.

[0096] As used herein, the term "CD4+ cells" refers to CD4-positive T lymphocytes in TIL cells, which represent helper T cells, including subtypes of Th-1, Th-2, Th-9, Th-17, regulatory T cells and follicular helper T cells, etc.

[0097] As used herein, the term "memory T cells" refers to a type of cell formed by initial cells after receiving antigen stimulation, which has the ability to survive for a long time and respond quickly to the same antigen again. It can be divided into effector memory T cells (T EM ), central memory T cells (T CM ), stem cell memory T cells (T SCM ) etc. SCM The proportion of T cells characterizes the stemness of the T cell population. SCM The higher the proportion, the stronger the stemness of the T cell population.

[0098] Culture of TIL cells

[0099] The culture of TIL cells refers to the process of isolating TIL cells from tumor tissues of tumor patients and amplifying them in large quantities to obtain a therapeutically effective amount of TIL cells. It usually requires two stages: primary culture and expansion culture.

[0100] As used herein, the term "primary culture" is also called "initial culture" or "pre-REP (before rapid expansion)", which refers to the isolation and first-stage culture of TIL cells in tumor tissue to obtain a certain number of TIL cells for subsequent rapid expansion.

[0101] The TIL cells before TIL cell culture begins are usually located in the patient's tumor tissue mass, and it is necessary to first obtain the tumor tissue from the patient, for example, by surgery, puncture, etc. The tumor is preferably a solid tumor. The tumor tissue block is cut into pieces, cleaned, etc., and placed in the primary culture medium for culture, for example, for about 7 days, to obtain the primary culture cell population. Usually the components of the primary culture medium include: cell basal culture medium, IL-2, serum substitute, L-glutamine or its substitute, buffer.

[0102] In the present invention, the method of the initial cultivation stage is not limited, and a conventional initial cultivation method known in the art can be used for the initial cultivation.

[0103] As used herein, the terms "expansion culture", "expanded culture", "rapid expansion culture" and "REP" are used interchangeably and refer to the expansion culture of initially cultured cells to obtain a large number of TIL cells in a shorter period of time to meet therapeutic purposes.

[0104] TIL cell expansion method of the present invention

[0105] In the present invention, a method for expanding TIL cells is provided, which can obtain TIL cells with hundreds of times of expansion in a short time. The expansion method of the present invention does not rely on activation antibodies, high-dose IL-2 and feeder cells, adopts reasonable factor concentrations and culture conditions, satisfies rapid cell proliferation, and obtains TIL cells with high expansion multiples.

[0106] The expansion culture of the present invention is carried out in the expansion culture complete medium of the present invention. The expansion culture complete medium of the present invention comprises the following components: IL-2, cell basal culture medium, serum replacement, L-glutamine or its replacement.

[0107] IL-2 is a potent T cell mitogenic cytokine that stimulates the growth and survival of effector T cells, and the addition of IL-2 to cultured lymphocytes promotes lymphocyte lysis of autologous tumor cells. In addition, the combination of IL-2 and TIL infusion can improve its therapeutic effect. However, the administration of high-dose IL-2 as standard therapy to support the growth and activity of infused TILs may inhibit the clinical application of TIL therapy.

[0108] High-dose IL-2 can promote regulatory T cells that inhibit the anti-tumor response of TILs. However, high-dose IL-2 is associated with serious adverse events, including capillary leak syndrome, which is characterized by hypotension, oliguria, edema, and even hypovolemic shock.

[0109] In the present invention, the dosage of IL-2 is reduced, and only 100-600 IU / ml is added to the complete culture medium for culturing. Preferably, the dosage of IL-2 is 200-400 IU / ml, and more preferably 300 IU / ml.

[0110] In a preferred embodiment, the cell basal medium of the present invention is a medium suitable for mammalian cell culture, such as RPMI 1640 medium, AIM-V TM In a preferred embodiment, the cell basal culture medium of the present invention is Advanced RPMI 1640 culture medium and CTS TM AIM-V TM culture medium 1:1 mixed with the resulting culture medium.

[0111] In a preferred embodiment, the contents of other components in the complete culture medium of the present invention are as follows: 0.5-2% of L-glutamine substitute, preferably the L-glutamine substitute is Gluta Max TM ; 1%-5% serum replacement, such as CTS TM Immune cell serum replacement.

[0112] In the present invention, the complete culture medium may or may not contain anti-CD137 antibodies; when CD137 antibodies are contained, the preferred concentration is 1-10 μg / ml, such as 5 μg / ml.

[0113] In the present invention, feeder cells may or may not be added during the expansion stage. Feeder cells include feeder cells commonly used in the art, especially irradiated feeder cells. Preferred feeder cells are PBMCs. When feeder cells are added, the ratio of feeder cells to TIL cells is (10-100):1.

[0114] In a preferred embodiment, the expansion culture is carried out for 6-14 days, preferably 6-12 days, more preferably 7-10 days, such as 7 days, 8 days, 9 days or 10 days.

[0115] In a preferred embodiment, the temperature of the expansion culture is 37°C±1°C, preferably 37°C±0.5°C.

[0116] In a preferred embodiment, the concentration of CO2 in the expansion culture is 3-8%, preferably 4-6%, and more preferably 5%.

[0117] The container used in the expansion stage of the present invention is not limited, and for example, the expansion stage can be carried out in a bioreactor.

[0118] The following is an example of the expansion step in a bioreactor to illustrate the expansion method of the present invention. The process includes: TIL expansion (divided into TIL static activation and TIL dynamic culture), cell washing, and cell cryopreservation preparation.

[0119] 1. TIL static activation

[0120] 1.1.1. Preparation of cell washing solution

[0121] 1.1.1.1. Prepare a compound electrolyte injection solution containing 0-1000 IU / ml recombinant human interleukin-2 for injection and 0.1-10% human serum albumin.

[0122] 1.1.1.2. After preparation, filter the solution through a 0.22μm capsule filter into a disposable liquid preparation bag and use it as cell washing solution.

[0123] 1.1.2. Preparation of complete culture medium for expansion

[0124] 1.1.2.1. Prepare a 1:1 mixed culture medium of Advanced RPMI 1640 and CTS AIM-VMedium containing 0.1-1% Gluta Max, 0.1-2.5% CTS Immune Cell Serum Replacement, and 100-300 IU / ml recombinant human interleukin-2 for injection.

[0125] 1.1.2.2. After preparation, filter the culture medium through a 0.22 μm capsule filter into a disposable liquid preparation bag and reserve it as complete culture medium for expansion.

[0126] 1.2. Feeder Cell Recovery

[0127] 1.2.1. Use a constant temperature water bath to revive the feeder cells.

[0128] 1.2.3. Reagents for preparation

[0129] 1.2.3.1. Aseptically aliquot cell washing solution and expansion complete culture medium as washing solution for Sepax C-Pro cell processor.

[0130] 1.2.3.2. Sterile packaging The complete culture medium is used as the final bag of the Sepax C-Pro cell processor for rapid dilution of washed feeder cells.

[0131] 1.2.4. Complete the kit assembly.

[0132] 1.2.5. Install the kit according to the Sepax C-Pro cell processor equipment requirements.

[0133] 1.2.6. Parameter settings

[0134] 1.2.6.1. Click the application and set key operating parameters. See Table 1 for specific parameters.

[0135] Table 1: Feeder cell washing parameters

[0136] parameter Numeric Initial volume (20-1200ml) 25-200ml Detect initial volume no Input bag rinsing yes Input bag rinsing volume Initial bag rinsing volume 25-100ml Pause input bag rinsing yes Optical cell detection yes Product filling speed Final product filling speed 17-120ml / min Intermediate volume 5-50ml Pre-wash cycles 1-3 times Pre-wash g-force 200-800g Pre-wash sedimentation time 120-600s Switch washing solution no Final volume 50-500ml

[0137] 1.2.7. Set up the cell washing program, enter the tracking information, suspend the instrument operation, and prepare the initial bag.

[0138] 1.2.8. Initial bag preparation

[0139] 1.2.8.1. Take the required feeder cells (PBMC, K562, etc.) from the liquid nitrogen tank, the ratio of feeder cells to TIL cells is (5-100):1, preferably (10-100):1, and place them in a 37°C digital constant temperature water bath for recovery.

[0140] 1.2.8.2. After thawing, connect the feeder cells to the cell washing solution in 1.2.3.1 and a new storage bag. Use the cell washing solution to dilute and transfer the feeder cells to the new storage bag as the initial bag for the Sepax C-Pro cell processor.

[0141] 1.2.9. Connect the initial bag to the kit and click on the Sepax C-Pro cell processor screen to confirm the program execution and start the feeder cell wash.

[0142] 1.2.10. Cleaning procedures include

[0143] 1.2.10.1. Prime the kit and fill the cavities with the initial bag contents.

[0144] 1.2.10.2. Rinse the initial bag. All the liquid in the initial bag and the pre-washing liquid enter the separation chamber for centrifugation, and then discharge the centrifugal supernatant to the initial bag to rinse the initial bag, and then all the liquid enters the separation chamber.

[0145] 1.2.10.3. Concentration: After centrifugation in the separation chamber for 2-10 minutes, discharge the supernatant except the intermediate volume into the waste liquid bag.

[0146] 1.2.10.4. Washing: The washing liquid enters the separation chamber, and after centrifugation for 2-10 minutes, the supernatant except the intermediate volume is discharged into the waste liquid bag. Before the last round of washing, the washing liquid is manually switched from the front washing liquid to the rear washing liquid.

[0147] 1.2.10.5. Resuspend cells and flush the cavity. The separation cavity discharges the remaining cell suspension (volume equal to the intermediate volume) into the final product bag. Then the post-washing solution enters the separation cavity to flush 1-5 times. After flushing, the suspension is transferred to the final product bag and replenished to the final volume.

[0148] 1.3.TIL activation

[0149] 1.3.1. Connect the permeable culture flask containing primary culture cells to the feeder cell product. Add the feeder cells into the permeable culture flask.

[0150] 1.3.2. Transfer the complete culture medium into a breathable culture bottle. Place the breathable culture bottle in an incubator at 37°C and 5% CO2 to start static activation.

[0151] 1.3.3. Every 2 days after activation, use a sterile pipetting machine to connect the breathable culture bottle to the liquid storage bag, pump out 0-800g of culture supernatant into the liquid storage bag, mix the remaining cell culture fluid, take samples and count them, and then add fluid to a total volume of 1kg (1L). This process lasts for 2-4 days.

[0152] 2.1. After static activation, use a sterile pipetting machine to connect the breathable culture bottle to the liquid storage bag, transfer the upper culture medium into the liquid storage bag, mix the remaining cell culture medium, and take samples for counting.

[0153] 2.2. Open the bioreactor (such as the Xuri cell expansion system or other bioreactor), install the cell culture bag and start operation. Connect the breathable culture flask to the cell culture bag and the expansion complete medium. Inoculate the cell suspension into the cell culture bag; calculate the total weight added by weighing, and use the expansion complete medium to fill the initial volume of the cell culture bag to its culture density of 0.1-1×10 6 cells / ml.

[0154] 2.3. Set the culture parameters as shown in Table 2, where the rocking speed is adjusted according to the culture volume, see Table 3 for details.

[0155] Table 2: Bioreactor parameter setting ranges

[0156] Swing speed 2-15rpm Swing Angle 2-10° <![CDATA[CO2 ratio]]> 3-8％ <![CDATA[O2 ratio]]> 15-50％ temperature 36.5-37.0℃ Gas flow rate 0.05-0.5L / min

[0157] 2.4. After entering the dynamic culture process, take samples and count them every day, and then add complete culture medium to the cell culture bag to a density of 0.1-1×10 6 cells / ml, and record the actual daily replenishment volume. When the maximum culture volume (10 L) is reached, no more replenishment will be performed.

[0158] 2.5. Dynamic amplification culture for 3-7 days, sample the cell suspension every day for testing, and decide whether to harvest the cells based on the test results. When the total cell count is greater than or equal to 1×10 10 cells, the expansion process can be ended and the cell washing process can be started.

[0159] 3. Cell Washing

[0160] 3.1. Cleaning and purification

[0161] 3.1.1. Take a 750-type or 500-type cryopreservation bag as preparation bag A, and weigh the empty weight for subsequent weighing of preparation bag A;

[0162] 3.1.2. Open the LOVO Cell Processing System (LOVO cell processing and purification instrument, hereinafter referred to as LOVO) and set the parameters. Wash and collect 60ml-300ml of cell suspension, freeze 1-5 bags, 100±10ml / bag;

[0163] 3.1.2.1. One mother liquid container, the volume of each container is 0-22000ml, the cell backlog of the mother liquid is 0-3%, the number of washes is 2-5 times, the rotator idle speed is 600rpm, and the consumables are pre-flushed with the selected cell washing solution;

[0164] 3.1.2.2. Mother liquor pre-washing times: 1 time, mother liquor pre-washing volume: 0-50ml, mother liquor pre-washing liquid injection speed: 50ml / min;

[0165] 3.1.2.3. The flushing volume of the mother liquid bag is 0-200ml, the pre-flushing volume of the top pipeline of bag No. 2 is 0-30ml, the pre-flushing volume of the bottom pipeline of bag No. 2 is 0-20ml, and the post-flushing volume of the bottom pipeline of bag No. 2 is 0-20ml;

[0166] 3.1.2.4. Final product volume 60-300ml, final product liquid inlet speed 150ml / min;

[0167] 3.1.2.5. No incubation stage needs to be set;

[0168] 3.1.2.6. Select cell washing solution, the liquid inlet speed of the rotator is 60-160ml / min, and the liquid retention speed is 7-100ml / min;

[0169] 3.1.2.7. The cell backlog at the rotor inlet is ≤20.0%, the rotor speed is 2000-4000rpm, the cell washing bag is selected for the washing and dilution solution, and the dilution volume of bag No. 2 is 200-800ml;

[0170] 3.1.3. Collect the cell suspension in preparation bag A and remove the heat-sealed tube for later use.

[0171] 4. Cell Cryopreservation Preparation

[0172] 4.1. Filling of preparations

[0173] 4.1.1. Connect the preparation bag A to a sampler and draw about 3 ml of cell suspension for testing;

[0174] 4.1.2. Dispense the same volume of CryoStor CS10 cell freezing solution (hereinafter referred to as freezing solution) into another freezing bag for standby use, with mass (g): volume (ml) = 1:1;

[0175] 4.1.3. Mix the cell suspension and cryopreservation solution in a 1:1 ratio to form the preparation solution. After mixing, divide the preparation solution into 1-5 cryopreservation bags according to the injection specifications (100±10ml / bag);

[0176] 4.1.4. After packaging, vent and seal each bag of preparation. After passing the light inspection, put it into an outer protective bag, evacuate and seal it, and then put it into a blood bag box pre-cooled at 2-8℃ for more than 30 minutes;

[0177] 4.1.5. The remaining preparation liquid is divided into 50 cryopreservation bags according to the sample specifications for inspection (10±2ml / bag) for post-cryopreservation resuscitation testing, and placed in the blood bag box as in 4.1.4. The remaining preparation liquid is taken out with a 10ml disposable sterile screw-mouth syringe for cell counting and inspection;

[0178] 4.1.6. Transfer the blood bag box containing the preparation liquid bag to the programmed cooling device for programmed cooling.

[0179] 4.2. Cooling and freezing

[0180] 4.2.1. Place the finished product in a programmed cooling device for programmed cooling. The cooling procedure is as follows;

[0181] 4.2.1.1. Step 1: Wait at 20.0℃;

[0182] 4.2.1.2. Step 2: Cool the sample to -6°C at a cooling rate of 1°C / min;

[0183] 4.2.1.3. Step 3: Cool the chamber to -50°C at a cooling rate of 25°C / min;

[0184] 4.2.1.4. Step 4: Raise the temperature of the chamber to -14°C at a rate of 10°C / min;

[0185] 4.2.1.5. Step 5: Cool the chamber to -45°C at a cooling rate of 1°C / min;

[0186] 4.2.1.6. Step 6: Cool the chamber to -90°C at a cooling rate of 10°C / min;

[0187] 4.2.2. After cooling, remove the blood bag box and transfer it to a gas phase liquid nitrogen tank (≤-150℃) for storage.

[0188] TIL cells of the present invention and their applications

[0189] The present invention also provides TIL cells cultured using the expansion method of the present invention, a pharmaceutical composition or preparation containing the TIL cells of the present invention, and uses thereof.

[0190] The TIL cells cultured by the expansion method of the present invention have high expansion multiples, high cell viability and good stemness (for example, T SCM The proportion of cells is high), which can meet the clinical application of TIL.

[0191] As used herein, the term "pharmaceutical composition" or "formulation" generally refers to a preparation that refers to a mixture of at least one cell and at least one and optionally more than one other pharmaceutically acceptable chemical component such as a carrier, stabilizer, diluent, dispersant, suspending agent, thickening agent and / or excipient.

[0192] In the present application, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the active ingredient. For example, a pharmaceutically acceptable carrier may not interfere with the biological activity of the active ingredient; for example, a pharmaceutically acceptable carrier may not interfere with the effectiveness of the biological activity possessed by the active ingredient. Such preparations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that may be used in the formulations described herein may include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), and the like. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art.

[0193] In the present invention, the TILs amplified using the method of the present invention can be administered to patients as a drug. In some embodiments, the pharmaceutical composition can be a suspension of TILs in a sterile buffer. The TILs obtained using the expansion method of the present invention can be administered by any suitable route known in the art.

[0194] The main advantages of the present invention include:

[0195] 1. The expansion method of the present invention shortens the time of TIL culture. The expansion stage can be expanded by about 500 times in 7-10 days, meeting clinical demand.

[0196] 2. The expansion culture method of the present invention optimizes and reduces the amount of IL-2 used in the culture process, reducing clinical adverse reactions caused by excessively high IL-2 concentrations.

[0197] 3. The expansion method of the present invention does not use CD3 monoclonal antibodies, thus avoiding excessive activation of TIL cells and improving the stemness and persistence of TIL cells.

[0198] 4. The expansion culture method of the present invention uses serum-free culture medium for culture, which reduces the risk of serum contamination, avoids the defects of poor serum batch stability, potential virus contamination and high cost, and improves the safety of cell culture.

[0199] 5. The present invention realizes the preparation of fully enclosed preparations by washing and filling. According to the total amount of cells cultured, it is divided into a variety of schemes for freezing 1-5 bags, so that the expanded cells meet the parameter requirements during washing and purification, and the density of frozen cells is guaranteed to be 1-30×10 7 range to avoid excessive infusion of cryopreservation fluid into the patient.

[0200] 6. The TIL cells prepared by the present invention have a high expansion multiple, a high viability and a high level of stemness, and the TIL cell preparation can be stored in liquid nitrogen for a long time and has a flexible use time.

[0201] 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 according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0202] Example 1: TIL expansion method with and without CD3 monoclonal antibody

[0203] In this example, the comparison of TIL cells obtained by culturing with or without adding CD3 monoclonal antibody during the expansion stage was verified under the same other conditions. The specific method is as follows:

[0204] Source of TIL cells: Three batches of TIL cells, LA2023062701, LA2023070501, and LA2023061401, were selected. Each batch of TIL cells came from a tumor patient, all of whom were lung cancer patients.

[0205] Initial culture of TIL cells: TIL cells were isolated from tumor tissues and cultured in a cell basal medium containing 3000 IU / ml IL-2 to obtain a number of 5×10 7 The above TIL cells are used for subsequent expansion.

[0206] Complete culture medium: Prepare a 1:1 mixed culture medium of Advanced RPMI 1640 and CTS AIM-VMedium containing 1% Gluta Max, 2.5% CTS Immune Cell Serum Replacement, 300 IU / ml recombinant human interleukin-2 for injection as complete culture medium.

[0207] Experimental group: cultured with the above-mentioned complete culture medium;

[0208] Control group: 30 ng / ml CD3 monoclonal antibody (OKT3) was added to the above culture medium. Other than that, the subsequent culture conditions of the experimental group and the control group were the same. The specific steps are as follows:

[0209] 1. Day 0 of expansion culture

[0210] 5.00×10 TIL cells were collected from each group after initial culture. 7 According to the culture conditions of the experimental group and the control group, static activation and expansion culture were performed respectively. The specific operations are as follows:

[0211] 1.1 Record the cells at the end of the initial culture: the total number of viable cells inoculated (5.00×10 7 individuals / group), inoculation volume, and cell viability.

[0212] 1.2 Feeder cells were washed. After washing feeder cells in the control group, 30 μg of CD3 monoclonal antibody (OKT3) was added to the final product bag, while no addition was made in the experimental group.

[0213] 1.3 After recording the feeder cell information, heat-seal the PVC tube of the feeder cell bag with the C-port long tube of the cell culture device G-Rex100M-CS. Open the Robert clamps of the PVC tube of the feeder cell bag and the C-port long tube of G-Rex 100M-CS, and place the 1.00×10 9 The feeder cells were respectively inoculated into G-Rex 100M-CS according to gravity.

[0214] 1.4 Fluid replenishment: Both the control group and the experimental group were replenished by gravity, so that the initial culture weight of G-Rex 100M-CS was 1000 g.

[0215] 1.5 Place two groups of G-Rex 100M-CS in a carbon dioxide incubator (37°C, 5% CO2) for culture.

[0216] 2. Day 2 of expansion

[0217] 2.1 Use a sterile pipetting machine to heat seal the disposable sampling bag with two sets of G-Rex 100M-CS C-port long tubes. Pump air in through a peristaltic pump and use the positive pressure in the tank to drain 500g of culture supernatant into the disposable sampling bag.

[0218] 2.2 Take samples and count the two groups of cells separately: Count the samples twice according to the "Standard Operating Procedures for Cell Sampling and Counting", and record the average of the two counts, including the viable cell density, viability and cell diameter. Calculate the volume of the cell suspension in the tank and the total number of viable cells.

[0219] 2.3 Use gravity to let REP-CM flow out of the liquid storage bag and replenish the liquid in G-Rex 100M-CS respectively, so that the weight of the two groups of cell culture is 1000g.

[0220] 2.4 The two groups of G-Rex 100M-CS were placed in a carbon dioxide incubator (37°C, 5% CO2) and cultured until the fourth day.

[0221] 3. Day 4 of expansion

[0222] 3.1 Use a sterile pipe connection machine to heat seal the disposable sampling bag with two sets of G-Rex 100M-CS C-port long tubes. Pump air in through a peristaltic pump and use the positive pressure in the tank to drain 700g of culture supernatant into the disposable sampling bag.

[0223] 3.2 Take samples and count the two groups of cells separately: Count the samples twice according to the "Standard Operating Procedures for Cell Sampling and Counting", and record the average of the two counts, including the viable cell density, viability and cell diameter. Calculate the volume of the cell suspension in the tank and the total number of viable cells.

[0224] 3.3 After completing the installation and initial setup of the culture bags of the two Xuri or other bioreactor equipment, connect the breathable culture flasks to the cell culture bags and the expansion culture complete medium. Place the two groups of cell suspensions after static activation into the two pre-grouped cell culture bags; then rinse the breathable culture flasks with the expansion culture complete medium, and then transfer all of them to the cell culture bags; calculate the total weight added by weighing, and use the expansion culture complete medium to fill the initial volume of the cell culture bag to a culture density of 0.1-0.5×106 cells / ml, and the initial culture volume is required to be ≥ the minimum culture volume of the cell culture bag. The setting ranges of the two groups of reactors are shown in Table 4.

[0225] Table 4 Bioreactor parameter settings

[0226] <![CDATA[CO2 ratio]]> 5％ <![CDATA[O2 ratio]]> 21％ temperature 37.0℃ Gas flow rate 0.20L / min

[0227] 4. Day 5-6 of expansion culture

[0228] After entering the dynamic culture process, samples were taken and counted every day, and then the complete culture medium was added to the cell culture bag to a density of 0.1-0.5×10 6 cells / ml, and record the actual daily replenishment volume. When the replenishment reaches the maximum culture volume (10 L), no more replenishment is performed.

[0229] 5. Day 7 of expansion training

[0230] After 7 days of co-culture, the expansion culture was terminated and the cells expanded for 7 days were taken for cell counting, cell phenotype and cell functional testing.

[0231] Among them, the cell phenotype was detected by flow cytometry, including the following steps: reagent preparation, sample pretreatment, surface staining, machine detection, and data analysis. The cell functional detection was performed by nonspecific stimulation + ELISA, including the following steps: magnetic bead activation of cells, supernatant collection, and ELISA detection of IFN-γ.

[0232] The detection data of cell count and cell viability after expansion culture are shown in Table 5 below, and the cell proliferation multiples are shown in Figure 1 The total number of cells is shown in Figure 2 :

[0233] Table 5 Cell detection data on the 7th day of expansion culture

[0234]

[0235] *Relative ratio of live cells = total number of live cells in the experimental group / total number of live cells in the control group

[0236] The results showed that for TIL cells from the same batch, the proliferation times of the experimental group were higher than those of the control group, and there was no significant difference in cell viability between the experimental and control groups. Compared with the control group, the number of live cells in the experimental group increased by 1.65, 1.69, and 1.69 times, indicating that not adding CD3 antibodies during the expansion stage is more conducive to the proliferation of TIL.

[0237] The flow cytometry results of the cells after expansion are shown in Table 6 below:

[0238] Table 6 Phenotype of cultured cells

[0239]

[0240]

[0241] The results showed that, unexpectedly, without adding CD3 antibodies, the proportion of CD3+ cells in the experimental group was basically the same or slightly increased compared with the control group, and the proportion of CD8+ and CD4+ cells was basically the same. More surprisingly, compared with the control group, the proportion of stem cell memory T cells (Tscm) in the experimental group increased significantly, with the three batches increasing by 2.6, 1.3, and 5.0 times, respectively, with an average increase of 2.9 times, indicating that the TIL cells cultured in the experimental group have better stemness.

[0242] The results of cell functional test after expansion are shown in Table 7 and Figure 3 :

[0243] Table 7 INF-γ secretion level of cultured cells

[0244]

[0245] The level of INF-γ secretion characterizes the killing ability of TIL cells. Unexpectedly, as shown in Table 7 and Figure 3 As shown, without adding CD3 antibodies during the expansion phase, the TIL cells in the experimental group actually had a higher level of INF-γ secretion, which was an average increase of about 1.2 times compared with the control group.

[0246] The above results show that the TIL cells cultured using the experimental group's culture method are superior to those of the control group in terms of cell proliferation ability, stemness, and INF-γ secretion level, indicating that the expansion culture scheme of the present invention without adding CD3 antibodies is more conducive to the culture of TIL cells.

[0247] Example 2: TIL expansion method with and without CD137 monoclonal antibody

[0248] In this example, the difference in TIL expansion methods with and without the addition of CD137 monoclonal antibody was verified using the method in Example 1.

[0249] Source of TIL cells: TIL cells with batch number MB2021112401 were used, which were derived from breast cancer patients;

[0250] Culture medium: the same complete culture medium as in Example 1;

[0251] Experimental group: cultured with the above medium;

[0252] Control group: 5 μg / ml of anti-CD137 (4-1BB) monoclonal antibody was added to the above culture medium.

[0253] The operation from day 0 to day 6 of the expansion culture is the same as in Example 1. The expansion culture is terminated on day 8. The cells expanded on day 8 are taken for cell counting and cell phenotype detection, and the detection method is the same as in Example 1.

[0254] The cell data after expansion culture are shown in Table 8 below:

[0255] Table 8 Expansion cell data (expansion day 8)

[0256]

[0257] The flow cytometry results of the cells after expansion are shown in Table 9 below:

[0258] Table 9 Phenotype of cultured cells (Day 8 of culture)

[0259]

[0260] The results showed that there was no significant difference in the proliferation ability, cell viability and cell phenotype of TIL cells with or without the addition of CD137 antibody during the expansion process.

[0261] Example 3: TIL expansion method without feeder cells

[0262] In this example, the method in Example 1 was used to compare the TIL expansion method with feeder cells and without feeder cells, and the method was as follows:

[0263] Source of TIL cells: TIL cells numbered LA2024031201 were derived from lung cancer patients;

[0264] Culture medium: the same complete culture medium as in Example 1;

[0265] Feeder cell group: culture conditions and steps were the same as those of the experimental group in Example 1;

[0266] No feeder cell group: No feeder cells were added on day 0 of culture, and other culture conditions and steps were the same as those in Example 1. Cells were taken after 8 days of culture for cell counting, cell phenotype and cell functionality detection, and the detection methods were the same as in Example 1.

[0267] The cell data after expansion culture are shown in Table 10 below:

[0268] Table 10 Expansion cell data (expansion day 8)

[0269]

[0270] *Relative ratio of living cells = Total number of living cells in the group without feeder cells / Total number of living cells in the group with feeder cells. The flow cytometry results of cells after expansion are shown in Table 11 below:

[0271] Table 11 Phenotype of cultured cells

[0272]

[0273] The results of cell functional test after expansion are shown in Table 12 below:

[0274] Table 12 INF-γ secretion level of cultured cells

[0275]

[0276]

[0277] The results showed that the TIL cells cultured without feeder cells were significantly better than those with feeder cells in terms of Tscm cell proportion and IFN-γ secretion level, while there was no significant difference in cell proliferation ability and cell viability.

[0278] 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 amplifying and culturing tumor infiltrating lymphocytes (TIL) cells, characterized in that: Includes steps: A. Provide TIL cells after initial culture; B. amplifying and culturing the TIL cells in an expansion culture medium, wherein the expansion culture medium contains IL-2; and the expansion culture medium does not contain or substantially does not contain anti-CD3 antibodies; C. Harvest TIL cells after expansion culture.

2. The method according to claim 1, characterized in that In step B, the amount of IL-2 in the expansion culture medium is 100-600 IU / ml.

3. The method according to claim 2, characterized in that The expansion medium further comprises the following components: cell basal medium, serum replacement, L-glutamine or its replacement.

4. The method according to claim 1, characterized in that In step B, the expansion culture is further divided into the following steps: B1. Static culture for 2-5 days; B2. Dynamic culture for 3-7 days.

5. The method according to claim 1, characterized in that In step C, the harvested TIL cells are expanded ≥ 200 times compared to the cells in step A.

6. The method according to claim 1, characterized in that In step C, the cell viability of the harvested TIL cells is ≥95%.

7. A tumor infiltrating lymphocyte (TIL) cell, characterized in that The TIL cells are obtained by culturing according to the method of claim 1.

8. A pharmaceutical composition or preparation, characterized in that The composition comprises: (a) the TIL cells according to claim 7; and (b) a pharmaceutically acceptable carrier, excipient or diluent.

9. The use of TIL cells according to claim 7, characterized in that: Used for preparing medicine for treating disease, wherein the disease is tumor.

10. A TIL cell expansion culture medium, characterized in that: The expansion medium contains IL-2; and the expansion medium does not contain or substantially does not contain anti-CD3 antibodies.

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