Tumor infiltrating lymphocyte culture medium and application thereof

Abstract

The application provides a tumor infiltrating lymphocyte culture medium and application thereof. The culture medium adopted by the application is composed of a basic culture medium, a serum substitute, a low-concentration interleukin, a stimulating factor and an antibiotic and the like. The application discloses a method for preparing high stemness tumor infiltrating lymphocytes (TIL) in vitro under serum-free and feeder-free conditions. Through optimized two-step culture (primary culture for 7-14 days + expansion culture for 7-14 days) and activation stimulation, high stemness TIL products with a CD3+ active cell ratio of greater than or equal to 95% and a Tcm ratio of greater than or equal to 70% can be obtained, and the total expansion multiple reaches 500-1000 times. The method of the application avoids the safety risk brought by feeder cells, reduces IL-2 related side effects, shortens the preparation period to 14-28 days, significantly improves the stem cell characteristics and sustained killing ability of TIL cells, and meets the quality control requirements of clinical-grade cell treatment products.

Description

Technical Field

[0001] This invention relates to the field of cell immunotherapy technology, specifically to a tumor-infiltrating lymphocyte culture medium and its application. Background Technology

[0002] Tumor-infiltrating lymphocyte (TIL) therapy is a novel adoptive cell immunotherapy that expands anti-tumor T cells from a patient's own tumor tissue in vitro and then reinfuses them into the body. It has shown significant efficacy in the treatment of solid tumors such as melanoma and cervical cancer. However, current TIL preparation technologies face three major bottlenecks:

[0003] First, traditional culture systems rely on high doses of IL-2 (usually above 6000 IU / mL) and feeder cells. High concentrations of IL-2 can easily induce terminal differentiation of T cells and activation-induced cell death (AICD), and increase the risk of side effects such as capillary leakage syndrome in vivo. Although feeder cells (such as irradiated PBMCs or NK cells) can promote expansion, they introduce the risk of exogenous cell contamination, increasing the difficulty of quality control and supervision.

[0004] Secondly, current culture systems generally rely on animal serum, which exhibits significant batch-to-batch variability, unstable sources, and the risk of contamination by unknown pathogens, making it difficult to meet GMP-grade cell product production standards. While existing serum-free culture protocols eliminate the need for serum, they still require feeder cells, failing to completely resolve safety concerns.

[0005] Third, the expanded TILs have insufficient stemness and weak effector function. TILs obtained through conventional methods are mostly effector memory T cells (Tem) or terminally differentiated effector T cells (Teff), with short in vivo survival time (<2 weeks), making it difficult to form durable anti-tumor immunity. Enriching stem cell memory T cells (Tscm) and central memory T cells (Tcm) with self-renewal and multi-lineage differentiation potential is key to improving clinical efficacy. Central memory T cells express high levels of molecules such as CD45RO, CD62L, CD44, and CCR7 on their surface, which play an important role in maintaining the stability and function of central memory T cells. T cell differentiation follows a progressive process from naive T cells (CD3, CD45RA, CCR7 positive, CD45RO negative) to stem cell-like memory T cells (Tscm, CD3, CD45RA, CD45RO, CCR7 positive), central memory T cells (Tcm, CD3, CD45RO, CCR7 positive), effector memory T cells (Tem, CD3, CD45RO positive, CCR7 negative), and effector T cells (Te, CD3 positive, CD45RO, CCR7 negative). In clinical practice, T cells need to be expanded in large quantities in vitro to meet therapeutic doses. Although some in vitro T cell expansion methods exist, these methods often lead to terminal differentiation of T cells into effector T cells. After reinfusion into the patient, these cells exhibit low proliferation and expansion capacity, severely impacting the efficacy of adoptive T cell therapy. Therefore, obtaining a large number of memory T cells in vitro and reducing terminal differentiation, thereby increasing the differentiation of T cells into Tcm, remains a challenge in cell immunotherapy.

[0006] To address the aforementioned issues, there is an urgent need in this field for a serum-free, trophoblast-free, and highly stem cell-resistant TIL preparation method that can ensure safety and efficacy while shortening the preparation cycle, reducing production costs, and enhancing the stem cell characteristics and sustained killing ability of TIL products. Summary of the Invention

[0007] The purpose of this invention is to address the problem of insufficient tumor-infiltrating lymphocytes in the prior art by providing a complete solution for preparing high-stem TILs under serum-free and trophoblast-free conditions through optimization of culture medium formulation, stimulating factor combination and culture protocol strategy.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] This invention discloses a tumor-infiltrating lymphocyte (TIL) culture medium, which includes: basal culture medium, supplementary components, serum substitutes and antibiotics; the culture medium does not contain animal serum or feeder cell components.

[0010] Preferably, the basal culture medium includes basic amino acids, inorganic salts, and vitamin components, including L-Asp, L-Glu, L-Ile, L-Ser, L-Gly, L-Pro, L-His hydrochloride monohydrate, L-Arg hydrochloride, L-Thr, L-Ala, L-Tyr disodium salt dihydrate, L-Val, L-Met, L-Leu, L-cysteine, L-Phe, L-Lys hydrochloride, L-Trp, L-Asn monohydrate, L-Cys hydrochloride monohydrate, L-hydroxyproline; NaCl, KCl, Na2HPO4, MgSO4, NaHCO3, CaCl2, folic acid, VB12, vitamin C, inositol, and biotin.

[0011] Preferably, the basal culture medium is selected from one or more of X-vivo™, T-vivo™, AIM-V™, CTS™ OpTmizer T (CTS), IMDM, DMEM, DMEM / F12, aMEM, RPMI1640, and M199.

[0012] Preferably, the serum substitute is selected from one or more of SuperGrow™, CTS™ Immune Cell SR, KnockOut™ Serum Replacement, NutriStem® T Cell XF Supplement, or PCT-approved.

[0013] Preferably, the supplementary component consists of albumin, glutamine, interleukin, and eugenol.

[0014] Preferably, the interleukins include interleukin-2, interleukin-7, and interleukin-15.

[0015] Preferably, the albumin is 20-50 g / L, the glutamine is 20-30 μg / mL, the interleukin-2 is 500-2000 IU / mL, the interleukin-7 is 100-2000 IU / mL, the interleukin-15 is 100-2000 IU / mL, and the eugenol is 5-35 μg / L.

[0016] Preferably, the albumin is 30 g / L, glutamine is 25 μg / mL, interleukin-2 is 2000 IU / mL, interleukin-7 is 1000 IU / mL, interleukin-15 is 1000 IU / mL, and eugenol is 20 μg / L.

[0017] Preferably, the culture medium contains the following antibiotics: penicillin-streptomycin: final concentration 2% V / V; gentamicin / amphotericidal B: final concentration 0.02% V / V;

[0018] This invention discloses a method for preparing serum-free, trophoblast-free, high-stem tumor-infiltrating lymphocytes (TILs), comprising:

[0019] a) Tumor tissue processing: After soaking and rinsing the tumor tissue, it is mechanically minced into a paste. Collagenase and nuclease can be used to assist in dissociation. The culture medium described in claims 1-6 is used and the tissue is fully suspended in a centrifuge tube. After standing to remove grease, impurities are removed by sieving to obtain tissue fragments for the separation of tumor-infiltrating lymphocytes.

[0020] b) Isolation and preliminary culture of tumor-infiltrating lymphocytes: The tissue fragments prepared in step a) are added at a concentration of 0.05~0.2g or 0.5~2×10⁻⁶ g / mL. 6 Cells were seeded at a density of 1 cell / mL into each well of a 24-well surface-treated polystyrene cell culture plate containing 1 mL of the aforementioned culture medium. Interleukin-2 was supplemented at 2000 IU / mL, and the cells were cultured at 37°C in 5% CO2 to obtain an initial TIL cell population. After 4–6 days, the cells were replenished with medium or transferred every 2–3 days to adjust the cell density to 1–1.5 × 10⁶ cells / mL. 6 Seeds were added at a rate of 100 cells / mL to new wells; initial TIL culture was complete when lymphocytes were clearly mature and there were no large numbers of adherent cells or dead cell debris. These initial TIL cultures could be collected for cryopreservation or used for further culture.

[0021] c) Activation stimulation of tumor-infiltrating lymphocytes: Add activating factors targeting CD3, CD28 or 4-1BB to the newly cultured TILs obtained in step b), and remove the activating factors after culturing for 48-72 h;

[0022] d) Expansion culture of tumor-infiltrating lymphocytes: The cells prepared in step c) are cultured at a rate of 1~3×10⁻⁶ cells / year. 6 The cells were re-inoculated into the culture medium at a density of cells / mL and cultured for 7-14 days. The expanded tumor-infiltrating lymphocytes were harvested for cell identification, quality testing, and cryopreservation.

[0023] Preferably, the method includes: a) tumor tissue processing: soaking and rinsing the tumor tissue, mechanically cutting it into a porridge-like form, selectively using enzyme-assisted dissociation, fully suspending it in a centrifuge tube using the culture medium, allowing it to stand to remove grease, removing impurities through a sieve, and obtaining tumor tissue fragments;

[0024] b) Isolation and preliminary culture of tumor-infiltrating lymphocytes: The tissue fragments prepared in step a) were seeded into the wells of a cell culture plate containing the aforementioned culture medium, with an additional supplement of interleukin-2 2000 IU / mL, to obtain an initial TIL cell population; after 4-6 days, the aforementioned culture medium was added every 2-3 days or the cells were transferred and the cell density was adjusted to 1-1.5 × 10⁻⁶ cells / mL. 6 Inoculate 1 / mL into a new well;

[0025] c) Activation stimulation of tumor-infiltrating lymphocytes: Add activating factors targeting CD3, CD28 or 4-1BB to the newly cultured TILs obtained in step b), and remove the activating factors after culturing for 48-72 h;

[0026] d) Expansion culture of tumor-infiltrating lymphocytes: The cells prepared in step c) are cultured at a rate of 1~3×10⁻⁶ cells / year. 6 The cells were re-inoculated into the culture medium at a density of cells / mL and cultured for 7-14 days to harvest the expanded tumor-infiltrating lymphocytes.

[0027] Preferably, the activating factor in step c) is selected from anti-human CD3, CD28, or 4-1BB antibodies, active microbeads with CD3, CD28, or 4-1BB antibodies conjugated to their surface, and Transact... TM and Dynabeads TM One or more of the following are used, wherein the antibody is coated on an untreated cell culture dish, and the active microbeads and activator are added directly to the culture medium for 48-72 hours.

[0028] Preferred,

[0029] Preferably, the continued culture step d) includes: counting and replacing the culture medium every 2-3 days, maintaining the cells at 1-5 × 10⁻⁶. 6 cells / mL density.

[0030] Preferably, the culture step can be performed using a breathable polystyrene cell culture box, a breathable culture bag, or a G-Rex culture flask, depending on the volume scale.

[0031] Preferably, the method further includes a step of identifying the cultured tumor-infiltrating lymphocytes.

[0032] Preferably, the method further includes the step of cryopreserving the cultured tumor-infiltrating lymphocytes.

[0033] Preferably, the cell cryopreservation step is performed using a programmed cooling system.

[0034] Preferably, the cryopreservation step involves using a programmed cooling device to lower the temperature to -80°C at a rate of -1°C / min, followed by transfer to liquid nitrogen for long-term storage.

[0035] Preferably, the tumor is selected from cervical cancer, ovarian cancer, endometrial cancer, breast cancer, melanoma, non-small cell lung cancer, gastric cancer, prostate cancer, kidney cancer, testicular cancer, or colorectal cancer.

[0036] Preferably, the product meets the following quality standards:

[0037] - Live cell ratio ≥90%;

[0038] - CD3+ T cell purity ≥90%;

[0039] - CD45+ T cell purity ≥90%;

[0040] - The proportion of Tcm (CD45RO+CD62L+) in CD45+ T cells is ≥70%;

[0041] - Killing efficiency against tumor cells ≥40% (E:T=4:1, 48h);

[0042] - Negative results for sterility, endotoxin, and mycoplasma testing.

[0043] This invention discloses the use of the TIL cell product in the preparation of medicaments for the prevention or treatment of solid tumors, including cervical cancer, ovarian cancer, endometrial cancer, breast cancer, melanoma, non-small cell lung cancer, gastric cancer, prostate cancer, kidney cancer, testicular cancer, or colorectal cancer.

[0044] The method of the present invention uses a low concentration of human interleukin-2 (IL-2) (500~2000 IU / mL) combined with a CD3 / CD28 / 4-1BB multifactor stimulation system to achieve efficient amplification of TILs in a complete culture medium without feeder cells.

[0045] This invention discloses a complete process for efficiently preparing tumor-infiltrating lymphocytes (TILs) with high stemness characteristics under serum-free and feeder-free conditions, including culture medium formulation, culture process, quality control standards, and its application in adoptive immunotherapy for tumors.

[0046] The first aspect of this invention provides a serum-free and trophoblast-free TIL-specific culture medium, the innovation of which lies in:

[0047] 1) Basic culture medium and supplementary culture medium: including basic amino acids, inorganic salts, vitamins and supplementary ingredients including but not limited to albumin, glutamine, interleukin, eugenol, etc., which can take into account both nutritional support and T cell specific optimization, such as maintaining high stemness.

[0048] 2) Low IL-2 concentration: 500~2000 IU / mL, lower than the traditional dosage, to avoid excessive differentiation;

[0049] 3) Multifactor synergistic stimulation: Activating factors targeting CD3, CD28 or 4-1BB, such as anti-human CD3, CD28 or 4-1BB antibodies, or active microbeads with CD3, CD28 or 4-1BB antibodies on their surface, are combined to simulate physiological TCR activation signals.

[0050] 4) Completely serum-free: Use 2-5% serum substitute to eliminate batch variations and pathogen risks;

[0051] 5) No feeder cells: The formula does not contain any exogenous cell components.

[0052] In another aspect, this invention provides a two-step culture process that eliminates the use of any feeder cells or animal serum throughout the entire process from isolation to harvest, simplifying the process and improving safety.

[0053] 1) Initial culture stage: Using a strategy of mechanically cutting up tumor tissue and stimulating T cell isolation and metastasis, tumor-infiltrating lymphocytes (TILs) are isolated, expanded, metastasized, and collected from tumor tissue in a complete culture medium containing serum substitutes, IL-2, and antibiotics;

[0054] 2) Expansion stage: First, activate TIL cells with activating factors. After culturing for 48-72 hours, remove the activator and continue culturing in a complete medium containing serum substitute, low IL2 and antibiotics for 7-14 days. During the process, count the cells every 2-3 days and replace and replenish the medium as needed to maintain the cells at a suitable density.

[0055] 3) Cell cryopreservation or cryopreservation uses a serum-free storage solution system.

[0056] In another aspect, this invention establishes quality standards for high-dryness TILs, requiring the final product to meet the following requirements:

[0057] 1) Tcm ratio (CD45RO+CD62L+) ≥70%: unconventional methods (usually <50%) ensure long-term survival in vivo and guarantee rapid response capability after secondary antigen stimulation;

[0058] 2) High purity: CD3+, CD45+ cells ≥90%, viability ≥90%;

[0059] 3) Strong killing ability: Killing efficiency against tumor cells ≥40% (E:T=4:1, 48h);

[0060] 4) Negative results for sterility, endotoxin, and mycoplasma testing.

[0061] Compared with the prior art, the present invention has the following significant advantages:

[0062] 1) Significantly improved safety: Completely eliminates the risk of exogenous contamination from feeder cells and animal serum, meeting GMP production standards for cell therapy products; IL-2 dosage is reduced by more than 80% during the expansion stage, significantly reducing systemic toxicity after reinfusion.

[0063] 2) Significantly enhanced stem cell characteristics: By optimizing the culture medium system, selecting the best serum substitute, shortening the overall culture time, adopting low IL-2 maintenance, and optimizing stimulating factors, the Tcm ratio was increased by 30-50%, giving TILs stronger in vivo persistence and self-renewal capacity (see [Appendix]). Figure 1 [Schematic diagram of T cell subset differentiation].

[0064] 3) Simplified process and reduced costs: No need to prepare and quality control feeder cells, the culture cycle is shortened to 14-28 days (traditional process requires 21-42 days), labor costs are reduced by 40%, making it more suitable for industrial production and clinical applications.

[0065] Durable and stable killing ability: Terminal TIL cells maintained a killing efficiency of >60% in three rounds of repeated killing experiments (see [Appendix]). Figure 3 []), with no obvious depletion phenomenon, which is superior to traditional methods (the third round of killing efficiency is usually <30%).

[0066] The method used in this invention avoids the safety risks associated with trophoblast cells, reduces IL-2-related side effects, shortens the preparation cycle, significantly enhances the stem cell characteristics and sustained killing ability of TIL cells, and meets the quality control requirements of clinical-grade cell therapy products. Attached Figure Description

[0067] Figure 1 Initial stage TIL viable cell count (left) and viability (right) (Pre is the experimental group, Con is the control group). TIL cells successfully completed the initial stage of isolation and expansion.

[0068] Figure 2 The initial flow cytometry results for TIL cell phenotype and stem cell (Tcm cell ratio) showed that the TIL cell phenotype was normal.

[0069] Figure 3 Comparison of the proliferation curves and viability (cell number vs. culture days, TIL as the experimental group and Con. as the control group) of TIL cells prepared by expansion culture.

[0070] Figure 4 Comparison of flow cytometry results of TIL cell phenotype and stemness (Tcm cell ratio) prepared by expansion culture (TIL is the experimental group, Con.1 and 2 are the control groups). The Tcm ratio of this system was significantly improved compared with the control system.

[0071] Figure 5The cytotoxicity of TIL cells in in vitro tumor cell killing experiments was compared. Figures A and B show the real-time cell index curves of SKOV3 ovarian cancer cells killed, and Figures C and D show the real-time cell index curves of HeLa cervical cancer cells killed. Figures A and C show an E:T ratio of 4:1, and Figures B and D show an E:T ratio of 1:1. Immune cells were added at the vertical line (TIL is the experimental group, Con.1 and 2 are the control groups, and Tumor Cell Only is the cancer cell growth group). The decrease in the vertical axis of the curves indicates that the tumor cells were killed by immune cells. The TIL cells prepared in this system have a stronger in vitro tumor cell killing function and can maintain it for a longer time compared with the control group. Detailed Implementation

[0072] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0073] Example 1: Preparation of serum-free and nutrient-free TIL medium and initial TIL isolation and culture

[0074] 1.1 Preparation of complete culture medium:

[0075] Prepare 1L of complete culture medium according to the following composition:

[0076] - Basic culture medium: X-Vivo TM (or AIM-V™), 1L;

[0077] - Serum substitute: SuperGrow™ 30 mL (3% V / V);

[0078] - Supplemental ingredients: glutamine 25μg / mL, albumin 30g / L, recombinant human interleukin-2 2000 IU / mL, recombinant human interleukin-7 1000 IU / mL, recombinant human interleukin-15 1000 IU / mL, eugenol 20μg / L;

[0079] - Antibiotics: Penicillin-Streptomycin: final concentration 2% V / V; Gentamicin / Amphotericin B: final concentration 0.02% V / V.

[0080] The above is the complete culture medium of this invention. The control complete culture medium is X-Vivo. TM Culture medium + 10% human serum + antibiotics (same as above).

[0081] 1.2 Tumor tissue acquisition and TIL isolation:

[0082] Tumor tissue (approximately 1g) was obtained from a surgical specimen removed from a patient with malignant cervical cancer and thoroughly immersed in phosphate-buffered saline or tissue transport fluid such as EzTissue Plus. TM Transported to the T-cell culture laboratory at 4℃ (<12h). After thoroughly soaking and rinsing with the culture medium described in 1.1, the cells were mechanically minced into a paste, added to 30mL of the culture medium in a centrifuge tube, thoroughly resuspended, allowed to stand to remove oil, and then sieved through a 70μm sieve to remove impurities.

[0083] 1.3 Initial TIL isolation and culture (days 0-12):

[0084] Tissue cells were administered at doses of 0.05–0.2 g or 0.5–2 × 10⁻⁶ g. 6 Cells were seeded into the wells of a 24-well surface-treated polystyrene cell culture plate containing 1 mL of the complete culture medium described in step 1.1. During the initial culture phase, interleukin-2 was supplemented at 2000 IU / mL. The cells were cultured at 37°C in 5% CO2 to obtain an initial TIL cell population (note the need to maintain low volatility; the supernatant can be carefully aspirated and fresh culture medium added after 1-2 days). After 4-6 days, the cells were replenished or transferred every 2-3 days to adjust the cell density to 1-1.5 × 10⁶ cells / well. 6 Cells / mL (Observe cells under a microscope; if there is no significant proliferation of suspended aggregated lymphocytes around the tissue block, and no decrease in adherent cancer cells or other cells, change the medium; if lymphocytes proliferate significantly and fill the entire well, carefully aspirate half of the culture medium from the top and discard it, then mix the cells at the bottom and count them. Adjust the cell density to 1~1.5×10⁶ cells / mL with complete culture medium). 6 Seeds were inoculated into new wells at a density of 100 cells / mL; if the lymphocyte density in most wells was high, such as greater than 2-3 × 10⁶ cells / mL... 6 / mL, the concentration can be adjusted to 1×10 after filtration through a sieve. 6 (Continue culturing at / mL) until lymphocytes are clearly mature and there are no large numbers of adherent cells and dead cell tissue debris; this completes the initial TIL culture. These initially cultured TIL cells can be collected for cryopreservation or used for further culture.

[0085] 1.4 Initial TIL cell count, viability, and phenotypic detection:

[0086] - Proliferation and viability: Trypan blue staining was used to count and assess viability: the cell count reached 4.5 × 10⁻⁶ on day 12. 7 The survival rate was 89% (control group size 3.0 × 10⁶). 7 (Survival rate 77%). Figure 1 ].

[0087] - Flow cytometry phenotyping: Cells were labeled with flow cytometry antibodies against CD3, CD4, CD8, CD45, CD45RO, CD62L, and CCR7, and analyzed by flow cytometry. The results are shown in […]. Figure 2 The initial TIL cells contained 46.6% CD4+ cells, 47.2% CD8+ cells, and 64.3% stem Tcm (CD45RO+, CD62L+).

[0088] Example 2: Preparation, Performance Verification, and Quality Testing of Expanded TILs

[0089] 2.1 Preparation of culture medium for the control culture system

[0090] Control conventional culture medium 1: X-vivo™ medium + 10% human AB serum + 2000 IU / mL IL-2, with the addition of irradiated feeder cells (PBMC source, TIL:Feeder=1:25).

[0091] Control medium 2: X-vivo™ medium + 10% human AB serum + 2000 IU / mL IL-2, without the addition of irradiated feeder cells.

[0092] 2.2 TIL activation stimulation and expansion (days 1-13 of this phase, days 12-24+ in total):

[0093] In Example 1, tumor cells (TILs) isolated from the initial culture stage of tumor tissue from the same cervical cancer patient were divided into several groups and cultured using different culture media as described above (the complete culture medium in Example 1, i.e., the experimental group, and control groups 1 and 2 in 2.1). The media were cultured at a rate of 2 × 10⁻⁶. 6 Re-inoculate cells / mL into fresh culture medium, and add Dynabeads active microbeads with CD3 and CD28 antibodies on their surface during the initial stage of amplification culture. TM After culturing for 48 hours, remove the activator using a magnetic plate / rack; continue culturing for 10-12 days, counting cells and replacing / replenishing the culture medium every 2-3 days to maintain cell density at 1-5 × 10⁶ cells / day. 6 Cells / mL density (depending on the volume, breathable polystyrene cell culture plates, culture boxes, culture bags or G-Rex culture flasks with untreated surfaces for tissue culture can be used).

[0094] 2.3 Harvesting and Freezing:

[0095] Terminal TIL cells were collected, counted, and viability was determined by trypan blue staining. Results are shown in [Appendix]. Figure 3 Cell expansion (at 5 × 10⁶ cells per sample). 5(Initial calculation): 7.36E7 cells were obtained in the experimental group, and 8.25E7 and 5.31E7 cells were obtained in the control group. The experimental group achieved higher cell expansion, but there was no statistical difference (p>0.05); Cell viability: 96.8% in the experimental group vs 94.2% in the control group (p<0.05), showing better survival.

[0096] Use cryopreservation solution such as Cellsaving TM Perform cryopreservation (resuspend to 1×10⁻⁶) 7 (cells / mL, 1 mL / vial aliquot, cooled to -80°C at a rate of -1°C / min using a programmed cooling device, then transferred to liquid nitrogen for long-term storage).

[0097] 2.4 Phenotypic and Quality Testing

[0098] Phenotypic analysis was performed using flow cytometry. Cells were labeled with flow cytometry antibodies against CD3, CD4, CD8, CD45, CD45RO, CD62L, and CCR7, and the results were analyzed by flow cytometry. See [Appendix]. Figure 4 ], where TIL is the experimental group of this scheme, and Con.1 and Con.2 are the traditional culture control groups 1 and 2 described in 2.1:

[0099] - Purity and typing: The experimental group had a final TIL cell (CD3+) percentage of >97% and a CD8+ percentage of 72.4% (the control groups 1 and 2 had a TIL cell (CD3+) percentage of >97% and a CD8+ percentage of 58.1% and 73.5%, respectively).

[0100] - Dryness markers: The proportion of dry Tcm (CD45RO+, CD62L+) in the experimental group was 74.6%; the proportion of dry Tcm in the control groups 1 and 2 was 32.6% and 56.5% respectively, which were much lower than those in the experimental group and showed a significant difference (p<0.05).

[0101] - Cost analysis: Compared with the control group, the experimental group reduced the culture cost by about 50% because it did not require the purchase of irradiated feeder cells and human AB serum was replaced with serum substitutes.

[0102] - Lethality: See Example 3;

[0103] - Safety: Tests for bacteria, fungi, mycoplasma, and endotoxins were all negative. Compared to the control group, the experimental group's culture system eliminated the need for irradiated feeder cells and replaced human AB serum with a serum substitute, significantly reducing the introduction of exogenous bioactive substances. The components were clearly defined and reliable, resulting in higher safety.

[0104] Example 3: Evaluation of in vitro tumor cell killing ability

[0105] 3.1. Preparation of tumor target cells (the day before yesterday):

[0106] HeLa and SKOV3 cancer cells were cultured normally in DMEM medium containing 10% FBS and were in the logarithmic division stage. They were then passaged normally after digestion. 1E6 target cells were resuspended in 1ml medium, counted, and the cell concentration was adjusted to 2E5 / ml after calculation based on the viable cell density. 50μL of DMEM containing 10% FBS was added to each well of the kill plate (E-plate 16). The kill plate was placed in the RTCA station located in the incubator. After setting the RTCA program, the baseline was detected, and 50μL of target cells at a concentration of 2E5 / ml was added to each well to continue overnight (24h) culture-monitoring.

[0107] 3.2. Immunization of cells (on the same day):

[0108] The TIL cells prepared by this invention (experimental group) were compared with TILs prepared by conventional methods (control group, such as conventional culture control groups 1 and 2 described in 2.1). The concentration of the immune cells to be tested was adjusted to equivalent effective target ratios of 1:1, 1:4, and 1:8 according to the positive rate and the ratio of effector cells to tumor cells (E:T). Cell suspensions were prepared. The killing plates were cultured for 24 hours and then removed when the Cell Index was around 1. 50 μL of immune cell culture medium (without IL-2) was added to several wells as a Tumoronly control. 50 μL of the above T cell suspension was added to each well of the experimental group. The killing plates were returned to the RTCA instrument for further detection.

[0109] 3.4. Data Analysis:

[0110] Approximately 120 hours later, the cell-killing plate was removed and the cell index was analyzed based on the exponential curve or by reading the cell index values ​​at selected time points (data was presented in two ways: exponential curve and %Cytolysis, where %Cytolysis = [CI(No effector) – CI(Effector)] / CI(No effector)*100%). The comparison results between the TIL cells prepared by this invention (experimental group) and TIL cells prepared by traditional methods (control group) are as follows, see [Appendix]. Figure 5Figures A and B show the real-time cell index curves of SKOV3 ovarian cancer cell killing, while figures C and D show the real-time cell index curves of HeLa cervical cancer cell killing. Figures A and C show an E:T ratio of 4:1, and figures B and D show an E:T ratio of 1:1. Immune cells were added at the vertical line and the killing time was recorded as the starting point (Tumor cell only means no immune cells were added; TIL means the experimental group with this protocol added; Con.1 and Con.2 are the traditional culture control groups described in 2.1). The decrease in the vertical axis of the curves indicates that the tumor cells were killed by immune cells. It can be seen that the expanded and cultured TIL has significant killing effect, which increases with the increase of E:T; the killing effect of the experimental group is stronger than that of the control group; at the same time, the killing time is maintained for a longer period of time on the right side of the horizontal axis.

[0111] - 48h: The expanded and cultured TILs showed significant killing effect, which increased with increasing E:T ratio. The killing rate against HeLa cervical cancer cells was 62% at E:T=4:1 (compared to 48% and 47% in the control group), and 48% at E:T=1:1 (compared to 36% and 18% in the control group, p>0.05). The killing rate against SKOV3 ovarian cancer cells was 93% at E:T=4:1 (compared to 69% and 52% in the control group, p>0.05), and 54% at E:T=1:1 (compared to 24% and 25% in the control group, p>0.05).

[0112] - After 72-96 hours: The expanded and cultured TILs maintained a longer killing effect on cancer cells. The experimental group (E:T=1:1) maintained a killing rate of ~40% against HeLa cervical cancer cells, while the control group decreased to 0-40% (p<0.001); against SKOV3 ovarian cancer cells, it maintained a killing rate of ~70%, while the control group decreased to ~30% (p<0.001). This indicates that the experimental group may have a significant exhaustion resistance advantage, enabling it to kill tumor cells for a prolonged period.

[0113] The present invention has been illustrated through the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A culture medium for tumor-infiltrating lymphocytes (TILs), characterized in that, The culture medium includes: basal culture medium, serum substitute, supplementary components, and antibiotics; the culture medium does not contain animal serum or feeder cell components.

2. The culture medium according to claim 1, characterized in that, The basal culture medium includes L-Asp, L-Glu, L-Ile, L-Ser, L-Gly, L-Pro, L-His hydrochloride monohydrate, L-Arg hydrochloride, L-Thr, L-Ala, L-Tyr disodium salt dihydrate, L-Val, L-Met, L-Leu, L-cysteine, L-Phe, L-Lys hydrochloride, L-Trp, L-Asn monohydrate, L-Cys hydrochloride monohydrate, L-hydroxyproline; NaCl, KCl, Na2HPO4, MgSO4, NaHCO3, CaCl2, folic acid, VB12, vitamin C, inositol, biotin; or selected from one or more of X-VIVO™, T-VIVO™, AIM-V™, CTS™ OpTmizer T (CTS), IMDM, DMEM, DMEM / F12, aMEM, RPMI1640, and M199.

3. The culture medium according to claim 1, characterized in that, The serum replacement is selected from one or more of SuperGrow™, CTS™ Immune Cell SR, KnockOut™ Serum Replacement, NutriStem® T Cell XFSupplement, or PCT-approved.

4. The culture medium according to claim 1, characterized in that, The supplementary components include albumin, glutamine, interleukin, and eugenol.

5. The culture medium according to claim 4, characterized in that, The interleukins include interleukin-2, interleukin-7, and interleukin-15.

6. The culture medium according to claim 5, characterized in that, The concentrations of albumin (20-50 g / L), glutamine (20-30 μg / mL), interleukin-2 (500-2000 IU / mL), interleukin-7 (100-2000 IU / mL), interleukin-15 (100-2000 IU / mL), and eugenol (5-35 μg / L) are specified.

7. A method for preparing serum-free, trophoblast-free, high-stem tumor-infiltrating lymphocytes (TILs), characterized in that, include: a) Tumor tissue processing: After soaking and rinsing the tumor tissue, it is mechanically minced into a paste. Enzyme-assisted dissociation can be selectively used. The tissue is fully suspended in a centrifuge tube using the culture medium described in claims 1-6, allowed to stand to remove grease, and impurities are removed through a sieve to obtain tumor tissue fragments. b) Isolation and preliminary culture of tumor-infiltrating lymphocytes: The tissue fragments prepared in step a) are seeded into the wells of a cell culture plate containing the culture medium described in claims 1-6, with additional interleukin-2 2000 IU / mL, to obtain an initial TIL cell population; after 4-6 days, the culture medium described in claims 1-6 is added every 2-3 days or the cell density is transferred and adjusted to 1-1.5 × 10⁻⁶ cells / mL. 6 Inoculate 1 / mL into a new well; c) Activation stimulation of tumor-infiltrating lymphocytes: Add activating factors targeting CD3, CD28 or 4-1BB to the newly cultured TILs obtained in step b), and remove the activating factors after culturing for 48-72 h; d) Expansion culture of tumor-infiltrating lymphocytes: The cells prepared in step c) are cultured at a rate of 1~3×10⁻⁶ cells / year. 6 The cells were re-inoculated at a density of cells / mL in the culture medium described in claims 1-6 and cultured for another 7-14 days to harvest the expanded tumor-infiltrating lymphocytes.

8. The method according to claim 7, characterized in that, The activating factor in step c) is selected from anti-human CD3, CD28, or 4-1BB antibodies, active microbeads with CD3, CD28, or 4-1BB antibodies conjugated to their surface, and the activator Transact. TM and activator Dynabeads TM One or more of them.

9. The method according to claim 7, characterized in that, The continued culture step described in step d) includes: counting cells every 2-3 days and replenishing or replacing the culture medium as described in claims 1-6 as needed, maintaining the cells at a temperature of 1-5 × 10⁻⁶. 6 The density is 10 cells / mL, and the culture period is 7-14 days.

10. The method according to claim 7, characterized in that, The tumor is selected from cervical cancer, ovarian cancer, endometrial cancer, breast cancer, melanoma, non-small cell lung cancer, gastric cancer, prostate cancer, kidney cancer, testicular cancer, or colorectal cancer.

Images