An expansion culture process of tumor infiltrating lymphocytes

Abstract

The present application relates to a kind of tumor infiltrating lymphocyte (TIL) cell expansion culture process. Specifically, the present application provides a kind of TIL cell expansion method, comprising: in expansion culture medium, preliminary culture TIL cell is carried out fast expansion culture. In the TIL cell expansion method of the present application, it is not dependent on activation antibody CD3 antibody, high-dose IL-2 and feeder cell, that is, the TIL cell number hundreds of times expansion can be realized in short time, the demand of TIL cell quantity in TIL cell therapy clinically can be satisfied, and it has broad application prospect in the field of TIL cell treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a process for expanding and culturing tumor-infiltrating lymphocytes. Background Technology

[0002] Tumor-infiltrating lymphocytes (TILs) can naturally recognize and target tumor cells. Compared to T cells in peripheral blood, TILs are typically rich in tumor antigen-specific T cell clones. TILs have shown promising therapeutic effects as an effective adoptive immunotherapy in various cancer types, especially solid tumors. Reports have shown positive effects of TIL therapy in the treatment of HPV-related cancers, melanoma, lung cancer, and breast cancer.

[0003] TIL therapy is a personalized adoptive cell immunotherapy, and the infusion product must be manufactured according to the patient's specific condition, resulting in relatively high costs. Furthermore, it requires highly specialized Good Manufacturing Practice (GMP) facilities and highly trained personnel, which are also both expensive and time-consuming. Although the future development of computerized bioreactors may at least partially replace some of the production and human handling work, the process cannot be fully automated due to the collection of heterogeneous tumor raw materials such as tumor fragments.

[0004] Secondly, the production time for TIL products is long; the total production time for a single TIL treatment can be up to 8 weeks. This highly personalized and labor-intensive production process is not conducive to controlling the disease in some patients with rapidly progressing cancers. The TIL production process typically begins with a pre-REP phase, in which TILs dissociate from or migrate from tumor fragments and undergo initial expansion. TILs then further expand in the REP phase in response to stimuli such as IL-2 or feeder cells. Traditional TIL production procedures, used for specific tumor identification, typically require 6-8 weeks.

[0005] Chinese patent application CN110785486A describes a manufacturing process for preparing TILs, which uses a G-Rex500MCS system to process 2.3 × 10⁻⁶ TILs. 10 -1.3×10 11 The production cycle for large-scale cell production has been reduced to 22 days. The TIL production protocol described in Chinese patent application CN114686430A uses different culture and amplification systems, enabling expansion to 1-2 × 10⁻⁶ cells within 27 days. 11 Each cell. Chinese patent application CN114908050A describes the amplification time at both ends of TILs as 27 days. How to reduce production time and lower production costs is a major challenge facing TIL therapy.

[0006] Furthermore, current TIL culture protocols use CD3 antibodies as activating antibodies, but the use of activating antibodies may lead to overactivation of TIL cells, making them prone to exhaustion. In addition, existing TIL cultures typically rely on high concentrations of IL-2; however, high doses of IL-2 are associated with serious adverse events and place higher demands on cell washing and other procedures.

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

[0008] The purpose of this invention is to provide a novel TIL cell culture protocol.

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

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

[0011] B. The TIL cells are expanded and cultured in an expansion culture medium containing IL-2; and the expansion culture medium contains little or no anti-CD3 antibody.

[0012] C. Harvest expanded and cultured TIL cells.

[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 hematoma, preferably a solid tumor.

[0015] In another preferred embodiment, the preliminary culture includes: dissociating or separating TIL cells from tumor tissue, and initially expanding them to obtain a certain order of magnitude of TIL cells, for example, greater than 1 × 10⁻⁶. 5 TIL cells, more preferably larger than 1 × 10 6 TIL cells.

[0016] In another preferred embodiment, the preliminary culture is carried out in a cell basal medium containing IL-2, wherein the amount of IL-2 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 culture medium is 100-600 IU / ml, preferably 200-400 IU / ml, and 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 substitute, L-glutamine or a substitute thereof.

[0019] In another preferred embodiment, the cell-based 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 medium, AIM-V medium, etc. TM Culture medium, SFM medium, X-VIVO medium, or combinations thereof.

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

[0022] In another preferred embodiment, the cell basal culture medium is Advanced RPMI 1640 medium and CTS. TM AIM-V TM The culture medium is obtained by mixing the culture media in a 1:1 ratio.

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

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

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

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

[0027] In another preferred embodiment, the expansion culture medium may or may not contain anti-CD137 antibody.

[0028] In another preferred embodiment, feeder cells may or may not be added during the amplification culture process.

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

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

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

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

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

[0034] In another preferred embodiment, the amplification culture lasts for 5-14 days, preferably 6-12 days, and more preferably 7-10 days.

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

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

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

[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 with or without feeder cells.

[0042] In another preferred embodiment, the dynamic culture is carried out without the addition of feeder cells.

[0043] In another preferred embodiment, the static culture is carried out in a container selected from the group consisting of: aerated culture flasks, cell culture bags, culture dishes, and cell plates.

[0044] In another preferred embodiment, the aerated culture flask is a bottom-permeable culture flask.

[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, and an automated cell processor, such as a fully automated cell processor.

[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 swing speed of 2-15 rpm and a swing angle of 2-10°.

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

[0049] In another preferred embodiment, the cell density during the amplification culture is 0.1-1×10⁻⁶. 6 More preferably, it is 0.1-0.5 × 10⁻⁶ cells / ml. 6 per ml.

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

[0051] In another preferred embodiment, compared with the control group with CD3 antibody added and other conditions the same, the total number of TIL live cells harvested by the method is increased by ≥1.4 times, preferably ≥1.5 times, and more preferably ≥1.6 times.

[0052] In another preferred embodiment, compared with the control group with CD3 antibody added and other conditions the same, the proportion of Tscm in TILs harvested by the method is increased by ≥1.2 times, preferably ≥1.25 times, and more preferably ≥1.3 times.

[0053] In another preferred embodiment, compared with the control group with CD3 antibody added and other conditions the same, the method increased the INF-γ secretion of TILs by ≥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 includes the steps of:

[0056] D. The harvested TIL cells are washed, purified, and cryopreserved.

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

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

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

[0060] In another preferred embodiment, the human serum albumin content 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 process is performed in 2-5 rounds, more preferably in 3 rounds.

[0062] In another preferred embodiment, in step D, the cryopreservation solution used for cryopreservation includes the following components: cell washing solution and CryoStor® CS10.

[0063] In another preferred embodiment, the cryopreservation solution is a cryopreservation solution obtained by mixing cell washing solution and CryoStor® CS10 in a 1:1 ratio.

[0064] In a second aspect of the invention, a tumor-infiltrating lymphocyte (TIL) cell is provided, said TIL cell being cultured by the method described in the first aspect of the invention.

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

[0066] In a fourth aspect of the invention, the use of TIL cells as described in the second aspect of the invention is provided for the preparation of medicaments for treating diseases.

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

[0068] In another preferred embodiment, the tumor is a solid tumor or a hematoma, 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, stomach cancer, colorectal cancer, and kidney cancer, or combinations thereof.

[0070] In a fifth aspect of the invention, a TIL cell expansion culture medium is provided, wherein the expansion culture medium contains IL-2; and the expansion culture 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 culture medium is 100-600 IU / ml, preferably 200-400 IU / ml, and 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 substitute, L-glutamine or a substitute thereof.

[0073] In another preferred embodiment, the cell-based 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 medium, AIM-V medium, etc. TM Culture medium, SFM medium, X-VIVO medium, or combinations thereof.

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

[0076] In another preferred embodiment, the cell basal culture medium is Advanced RPMI 1640 medium and CTS. TM AIM-V TM The culture medium is obtained by mixing the culture media in a 1:1 ratio.

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

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

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

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

[0081] In another preferred embodiment, the expansion culture medium may or may not contain anti-CD137 antibody.

[0082] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0083] Figure 1 The graph shows a comparison of cell proliferation folds on day 7 of expansion culture in Example 1.

[0084] Figure 2 The image shows a comparison of the total number of cells on day 7 of expansion culture in Example 1.

[0085] Figure 3 The INF-γ secretion level of cells on day 7 of expansion culture in Example 1 is shown. Detailed Implementation

[0086] Through extensive and in-depth research, the inventors have developed a method for expanding TIL cells, enabling large-scale expansion of TIL cells in a short period of time. This method does not use the CD3 activating antibody to avoid over-activation of TIL cells; it also reduces the dosage of IL-2, thus lowering side effects and safety risks; furthermore, it does not rely on feeder cells. Using this method, TIL cells can be expanded hundreds of times in as little as 7-8 days, meeting the clinical demand for TIL cell numbers in TIL cell therapy, and the resulting TIL cells exhibit high levels of stemness and INFγ secretion capacity. This invention was completed based on these findings.

[0087] definition

[0088] Unless otherwise defined, 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 pertains.

[0089] As used in this article, the term "tumor-infiltrating lymphocytes" or "TIL cells" refers to a type of immune cell that exists in the tumor microenvironment. TIL cells offer significant advantages in cancer treatment. First, they can be extracted directly from the patient, expanded and activated in vitro, and then re-injected, enabling personalized treatment. Second, because TIL cells originate from the patient's own body, there is no risk of allogeneic rejection, resulting in a high safety profile. Furthermore, TIL cell therapy has demonstrated significant efficacy in clinical trials against certain refractory or advanced tumors.

[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. The IL-2 used in this invention includes all forms of IL-2, such as IL-2 derived from humans or other mammals, and comprises wild-type and mutant IL-2, provided that the mutant has similar activity to the wild-type. In this invention, the IL-2 comprises recombinant human IL-2.

[0091] In some embodiments of the present invention, the term "anti-CD3 antibody" refers to any form of antibody targeting CD3, such as an anti-CD3 monoclonal antibody, the source of which 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" generally refers to a cultured cell that grows in vitro and secretes at least one factor into a culture medium and can be used to support the growth of another cell of interest. In one embodiment, the feeder cell may include PBMCs.

[0094] As used in this article, 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 cytotoxic T cells.

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

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

[0097] As used in this article, the term "memory T cell" refers to a type of cell that, after receiving antigen stimulation, forms from naïve cells and possesses the ability to survive long-term and rapidly respond to the same antigen again. These cells can be divided into effector memory T cells (T cells). EM ), central memory T cells (T CM ), stem cell memory T cells (T) SCM ) etc. T SCM The proportion of T cells characterizes the stemness of the T cell population. SCM The higher the proportion of T cells, the stronger the stemness of the T cell population.

[0098] Culture of TIL cells

[0099] TIL cell culture refers to the process of isolating TIL cells from tumor tissue of cancer patients and expanding them in large quantities to obtain a therapeutically effective amount of TIL cells. It usually involves two stages: initial culture and expansion culture.

[0100] As used in this article, the term "primary culture" also refers to "pre-REP" or "pre-expansion," which refers to the isolation and first-stage culture of TILs in tumor tissue to obtain a certain number of TILs for subsequent rapid expansion.

[0101] Before starting TIL cell culture, TIL cells are typically located within tumor tissue masses in patients. This requires first obtaining tumor tissue from the patient, such as through surgery or biopsy. The tumor is preferably a solid tumor. After cutting and washing, the tumor tissue mass is placed in a primary culture medium for culture, for example, for about 7 days, to obtain a primary cell population. The primary culture medium typically includes: basal cell culture medium, IL-2, serum substitute, L-glutamine or its substitute, and buffer.

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

[0103] As used in this article, the terms “expansion culture,” “rapid expansion culture,” “rapid expansion culture,” and “REP” are used interchangeably to refer to the expansion culture of cells after initial culture in order to obtain a large number of TIL cells in a short period of time for therapeutic purposes.

[0104] The TIL cell expansion method of the present invention

[0105] This invention provides a method for expanding TIL cells, which can obtain TIL cells with a multiplicity of several hundredfold increase in a short time. The expansion method of this invention does not rely on activating antibodies, high doses of IL-2, or feeder cells. It uses appropriate factor concentrations and culture conditions to achieve rapid cell proliferation and obtain TIL cells with a high multiplicity.

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

[0107] IL-2 is a potent T-cell mitotic cytokine that stimulates the growth and survival of effector T cells. Adding IL-2 to cultured lymphocytes promotes lymphocyte lysis of autologous tumor cells. Furthermore, the combination of IL-2 and TIL infusion can improve therapeutic efficacy. However, administering 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 doses of IL-2 can promote the development of regulatory T cells that suppress the antitumor response of TILs. However, high doses of IL-2 are associated with serious adverse events, including capillary leak syndrome, characterized by hypotension, oliguria, edema, and even hypovolemic shock.

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

[0110] In a preferred embodiment, the cell-based culture medium of the present invention is a culture medium suitable for mammalian cell culture, such as RPMI 1640 medium or AIM-V medium. TM Culture media, SFM medium, X-VIVO medium, and other culture media known in the art. In a preferred embodiment, the cell-based culture medium of the present invention is Advanced RPMI 1640 medium and CTS. TM AIM-V TM The culture medium is obtained by mixing the culture media in a 1:1 ratio.

[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 L-glutamine substitute is Gluta Max. TM ; 1%-5% serum replacement therapy, such as CTS TM Immune cell serum substitutes.

[0112] In the invention, the complete culture medium for expansion may optionally contain or not contain anti-CD137 antibody; when CD137 antibody is present, the preferred concentration is 1-10 μg / ml, for example 5 μg / ml.

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

[0114] In a preferred embodiment, the amplification culture lasts for 6-14 days, preferably 6-12 days, more preferably 7-10 days, for example 7 days, 8 days, 9 days or 10 days.

[0115] In a preferred embodiment, the amplification culture temperature is 37℃±1℃, preferably 37℃±0.5℃.

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

[0117] The containers used in the expansion stage of this invention are not limited; for example, they can be carried out in a bioreactor.

[0118] The following example, using the expansion process in a bioreactor, illustrates 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 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 through a 0.22μm capsule filter into a disposable preparation bag for use 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 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 in Advanced RPMI 1640 and CTS AIM-VMedium.

[0125] 1.1.2.2. After preparation, filter through a 0.22μm capsule filter into a disposable preparation bag for use as a complete culture medium for expansion.

[0126] 1.2. Feeder cell resuscitation

[0127] 1.2.1. Use a constant temperature water bath for cell resuscitation.

[0128] 1.2.3. Reagent dispensing

[0129] 1.2.3.1. Aseptically dispense cell washing solution and expanded complete culture medium as washing solutions for Sepax C-Pro cell processing instrument.

[0130] 1.2.3.2. Aseptic dispensing of complete culture medium for expansion as the final packaging for Sepax C-Pro cell processing instrument for rapid dilution of washed feeder cells.

[0131] 1.2.4. Complete kit assembly.

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

[0133] 1.2.6. Parameter Settings

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

[0135]

[0136] 1.2.7. Set up the cell washing program, enter the tracking information, pause the instrument operation, and prepare the initial bag.

[0137] 1.2.8. Initial Packaging Preparation

[0138] 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. Place them in a 37°C digital display constant temperature water bath for resuscitation.

[0139] 1.2.8.2. Connect the revived feeder cells to the cell washing solution from 1.2.3.1 and a new reservoir bag. Dilute and transfer the feeder cells into the new reservoir bag using the cell washing solution as the initial bag for the Sepax C-Pro cell processing instrument.

[0140] 1.2.9. Connect the initial bag to the kit, click on the Sepax C-Pro cell processor screen to confirm the program execution, and begin cell culture and washing.

[0141] 1.2.10. Cleaning procedure includes the following contents

[0142] 1.2.10.1. Use the initial bag fluid for kit filling and cavity filling.

[0143] 1.2.10.2. Initial bag washing: All liquid in the initial bag and the pre-wash liquid are centrifuged in the separation chamber. Then the supernatant is discharged to the initial bag to wash the initial bag. Then all liquid is fed into the separation chamber.

[0144] 1.2.10.3. Concentrate: After centrifuging in the separation chamber for 2-10 min, discharge the supernatant (excluding the intermediate volume) into the waste liquid bag.

[0145] 1.2.10.4. Washing: The washing liquid enters the separation chamber and is centrifuged for 2-10 minutes. The supernatant, except for the intermediate volume, is discharged into the waste liquid bag. Before the last round of washing, the washing liquid is manually switched from the pre-washing liquid to the post-washing liquid.

[0146] 1.2.10.5. Cell resuspension and chamber rinsing: The remaining cell suspension (volume equal to the intermediate volume) is drained from the separation chamber into the final product bag. Then, the washing solution is introduced into the separation chamber to rinse 1-5 times. After rinsing, the suspension is transferred to the final product bag and replenished to the final volume.

[0147] 1.3. TIL Activation

[0148] 1.3.1. Connect the aerated culture flask containing the primary cultured cells to the feeder cell product. Add the feeder cells to the aerated culture flask.

[0149] 1.3.2. Transfer the complete culture medium to an aerated culture flask. Place the aerated culture flask in an incubator at 37°C and 5% CO2 to begin static activation.

[0150] 1.3.3. Every two days after activation, use a sterile connector to connect the aerated culture flask to the storage bag, pump out 0-800g of culture supernatant into the storage bag, mix the remaining cell culture medium, take a sample, count the cells, and then add fluid to bring the total volume to 1kg (1L). This process should be repeated for 2-4 days.

[0151] 2.1. After static activation, use a sterile connector to connect the aerated culture bottle to the storage bag, transfer the upper culture medium into the storage bag, mix the remaining cell culture medium, and take samples for counting.

[0152] 2.2. Turn on the bioreactor (e.g., the Xuri cell expansion system or other bioreactors), install the cell culture bag, and start operation. Connect the aerated culture flask to the cell culture bag and the complete expansion medium. Introduce the cell suspension into the cell culture bag; after weighing and calculating the total weight added, use the complete expansion medium to fill the initial volume of the cell culture bag to achieve a culture density of 0.1-1 × 10⁻⁶ cells / year. 6 cells / ml.

[0153] 2.3. Set the culture parameters as shown in Table 2, where the swing speed is adjusted according to the culture volume, as detailed in Table 2.

[0154]

[0155] 2.4. After entering the dynamic culture process, take samples and count them daily, and based on the culture results, replenish the cell culture bag with complete expansion culture medium until the density is 0.1-1 × 10⁻⁶. 6The cells / ml was recorded, and the actual daily replenishment volume was recorded. When the maximum culture volume (10L) was reached, no further replenishment was performed.

[0156] 2.5. Dynamically expand and culture for 3-7 days, sampling and testing the cell suspension daily. Determine whether to harvest cells based on the test results. Harvest cells when the total cell count is greater than or equal to 1×10⁻⁶. 10 When cells are in the range of 'cells', the expansion culture can be ended and the cell washing process can begin.

[0157] 3. Cell washing

[0158] 3.1. Cleaning and purification

[0159] 3.1.1. Take a 750 or 500 type cryopreservation bag as preparation bag A, weigh the empty bag for subsequent dispensing of preparation bag A;

[0160] 3.1.2. Turn on the LOVO Cell Processing System (LOVO cell processing and purification instrument, hereinafter referred to as LOVO), set the parameters, wash and collect 60ml-300ml of cell suspension, freeze 1-5 bags, 100±10ml / bag;

[0161] 3.1.2.1. One mother liquor container, each container has a mother liquor volume of 0-22000ml, a mother liquor cell accumulation of 0-3%, 2-5 washing cycles, a rotary oscillation speed of 600rpm, and cell washing solution for pre-rinsing consumables.

[0162] 3.1.2.2. The number of times the mother liquor is pre-flushed is once, the volume of the mother liquor pre-flushed is 0-50ml, and the mother liquor pre-flushed inlet rate is 50ml / min;

[0163] 3.1.2.3. The mother liquor bag rinse volume is 0-200ml, the top tubing of bag 2 is pre-rinsed with 0-30ml, the bottom tubing of bag 2 is pre-rinsed with 0-20ml, and the bottom tubing of bag 2 is rinsed with 0-20ml.

[0164] 3.1.2.4. Final product volume 60-300ml, final product infusion rate 150ml / min;

[0165] 3.1.2.5. No settings are required for the incubation stage;

[0166] 3.1.2.6. Use cell washing solution as the washing buffer. The feed rate of the rotary vortex is 60-160 ml / min, and the liquid retention rate is 7-100 ml / min.

[0167] 3.1.2.7. Cell accumulation at the rotator inlet ≤20.0%, rotator speed 2000-4000rpm, use cell washing bags for rinsing and dilution, and dilute 200-800ml in size 2 bags;

[0168] 3.1.3. The cell suspension is collected in formulation bag A, and the heat-sealed tubing is removed for later use.

[0169] 4. Cell cryopreservation preparations

[0170] 4.1. Formulation filling

[0171] 4.1.1. Connect the preparation bag A to a sampler and draw approximately 3 ml of cell suspension for testing;

[0172] 4.1.2. Aliquot the same volume of CryoStor CS10 cell cryopreservation solution (hereinafter referred to as cryopreservation solution) into another cryopreservation bag for later use, with a mass (g): volume (ml) ratio of 1:1;

[0173] 4.1.3. Mix the cell suspension and cryopreservation solution 1:1 to form a preparation solution. After mixing, dispense the preparation solution into 1-5 cryopreservation bags according to the injection specification (100±10ml / bag).

[0174] 4.1.4. After dispensing, each bag of preparation is vented and sealed. After passing the light inspection, it is placed into an outer protective bag, vacuum sealed, and then placed in a blood bag box that has been pre-cooled at 2-8℃ for more than 30 minutes.

[0175] 4.1.5. The remaining preparation solution is dispensed into 50 cryopreservation bags according to the sample specifications (10±2ml / bag) for post-freezing thawing and testing. It is placed in the blood bag box in the same way as in 4.1.4. The remaining preparation solution is taken out using a 10ml disposable sterile screw-top syringe for cell counting and testing.

[0176] 4.1.6. Transfer the blood bag containing the preparation solution to the programmed cooling device for programmed cooling.

[0177] 4.2. Cooling and freezing

[0178] 4.2.1. Place the finished product in a programmed cooling device for programmed cooling. The cooling program is as follows;

[0179] 4.2.1.1. Step one: Wait at 20.0℃;

[0180] 4.2.1.2. Step 2: Cool the sample to -6℃ at a cooling rate of 1℃ / min;

[0181] 4.2.1.3. Step 3: Cool the chamber to -50°C at a cooling rate of 25°C / minute;

[0182] 4.2.1.4. Step four: Heat the chamber to -14°C at a heating rate of 10°C / minute;

[0183] 4.2.1.5. Step five: Cool the chamber to -45°C at a cooling rate of 1°C / minute;

[0184] 4.2.1.6. Step Six: Cool the chamber to -90°C at a cooling rate of 10°C / minute;

[0185] 4.2.2. After cooling is complete, remove the blood bag box and transfer it to a gas phase liquid nitrogen tank (≤-150℃) for storage.

[0186] The TIL cells of the present invention and their applications

[0187] In this invention, TIL cells cultured using the expansion culture method of this invention, pharmaceutical compositions or preparations containing TIL cells of this invention, and their uses are also provided.

[0188] The TIL cells cultured using the expansion method of this invention exhibit high expansion fold, high cell viability, and good stemness (e.g., T cells). SCM (High proportion of cells) can meet the clinical application requirements of TIL.

[0189] As used herein, the terms “pharmaceutical composition” or “formulation” generally refer to a preparation that is a mixture of at least one cell and at least one and optionally more than one other pharmaceutically acceptable chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients.

[0190] In this application, the term "pharmaceutically acceptable carrier" generally refers to one or more nontoxic 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 formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents, or encapsulating substances 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 ions (such as sodium), etc. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art.

[0191] In this invention, TILs amplified using the method of this invention can be administered to patients as a drug. In some embodiments, the pharmaceutical composition may be a suspension of TILs in a sterile buffer. TILs obtained using the amplification method of this invention can be administered via any suitable route known in the art.

[0192] The main advantages of this invention include:

[0193] 1. The expansion method of the present invention shortens the TIL culture time, and the expansion stage can be expanded by about 500 times in 7-10 days to meet the clinical demand.

[0194] 2. The expansion culture method of the present invention optimizes and reduces the amount of IL-2 used in the culture process, thereby reducing adverse clinical reactions caused by excessively high IL-2 concentrations.

[0195] 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.

[0196] 4. The expansion method of the present invention uses serum-free culture medium, which reduces the risk of serum contamination, avoids the defects of poor batch stability of serum, potential viral contamination and high cost, and improves the safety of cell culture.

[0197] 5. This invention achieves fully sealed formulation washing and filling preparation. Based on the total number of cells in the expansion culture, it offers multiple freezing schemes of 1-5 bags, ensuring that the expanded cells meet parameter requirements during washing and purification, and guaranteeing a frozen cell density of 1-30 × 10⁻⁶. 7 Within a certain range, avoid administering too much cryopreserved fluid to the patient.

[0198] 6. The TIL cells prepared by this invention have high expansion rate, high viability and high level of stemness, and the TIL cell preparation can be stored in liquid nitrogen for a long time and can be used flexibly.

[0199] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally 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 as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0200] Example 1: TIL expansion methods with and without CD3 monoclonal antibody

[0201] In this embodiment, the comparison of TIL cells obtained by adding CD3 monoclonal antibody or not adding CD3 monoclonal antibody during the expansion stage was verified under the same conditions. The specific method is as follows:

[0202] TIL cell source: Three batches of TIL cells, LA2023062701, LA2023070501, and LA2023061401, were selected. Each batch of TIL cells came from a single cancer patient, all of whom were lung cancer patients.

[0203] Preliminary culture of TIL cells: TIL cells were isolated from tumor tissue and initially cultured in a cell basal medium containing 3000 IU / ml IL-2, yielding a number of 5 × 10⁶ cells / ml. 7 The TIL cells mentioned above were used for subsequent expansion culture.

[0204] Complete culture medium for expansion: Prepare a 1:1 mixture 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 the complete culture medium for expansion.

[0205] Experimental group: cultured using the above-mentioned expanded complete culture medium;

[0206] Control group: An additional 30 ng / ml of CD3 monoclonal antibody (OKT3) was added to the above culture medium. Otherwise, the subsequent culture conditions for both the experimental and control groups were identical. The specific operating procedures are as follows:

[0207] 1. Day 0 of the expansion culture

[0208] 5.00 × 10⁶ TIL cells were collected from each group after initial culture. 7 Each group was subjected to static activation and amplification culture under the same conditions as the experimental and control groups, respectively. The specific procedures are as follows:

[0209] 1.1 Record the number of cells at the end of the initial culture: total number of viable cells inoculated (5.00 × 10⁻⁶). 7 (number / group), inoculation volume, and cell viability.

[0210] 1.2 Feeder cells were washed. After washing, 30 μg of CD3 monoclonal antibody (OKT3) was added to the final product bag of the control group, while no such addition was made to the experimental group.

[0211] 1.3 After recording the feeder cell information, heat-seal the PVC tubing of the feeder cell bag to the C-port long tube of the G-Rex 100M-CS cell culture apparatus. Open the Robert clamps on the PVC tubing of the feeder cell bag and the C-port long tube of the G-Rex 100M-CS, and transfer the 1.00×10⁶ cells from the bag... 9 Each feeder cell was introduced into a G-Rex 100M-CS cell according to gravity.

[0212] 1.4 Fluid replenishment: Both the control and experimental groups were replenished with fluids by gravity to ensure that the initial culture weight of G-Rex 100M-CS was 1000g.

[0213] 1.5 Place the two groups of G-Rex 100M-CS in a carbon dioxide incubator (37℃, 5% CO2) for incubation.

[0214] 2. Day 2 of the extended culture

[0215] 2.1 Using a sterile connector, heat-seal the disposable sampling bags to the C-port long tubes of the two sets of G-Rex 100M-CS containers. Air is pumped in using a peristaltic pump, and the positive pressure inside the container is used to drain 500g of culture supernatant into the disposable sampling bags.

[0216] 2.2 Sampling and counting of cells in both groups: The samples were counted twice according to the "Standard Operating Procedure for Cell Sampling and Counting," and the average of the two counts was recorded, including the viable cell density, viability, and cell diameter. The total number of viable cells was calculated by calculating the volume of the cell suspension in each container.

[0217] 2.3 Using gravity, REP-CM was drained from the storage bag into G-Rex 100M-CS for replenishment, so that the cell culture weight of the two groups was 1000g.

[0218] 2.4 The two groups of G-Rex 100M-CS were placed in a carbon dioxide incubator (37℃, 5% CO2) and cultured until day 4.

[0219] 3. Day 4 of extended culture

[0220] 3.1 Using a sterile connector, heat-seal the disposable sampling bags to the C-port long tubes of the two sets of G-Rex 100M-CS. Air is pumped in using a peristaltic pump, and the positive pressure inside the container is used to drain 700g of culture supernatant into the disposable sampling bags.

[0221] 3.2 Sampling and counting of cells in both groups: The samples were counted twice according to the "Standard Operating Procedure for Cell Sampling and Counting," and the average of the two counts was recorded, including the viable cell density, viability, and cell diameter. The total number of viable cells was calculated by calculating the volume of the cell suspension in each container.

[0222] 3.3 After completing the installation and initial setup of the culture bags for the two Xuri or other bioreactor devices, connect the aerated culture flasks to the cell culture bags and the complete expansion medium. Transfer all of the two statically activated cell suspensions into the two pre-grouped cell culture bags; then rinse the aerated culture flasks with complete expansion medium and transfer them all to the cell culture bags; weigh and calculate the total weight added, and use complete expansion medium to fill the initial volume of the cell culture bags to a culture density of 0.1-0.5 × 10⁻⁶. 6 Cells / ml, and the initial culture volume must be greater than or equal to the minimum culture volume of the cell culture bag. The setup ranges for the two reactors are shown in Table 3.

[0223]

[0224] 4. Days 5-6 of the extended culture.

[0225] After entering the dynamic culture process, samples are taken and counted daily. Based on the culture results, complete culture medium is added to the cell culture bag until the density reaches 0.1-0.5 × 10⁻⁶. 6 Cells / ml, and record the actual daily replenishment volume. When the maximum culture volume (10L) is reached, no further replenishment is required.

[0226] 5. Day 7 of extended culture

[0227] The cells were cultured for 7 days, and after the expansion culture was completed, cells were taken for cell counting, cell phenotype and cell function testing.

[0228] Cell phenotype was detected using flow cytometry, including the following steps: reagent preparation, sample pretreatment, surface staining, flow cytometry detection, and data analysis. Cell functional assays were performed using non-specific stimulation + ELISA, including the following steps: magnetic bead activation of cells, collection of supernatant, and ELISA detection of IFN-γ.

[0229] The cell count and cell viability data after expansion culture are shown in Table 4 below, and the cell proliferation fold is shown in [Table 4]. Figure 1 Total cell count Figure 2 :

[0230] Table 4. Cell detection data on day 7 of expansion culture

[0231]

[0232] *Relative ploidy rate of viable cells = Total number of viable cells in the experimental group / Total number of viable cells in the control group

[0233] The results showed that for TIL cells from the same batch, the proliferation rate of the experimental group was higher than that of the control group, and there was no significant difference in cell viability between the two groups. Compared with the control group, the number of viable cells in the experimental group increased by 1.65, 1.69, and 1.69 times, indicating that not adding CD3 antibody during the expansion stage is more conducive to TIL proliferation.

[0234] The flow cytometry results of the expanded cells are shown in Table 5 below:

[0235]

[0236] The results showed that, unexpectedly, without the addition of CD3 antibody, the proportion of CD3+ cells in the experimental group was basically the same or slightly increased compared to the control group, while the proportions of CD8+ and CD4+ cells were basically the same. Even more surprisingly, compared to the control group, the proportion of stem cell memory T cells (Tscm) in the experimental group was significantly increased, with increases of 2.6, 1.3, and 5.0 times in the three batches, respectively, averaging 2.9 times, indicating that the TIL cells cultured in the experimental group exhibited better stem cell characteristics.

[0237] The results of functional assays of cells after expansion culture are shown in Table 6 below. Figure 3 :

[0238]

[0239] INF-γ secretion levels characterize the cytotoxic ability of TIL cells. Surprisingly, as shown in Table 6 and... Figure 3 As shown, without the addition of CD3 antibody during the expansion stage, the TIL cells in the experimental group actually had a higher INF-γ secretion level, which was about 1.2 times higher on average than the control group.

[0240] The results above show that the TIL cells cultured using the experimental method are superior to the control group in terms of cell proliferation, stemness, and INF-γ secretion level, indicating that the CD3 antibody-free expansion culture protocol of the present invention is more conducive to the culture of TIL cells.

[0241] Example 2: TIL expansion methods with and without CD137 monoclonal antibody

[0242] In this embodiment, the difference between the TIL amplification methods with and without CD137 monoclonal antibody was verified using the same method as in Example 1.

[0243] TIL cell source: TIL cells with batch number MB2021112401 were derived from breast cancer patients;

[0244] Culture medium: the same complete culture medium as in Example 1;

[0245] Experimental group: cultured using the above-mentioned culture medium;

[0246] Control group: 5 μg / ml of anti-CD137(4-1BB) monoclonal antibody was added to the culture medium described above.

[0247] The procedures for expansion culture from day 0 to day 6 are the same as in Example 1. Expansion culture is terminated on day 8. Cells cultured for 8 days are used for cell counting and cell phenotype detection, using the same methods as in Example 1.

[0248] The cell data after expansion culture are shown in Table 7 below:

[0249]

[0250] The flow cytometry results of the expanded cells are shown in Table 8 below:

[0251]

[0252] The results showed that adding or not adding CD137 antibody during the expansion process did not significantly affect the proliferation capacity, cell viability, or cell phenotype of TIL cells.

[0253] Example 3: TIL expansion method without feeder cells

[0254] In this embodiment, the method described in Example 1 is used to compare the TIL expansion methods with and without feeder cells. The method is as follows:

[0255] TIL cell origin: TIL cells with the code LA2024031201 were derived from a lung cancer patient;

[0256] Culture medium: the same complete culture medium as in Example 1;

[0257] Feeder cell group: The culture conditions and procedures are the same as those in the experimental group in Example 1;

[0258] No feeder cells group: No feeder cells were added on day 0 of the expansion culture, and other culture conditions and steps were the same as in Example 1. Cells were collected after 8 days of expansion culture for cell counting, cell phenotype and cell function testing, and the testing methods were the same as in Example 1.

[0259] The cell data after expansion culture are shown in Table 9 below:

[0260]

[0261] *Relative ploidy rate = Total number of viable cells in the group without feeder cells / Total number of viable cells in the group with feeder cells

[0262] The flow cytometry results of the expanded cells are shown in Table 10 below:

[0263]

[0264] The results of functional testing of cells after expansion culture are shown in Table 11 below:

[0265]

[0266] The results showed that TILs cultured without feeder cells were significantly superior to the group with feeder cells in terms of Tscm cell ratio and IFN-γ secretion level, while there was no significant difference in cell proliferation capacity and cell viability between the two groups.

[0267] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for expanding and culturing tumor-infiltrating lymphocytes (TILs) without relying on activating antibodies or serum, characterized in that, Including the following steps: A. Provide TIL cells after initial culture; B. The TIL cells are expanded and cultured in an expansion culture medium comprising the following components: IL-2, cell basal medium, serum substitute, and L-glutamine or a substitute thereof; In the expansion culture medium, IL-2 is the only T cell mitotic cytokine, and the dosage of IL-2 is 200-400 IU / ml; the content of the serum substitute is 1%-5%; the expansion culture medium does not contain anti-CD3 antibody, and there is no serum in the expansion culture medium; C. Harvest and expand TIL cells after culture; In step C, the harvested TIL cells are expanded by ≥400 times compared to the cells in step A.

2. The method as described in claim 1, characterized in that, In step B, the amount of IL-2 used in the expansion culture medium is 300±50 IU / ml.

3. The method as described in claim 2, characterized in that, The expansion culture medium consists of: IL-2, cell basal medium, serum substitute, and L-glutamine or its substitute.

4. The method as described in claim 2, characterized in that, The expansion culture medium consisted of: 300 IU / ml IL-2, Advanced RPMI 1640 and CTS AIM-V Medium 1:1 mixed culture medium as the cell basal medium, 2.5% CTS immune cell serum substitute, and 1% Gluta Max.

5. The method as described in claim 1, characterized in that, The expansion culture medium does not contain anti-CD137 antibody.

6. The method as described in claim 1, characterized in that, In step B, the amplification culture lasts for 6-12 days.

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

8. The method according to any one of claims 1-7, characterized in that, No feeder cells are added during the amplification culture process.

9. The method as described in claim 1, characterized in that, In step C, the harvested TIL cells are expanded by ≥500 times compared to the cells in step A.

10. The method as described in claim 1, characterized in that, In step C, the cell viability of the harvested TIL cells is ≥95%.

11. A TIL cell expansion culture medium, characterized in that, The expansion culture medium includes the following components: IL-2, cell basal culture medium, serum substitute and L-glutamine or its substitute; in the expansion culture medium, IL-2 is the only T cell mitotic cytokine, and the amount of IL-2 is 200-400 IU / ml; the content of serum substitute is 1%-5%; the expansion culture medium does not contain anti-CD3 antibody and is serum-free.

12. The culture medium as described in claim 11, characterized in that, The expansion culture medium consisted of: 300 IU / ml IL-2, Advanced RPMI 1640 and CTS AIM-V Medium 1:1 mixed culture medium as the cell basal medium, 2.5% CTS immune cell serum substitute, and 1% Gluta Max.

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