Method for improving TIL production quality by depriving aspartic acid to regulate immune metabolism

Abstract

The invention provides a method for improving TIL production quality by depriving aspartic acid to regulate immune metabolism. The method comprises an initial amplification stage and a rapid amplification stage, wherein in the later culture stage of the initial amplification stage and / or the rapid amplification stage, the cells are transferred into a deprived aspartic acid culture medium for culture until the cells of each stage are harvested. By adopting the method disclosed by the invention, the TIL multiplication capacity can be enhanced on the basis of the existing TIL amplification process, the risks of insufficient multiplication power and limited amplification in the amplification process are reduced, and a TIL amplification product meeting the feedback requirement is obtained. The method can also improve the immune effect function of an amplified final product, so that the anti-tumor immune effect of the TIL preparation for reinfusion is improved; the TIL metabolic state can be improved, the mitochondrial function can be improved, the oxidative stress level can be reduced, and the effective and continuous anti-tumor reaction can be maintained in vivo after reinfusion.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for improving TIL production quality by depriving aspartic acid to regulate immune metabolism. Background Technology

[0002] Tumor-infiltrating lymphocyte (TIL) therapy is a novel cellular immunotherapy that has made significant progress in the field of cancer treatment in recent years. This technology is based on the patient's own immune system, extracting, expanding, and reinfusing tumor-infiltrating lymphocytes (TILs) to attack tumor cells, exhibiting high specificity and safety. 1. Technical Principle: TIL therapy extracts TILs that have infiltrated and exhibit anti-tumor activity from the patient's tumor tissue. After laboratory culture and expansion, these TILs are reinfused to activate and enhance the immune response targeting tumor cells. 2. Development History: Research on this therapy began in the 1980s when Professor Steven A. Rosenberg first discovered that TILs could inhibit the metastasis of malignant melanoma cells. With technological advancements, it has gradually moved towards clinical application and achieved significant results. 3. Technical Characteristics: These include high specificity (able to recognize and attack tumor cells), high safety (low risk of immune rejection), and significant efficacy (showing good performance in various solid tumors such as melanoma, lung cancer, and breast cancer). 4. Clinical Application: TIL therapy has undergone multiple clinical trials worldwide and has received FDA approval for unresectable or metastatic melanoma, showing promising application prospects. 5. Future Outlook: With continuous technological advancements, TIL therapy is expected to achieve breakthroughs in the treatment of more tumor types, becoming an important means of tumor immunotherapy. Researchers will continue to optimize its preparation process and treatment methods to improve efficacy and safety, reduce costs, and benefit more patients. In conclusion, TIL therapy has significant application value and broad development prospects in the field of tumor treatment.

[0003] Regarding methods for expanding tumor-infiltrating lymphocytes (TILs), a novel method for culturing and in vitro expanding TILs from primary liver cancer is disclosed in patent application number CN202110292616. This method includes the following steps: 1. Cleaning the surface of the liver cancer tissue specimen and removing adipose tissue; 2. Cutting the tissue into small pieces and seeding them into wells, culturing them in primary culture medium; 3. After a certain number of tumor-infiltrating lymphocytes have emerged from the wells, mixing them with feeder cells and adding them to complete culture medium for further culture; 4. After culturing until at least some of the feeder cells have died, collecting the cells and cryopreserving them to obtain tumor-infiltrating lymphocytes. This method is rapid, reliable, and cost-effective, enabling the acquisition of a sufficient number of highly active lymphocytes in a short time, making it suitable for cell therapy (such as ACT) and providing a good foundation for treatment methods such as CAR-T and TCRT. Patent application number CN202380015383 discloses a method for obtaining tumor-derived intracellular lipid cells (TILs) through the following steps: First, TILs derived from tumor tissue undergo at least one stage of in vitro expansion; in each in vitro expansion stage, the in vitro expanded and / or unexpanded TILs are contacted with T cell activators and / or T cell growth factors for a certain period of time, followed by co-culturing with feeder cells. This method can effectively increase the quantity and activity of TILs, providing a new drug basis for tumor treatment and possessing significant clinical application value.

[0004] However, existing TIL culture methods still have the following shortcomings: 1) The amplification process is highly dependent on the starting material, and insufficient proliferation activity easily leads to limited amplification: Existing TIL preparation procedures typically include two stages: "initial amplification (pre-REP, approximately 2–4 weeks)" and "rapid amplification (REP, approximately 14 days)." The overall culture cycle is relatively long, and the amplification effect largely depends on the quality of the tumor sample, the amount of tumor tissue, and the quantity and functional status of the initial TILs. For samples with limited tumor tissue or low TIL infiltration, insufficient proliferation kinetics, slow amplification rate, or limited amplification often occur during the amplification process, making it difficult to consistently obtain cell products that meet the reinfusion requirements.

[0005] 2) The amplification end products are prone to functional limitation / exhaustion-like phenotypes and insufficient immune effect: Existing TIL amplification systems mostly rely on continuous stimulation with high doses of cytokines (such as IL-2) to promote rapid cell proliferation. However, such strong stimulation conditions can also induce T cells to develop towards terminal differentiation and exhibit functional limitation-related characteristics such as increased expression of inhibitory checkpoint molecules (such as PD-1). This is accompanied by cytotoxic effects and decreased or unstable cytokine secretion capacity, which affects the anti-tumor immune effect of TIL preparations reinfused.

[0006] 3) Limited metabolic adaptability and antioxidant capacity of the amplified end products affect the effectiveness and sustainability of the response after reinfusion: The tumor microenvironment is generally characterized by nutritional constraints and enhanced oxidative stress, requiring high metabolic adaptability and antioxidant capacity of the reinfused TIL. Currently amplified TILs often have insufficient metabolic adaptability and limited antioxidant capacity, making them more prone to functional failure in the unfavorable tumor microenvironment, thus limiting the effectiveness and sustainability of the in vivo antitumor response after reinfusion. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for improving TIL production quality by depriving aspartic acid to regulate immune metabolism.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for improving the production quality of TILs by regulating immune metabolism through aspartate deprivation, the method comprising an initial expansion phase and a rapid expansion phase; wherein, in the later stages of the initial expansion phase and / or the rapid expansion phase, the cells are cultured in an aspartate-deprived medium until the cells at each stage are harvested.

[0009] Preferably, the late stage of culture refers to 3 days before the expected harvest date, at which time changing the medium to aspartate-deprived medium will yield TIL cells of the best quality. If aspartate-deprived medium is cultured at an earlier stage (e.g., 4-5 days before harvest) or a later stage (e.g., 1-2 days before the expected harvest), the quality of the obtained TILs will decrease.

[0010] Preferably, the method involves changing the cell medium to an aspartate-deprived medium during the later stage of the initial expansion phase. The initial expansion phase includes the following steps: A1. Take isolated tumor tissue, cut it into small pieces, and inoculate it into intact culture medium for culture; A2. Three days before the expected harvest date, change the medium to aspartic acid-deprived medium and continue culturing. A3. TIL cells were harvested after culturing in an aspartate-deprived medium for 3 days. The rapid expansion phase includes the following steps: B1. The TIL cells and feeder cells harvested in the initial expansion phase were added to a complete culture medium and stimulants were added. The culture was then recorded as day 0. B2. After culturing for 6 days, use complete cell culture medium for scale-up culture until TIL cells are harvested.

[0011] Preferably, the method involves changing the cell medium to aspartate-deprived medium during the later stages of rapid amplification, and the initial amplification stage includes the following steps: A11. Take isolated tumor tissue, cut it into small pieces, and inoculate it into intact culture medium for culture until the harvest day to harvest TIL cells; The rapid expansion phase includes the following steps: B11. The TIL cells and feeder cells harvested in the initial expansion phase were added to a complete culture medium and stimulants were added. The culture was then recorded as day 0. B12. After culturing to day 6, the cells were scaled up using intact cell culture medium. Three days before the expected harvest date, the cells were changed to medium containing aspartic acid deprivation for continued scale-up culture. B13. After culturing in aspartic acid-deprived medium for 3 days, TIL cells were harvested.

[0012] Preferably, the method involves changing the cell medium to aspartate-deprived medium during the initial amplification phase and the later stages of the rapid amplification phase. The initial amplification phase includes the following steps: A1. Take isolated tumor tissue, cut it into small pieces, and inoculate it into intact culture medium for culture; A2. Three days before the expected harvest date, change the medium to aspartic acid-deprived medium and continue culturing. A3. TIL cells were harvested after culturing in an aspartate-deprived medium for 3 days. The rapid expansion phase includes the following steps: B11. The TIL cells and feeder cells harvested in the initial expansion phase were added to a complete culture medium and stimulants were added. The culture was then recorded as day 0. B12. After culturing to day 6, the cells were scaled up using intact cell culture medium. Three days before the expected harvest date, the cells were changed to medium containing aspartic acid deprivation for continued scale-up culture. B13. After culturing in an aspartate-deprived medium for 3 days, TIL cells were harvested.

[0013] Preferably, in the initial expansion phase, the total cell culture time is 2 to 4 weeks; in the rapid expansion phase, the total cell culture time is 13 to 14 days.

[0014] Preferably, the complete culture medium comprises: basal culture medium, 6000 IU / ml IL-2, 10% human AB serum and 1% gentamicin.

[0015] Preferably, the aspartate-deprived culture medium comprises: a basal medium with an L-aspartate content of 0–2 mg / L, 6000 IU / ml IL-2, 10% human AB serum, and 1% gentamicin. The basal medium with an L-aspartate content of 0–2 mg / L refers to a culture medium obtained by reducing the L-aspartate content to 0–2 mg / L from a conventional L-aspartate-containing basal medium.

[0016] More preferably, the aspartic acid deprivation medium includes a basal medium with an L-aspartic acid content of 0.

[0017] Preferably, the basal culture medium is selected from RPMI-1640 medium.

[0018] Preferably, in step B1 or B11, the feeder cells are irradiated peripheral blood mononuclear cells.

[0019] Preferably, the ratio of TIL cells to feeder cells is: 2-3 × 10⁻⁶ cells per 2 million TIL cells. 8 Each feeder cell.

[0020] Preferably, the stimulant includes at least one of anti-CD3 antibody and IL-2.

[0021] Preferably, the final concentration of the added stimulant is 30 ng / ml.

[0022] Preferably, in step B2 or B12, maintaining a cell density of 1 million / ml during the scale-up culture is beneficial for amplification and quality.

[0023] Preferably, in step A2 or B12, the expected harvest date is determined based on the time required for the TIL to grow to fill the wells of a 24-well plate (approximately 2 million); for example, if the day when the complete culture medium is added in step A1 is taken as day 0 of the culture, and based on the observation of TIL growth, it is estimated that it will take 20 days of culture to fill the wells of a 24-well plate, then the expected harvest date is day 20 of the culture.

[0024] In step B12, the time required for the number of TILs to meet the patient's infusion dose requirement is determined. For example, after scale-up culture in step B12, if the number of TILs is expected to reach the patient's infusion dose requirement based on observation of TIL growth, it is estimated that the culture will continue until day 13. Therefore, the expected harvest day is day 13 of culture.

[0025] Secondly, the present invention provides a method for obtaining TIL according to the aforementioned method, wherein the TIL CD8 + T cells accounted for more than 80%, CD4 +The percentage of T cells is over 14%, the percentage of GZMB+ expression is over 32%, the percentage of TNFα+ expression is over 610%, the percentage of IFNγ+ expression is over 50%, and the percentage of PD-1+ and TCF-1+ expression is over 310%.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a method to enhance the proliferation capacity of TILs based on existing TIL amplification processes, thereby reducing the risk of insufficient proliferation kinetics and limited amplification during the amplification process, and obtaining TIL amplification products that meet the reinfusion requirements.

[0027] 2. The method of the present invention can improve the immune effector function of the amplified end product, increase the proportion of effector T cells with cytotoxic effects and cytokine secretion capabilities in the amplified TIL or enhance the effector intensity, while reducing the increase in inhibitory checkpoint expression and functionally limited / exhausted phenotypic characteristics, thereby enhancing the anti-tumor immune effect of the reinfused TIL preparation.

[0028] 3. The method of the present invention can improve the metabolic state of TIL, improve mitochondrial function and reduce oxidative stress level, which is beneficial to maintaining an effective and sustained anti-tumor response in vivo after reinfusion. Attached Figure Description

[0029] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The results of CFSE dilution experiments were performed on the TILs harvested in Example 1 and Comparative Example 1; wherein, Figure 1 A is a flow cytometry plot of CFSE staining; Figure 1 B is its MFI (mean fluorescence intensity) value; Figure 2 The results of flow cytometry analysis of the TILs harvested in Example 1 and Comparative Example 1 are shown; wherein, Figure 2 A is a flow cytometry chromatogram of CD4 / CD8 staining; Figure 2 B is CD4 + Cell percentage results; Figure 2 C is CD8 + Cell percentage results; Figure 3 The results show the cytotoxic effects and cytokine secretion capacity of TILs harvested in Example 1 and Comparative Example 1; wherein, Figure 3 A is a flow cytometry analysis of GAMB / TNFα / IFNγ staining; Figure 3 B is GZMB + Cell percentage results; Figure 3 C is TNFα +Cell percentage results; Figure 3 D is IFNγ + Cell percentage results; Figure 4 The results of TILs obtained in Example 1 and Comparative Example 1 were obtained from measurements of phenotypic characteristics related to reduced inhibitory checkpoints and functional limitation; wherein, Figure 4 A is a flow cytometry analysis chromatogram of PD-1 / TCF-1 staining; Figure 4 B is PD-1 + and TCF-1 + Results of the percentage of double-positive cells; Figure 4 C is PD-1 + Cell percentage results; Figure 5 The results of the assays conducted on TILs harvested in Example 1 and Comparative Example 1 regarding their effects on improving mitochondrial structure and mitochondrial-related metabolic characteristics, and reducing oxidative stress levels; wherein, Figure 5 A represents the results observed using transmission electron microscopy; Figure 5 B shows the comparison between the number of mitochondria in a single cell (left image) and the number of cristae in a single mitochondria (right image); Figure 5 C represents the results of Mito-Tracker Green staining; the left figure is the flow cytometry analysis plot, and the right figure is its MFI value. Figure 5 D represents the results of Mito SOX Red staining. The left figure is the flow cytometry analysis plot, and the right figure is its MFI value. Figure 6 The results of the assays performed on the TILs harvested in Example 1 and Comparative Example 1 in reducing lipid peroxidation-related damage and enhancing antioxidant stress capacity are as follows; wherein, Figure 6 A is a flow cytometry chromatogram of BODIPY 581 / 591 staining; Figure 6 B is its MFI value; Figure 6 C represents the MDA ELISA test result. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0031] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0032] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0033] 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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] In the following examples, all raw materials, reagents, and biological materials used were obtained through purchase or conventional methods, and the present invention does not impose any particular limitations. For example, the RPMI-1640 medium described in the following examples was purchased from Gibco (a subsidiary of Thermo Fisher Scientific); the L-aspartic acid-free RPMI-1640 medium was prepared by Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., which, compared with the conventional RPMI-1640 medium, only lacked the addition of L-aspartic acid; the anti-CD3 antibody (OKT3) was purchased from eBioscience (a subsidiary of Thermo Fisher Scientific).

[0035] Example 1 This embodiment provides a method for improving TIL production quality by regulating immune metabolism through aspartate deprivation, including the following steps: I. Initial Amplification (pre-REP) 1. Starting Material Acquisition. The starting material for TIL amplification was surgically removed tumor tissue (from Union Hospital affiliated with Tongji Medical College of Huazhong University of Science and Technology, with the patient's signed consent). After observing the gross tumor specimen, the sampling site was determined, prioritizing highly vascularized areas near the tumor margin and avoiding areas with obvious necrosis. The sampling site was rinsed and disinfected with 0.5% povidone-iodine solution. After wearing sterile gloves, a piece of tumor tissue at least the size of a soybean (approximately 0.5cm × 0.5cm × 0.5cm) was cut using sterile surgical instruments. The tumor tissue was placed in a sterile transport medium and transported to the laboratory under ice-cold conditions. The transport medium consisted of DMEM high-glucose medium + 1% gentamicin.

[0036] 2. Culture system preparation. Add 2 ml of complete culture medium to each well of a 24-well culture plate. The complete culture medium includes RPMI-1640 medium, 6000 IU / ml IL-2, 10% human AB serum, and 1% gentamicin. The number of culture wells is determined based on the amount of tumor tissue.

[0037] 3. Tumor Tissue Processing. Using sterile forceps, remove the tumor tissue from the transport container and place it in a sterile culture dish. From this step onward, minimize the time the tissue is exposed to air to avoid drying. Remove as much necrotic and fatty tissue as possible around the tumor tissue, as these are often high-risk sources of microbial contamination.

[0038] 4. Inoculation. Cut the tumor tissue into small tissue blocks of approximately 1–3 mm³, and place each tissue block into a well of a 24-well plate pre-filled with complete culture medium. Incubate the 24-well plate in a cell culture incubator (37 °C, 5% CO2).

[0039] 5. Contamination Monitoring. Observe the culture status of each well daily using an inverted microscope to monitor microbial contamination and TIL growth. For culture wells showing microbial contamination, aspirate and discard the culture medium, and clean the well with PBS solution containing 1% gentamicin. Contamination usually manifests as abnormal color or turbidity of the culture medium, and in some cases can only be observed under a microscope. Cross-contamination should be avoided during operation, and pipette tips should not be shared between different wells until contamination is confirmed to be completely eliminated.

[0040] 6. TILs are typically round or slightly elongated in shape, gradually dispersing after detaching from the tissue block and tending to form regular round cell clusters. Change the medium every 3 days, aspirating and discarding 1 ml of the supernatant from each well, and then adding 1 ml of intact culture medium. When the TILs grow to cover the bottom of the well, gently pipette 1 ml to resuspend the cells, and transfer 1 ml of the cell suspension to a new culture well. Then add 1 ml of intact culture medium to both the original and new wells.

[0041] 7. The pre-REP phase typically takes 2–4 weeks. The harvest time is determined based on cell growth: the estimated culture time required when a well is fully colonized (approximately 2 million TIL cells per well), at which point the expected number of wells to be harvested will meet the initial requirements for rapid expansion (REP), is designated as the expected harvest date. Three days before the expected harvest date, aspirate and combine the cell suspensions from each well, centrifuge at 2100 rpm for 8 min, and discard the supernatant. Resuspend the cells in aspartate-deprived medium and thoroughly mix. The aspartate-deprived medium consists of: RPMI-1640 medium without L-aspartate, 6000 IU / ml IL-2, 10% human AB serum, and 1% gentamicin. Seed the resuspended TILs at 2 ml per well into new 24-well plates for further culture.

[0042] 8. Harvest cells. Harvest cells 3 days after changing to aspartate-deprived medium. Collect TILs by pipetting 1 ml up and down, and combine TILs from multiple wells, mix well, and count cells. Simultaneously, assess cell viability using trypan blue staining. Do not combine the following wells: wells with no TILs or very few TILs, wells containing a large amount of debris or obvious cell death, or wells suspected of contamination. If subsequent REP is initiated using fresh TILs, all REP-related reagents should be prepared before harvesting, including irradiated peripheral blood mononuclear cells (PBMCs) as feeder cells.

[0043] 9. Optional: If REP cannot be started immediately, TILs can be cryopreserved. After harvesting TILs as described above, add 1 ml of cryopreservation solution to every 20 million TILs, aliquot into cryovials and place them in a programmed cooling box. Store in a -80 ℃ freezer for at least 24 h, and then transfer to a -150 ℃ freezer or liquid nitrogen for long-term storage.

[0044] II. Rapid Amplification (REP) 1. Optional: If the TIL obtained in step one has been cryopreserved, thaw the cryopreserved TIL rapidly in a 37°C water bath before starting REP, resuspend it in intact culture medium, adjust the cell concentration to 1 million / ml, seed 2ml per well in a 24-well plate, and incubate statically in a cell culture incubator for 1–2 days. Harvest the TIL after the cells have recovered and start REP.

[0045] 2. Before the start of the REP phase, feeder cells are prepared using irradiated peripheral blood mononuclear cells (PBMCs). PBMCs can be autologous or allogeneic; allogeneic PBMCs can be obtained by mixing PBMCs from at least three donors who have been appropriately tested and excluded from hepatitis B, hepatitis C, HIV, and syphilis infections. In this example, the PBMCs used are typically approximately 2–3 × 10⁻⁶ cells from three donors (collected from Union Hospital affiliated with Tongji Medical College of Huazhong University of Science and Technology, with signed consent from the donors). 8 2~3×10 8 Each PBMC was resuspended in 40 ml of intact culture medium and placed in a 50 ml centrifuge tube for irradiation at 40 Gy. After irradiation, the cells were centrifuged at 2100 r / min for 5 min, and the supernatant was discarded. The resulting cells were the feeder cells.

[0046] 3. Add 2-3 × 10 at a rate of 10000 TIL per 2 million TILs. 8 The ratio of feeder cells was determined using a T175 culture flask. TILs and feeder cells were resuspended sequentially in 150 ml of complete culture medium, and anti-CD3 antibody (OKT3) with a final concentration of 30 ng / ml was added. The mixture was then thoroughly pipetted and mixed.

[0047] 4. TILs are suspension cells. Place the culture flask vertically in the cell culture incubator. The above time point is recorded as REP Day 0.

[0048] 5. Day 3: Change the medium. Transfer the cell suspension from the culture flask to a centrifuge tube and centrifuge at 2100 r / min for 10 min. Retain approximately 30 ml of culture medium, discard the remaining supernatant, resuspend the TILs, transfer them to a T175 culture flask, add complete culture medium to a final volume of 150 ml, and continue culturing.

[0049] 6. Day 6: Transfer to culture bags for culture. Transfer the cell suspension from the culture flask to a centrifuge tube, centrifuge at 2100 r / min for 10 min, and discard the supernatant. Resuspend the TILs in intact culture medium and count the cells, adjusting the cell concentration to 1 million TILs / ml. Then transfer the cell suspension to a Takara GT-T610 cell culture bag (maximum volume 1800 mL), and place the culture bag horizontally in a cell culture incubator for continued culture.

[0050] 7. Day 8: Add culture medium. Add the same volume of complete culture medium as on Day 6 to the culture bag, and supplement with IL-2 to bring the final concentration in the culture system to 6000 IU / mL. When the total volume of the solution in the culture bag exceeds the maximum volume of a single culture bag, the cell suspension can be aliquoted into multiple culture bags as needed for continued culture.

[0051] 8. Day 10 (3 days before the expected harvest date): Replace the aspartate-deprived medium. Based on the patient's infused dose, the expected time for the number of TILs in the culture bag to meet the patient's infused dose is Day 13, which is the expected harvest date. Transfer the cell suspension in the culture bag to a centrifuge tube, centrifuge at 2100 r / min for 10 min, and discard the supernatant; resuspend the cells in twice the culture volume of aspartate-deprived medium from Day 8, and then transfer them back to the culture bag for continued culture.

[0052] 9. Harvest cells on Day 13 (the latest harvest date is Day 14; if the expected harvest date is Day 14, then in step 8, replace the aspartate-deprived medium on Day 11). Transfer the cell suspension from the culture bag to centrifuge tubes and centrifuge at 2100 rpm for 10 min, discarding the supernatant. Count and assess cell viability using trypan blue staining. Reserve a small amount of cell sample for quality control and flow cytometry analysis; resuspend the remaining cells according to dosage requirements and prepare infusion buffer.

[0053] Comparative Example 1 This embodiment provides a method for amplifying TILs, the steps of which are basically the same as those in Example 1, the only difference being that: in this comparative example, conventional RPMI-1640 medium containing L-aspartic acid is used instead of RPMI-1640 medium without L-aspartic acid, that is, complete medium is used throughout the process, and aspartic acid-deprived medium is not used.

[0054] Effect detection: 1) A CFSE dilution experiment was performed on the TILs harvested in Example 1 and Comparative Example 1. The specific steps were as follows: 1.1 Transfer an appropriate amount of the cell suspension harvested from Example 1 and Comparative Example 1 to a 15 ml centrifuge tube, count the cells, centrifuge at 2100 r for 5 min, and discard the supernatant; 1.2 Resuspend TILs in PBS to a concentration of 5-10 × 10⁻⁶. 6 Add cells / ml to CFSE working solution and mix well to make a final concentration of 1μM. Incubate at room temperature in the dark for 10 min. Mix the cells every 2-3 min during the labeling process (to prevent local high concentration from causing cytotoxicity). 1.3 After the TIL of Comparative Example 1 was treated in step 1.2, it was added to 5 times the volume of intact culture medium and mixed well. After the TIL of Example 1 was treated in step 1.2, it was added to 5 times the volume of aspartic acid deprivation culture medium and mixed well. The label was terminated, and the mixture was centrifuged at 2100 r for 5 min and the supernatant was discarded. 1.4 After the TIL of Comparative Example 1 was treated in step 1.3, it was washed again with 5 times the volume of intact culture medium. After the TIL of Example 1 was treated in step 1.3, it was washed again with 5 times the volume of aspartic acid deprivation culture medium. The mixture was centrifuged at 2100 r for 5 min and the supernatant was discarded. 1.5 The TIL of Comparative Example 1 was treated in step 1.4 and then resuspended in intact culture medium; the TIL of Example 1 was treated in step 1.4 and then resuspended in aspartic acid-deprived culture medium, and the samples were counted separately. 1.6 Inoculate into 24-well plates and incubate for 3 days (37 ℃, 5% CO2); 1.7 Take an appropriate amount of each cell suspension obtained after culture in step 1.6 and transfer it to a centrifuge tube for counting; 1.8 Add 1 million TIL to a flow cytometer, centrifuge at 2100 r for 5 min, and discard the supernatant; 1.9 Resuspend in 200 μl PBS and perform flow cytometry analysis.

[0055] The results are as follows Figure 1 As shown. The results show that, compared with the TIL(Asn-) harvested by the method in Comparative Example 1, the CFSE peak of the TIL(Asn+) harvested by the method in Example 1 is generally shifted to the left ( ). Figure 1 A) and MFI decreased significantly ( Figure 1 B) suggests that treatment with aspartic acid-deprived medium resulted in more thorough dye dilution of TILs within the observation time window, leading to increased cell division generations and enhanced proliferation activity.

[0056] 2) Flow cytometry analysis was performed on the TILs harvested in Example 1 and Comparative Example 1, and the results are as follows: Figure 2 As shown. Figure 2 Results A show that the CD8+ of TIL(Asn+) harvested by the method in Example 1 is... + T cells accounted for 80.8%, CD4 + The proportion of T cells was 14.6%; Comparative Example 1 showed that the CD8+ of TILs (Asn-) harvested using this method... + T cells accounted for 81%, CD4 + T cells accounted for 14.6%. Figure 2 B and Figure 2 The comparison results of C showed that the two groups of CD8 + / CD4 + The lack of significant differences in composition indicates that the method of the present invention enhances function and proliferation without relying on significant changes in the proportion of major T cell subsets.

[0057] 3) Determination of the cytotoxic effects and cytokine secretion capacity of TILs harvested in Example 1 and Comparative Example 1. The specific method is as follows: 3.1 Transfer an appropriate amount of the cell suspension harvested from Example 1 and Comparative Example 1 into a centrifuge tube and count the cells; 3.2 Add 1 million TIL to a flow cytometer, centrifuge at 2100 r for 5 min, and discard the supernatant; 3.3 Add 2 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 3.4 Add 500 μl of 4% PFA solution, mix well, and fix at room temperature for 15 min; 3.5 Add 2 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 3.6 Take another test tube, dilute 10×BD Perm / Wash buffer with PBS at a ratio of 1:10 to prepare 2 ml of 1×BD Perm / Wash working solution, add it to the flow cytometry tube after step 3.5, incubate at room temperature for 15 min to break the membrane, centrifuge at 2100 r for 5 min, and discard the supernatant. 3.7 Take a 1.5 ml Ep tube, add 100 μl of 1×BD Perm / Wash working solution, then add 1 μl each of GZMB antibody / TNFα antibody / IFNγ antibody, mix well, transfer the solution in the Ep tube to the flow cytometer tube processed in step 3.6, mix well, and incubate in a refrigerator at 4 ℃ in the dark for 20 min. 3.8 Remove the flow cytometry tube, add 2 ml of 1×BD Perm / Wash working solution to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 3.9 Add 2 ml of 1×BD Perm / Wash working solution to rinse again, centrifuge at 2100 r for 5 min, and discard the supernatant; 3.10 Resuspend in 200 μl PBS and perform flow cytometry analysis.

[0058] Flow cytometry results as follows Figure 3 As shown, Figure 3 The results of Example A showed that the positive rate of GZMB in TIL(Asn+) harvested by the method of Example 1 was 32.8%, while that of TIL(Asn-) harvested by Comparative Example 1 was 25.5%; the positive rate of TNFα was 61.8%, while that of TIL(Asn-) harvested by Comparative Example 1 was 50.6%; the positive rate of IFNγ in TIL(Asn+) harvested by the method of Example 1 was 50.7%, while that of TIL(Asn-) harvested by Comparative Example 1 was 41.3%. Figure 3 The comparison results of B, 3C, and 3D show that the two groups of GZMB + TNFα + IFNγ +Significant differences were observed. The above results indicate that the method of the present invention can increase the proportion of effector T cells with cytotoxic effects and cytokine secretion capabilities in expanded TILs, thereby enhancing the immune effect of the reinfused TIL preparation.

[0059] 4) Determination of phenotypic traits associated with reduced inhibitory checkpoints and functional limitations. The specific method is as follows: 4.1 Transfer an appropriate amount of the cell suspension harvested from Example 1 and Comparative Example 1 into a centrifuge tube and count the cells; 4.2 Add 1 million TIL to a flow cytometer, centrifuge at 2100 r for 5 min, and discard the supernatant; 4.3 Add 2 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 4.4 Take a 1.5 ml Ep tube, add 100 μl PBS, then add 1 μl PD-1 antibody, mix well, transfer the solution in the Ep tube to the flow cytometry tube treated in step 4.3, mix well, and incubate in the dark at 4°C for 20 min. 4.5 Add 2 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 4.6 Add 500 μl of 4% PFA solution, mix well, and fix at room temperature for 15 min; 4.7 Add 2 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 4.8 Take another test tube, dilute 10×BD Perm / Wash buffer with PBS at a ratio of 1:10 to prepare 2 ml of 1×BD Perm / Wash working solution, add it to the flow cytometry tube after step 3.7, incubate at room temperature for 15 min to break the membrane, centrifuge at 2100 r for 5 min, and discard the supernatant. 4.9 Take a 1.5 ml Ep tube, add 100 μl of 1×BD Perm / Wash working solution, then add 1 μl of TCF-1 antibody, mix well, transfer the solution in the Ep tube to the flow cytometry tube processed in step 3.8, mix well, and incubate in a refrigerator at 4 ℃ in the dark for 60 min. 4.10 Remove the flow cytometry tube, add 2 ml of 1×BD Perm / Wash working solution to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 4.11 Add 2 ml of 1×BD Perm / Wash working solution and rinse again, centrifuge at 2100 r for 5 min, and discard the supernatant; 4.12 Resuspend in 200 μl PBS and perform flow cytometry analysis.

[0060] Flow cytometry results as follows Figure 4 As shown, Figure 4 A and Figure 4 The results of C showed that the proportion of PD-1 positive TILs (Asn+) harvested by the method of Example 1 was significantly lower than that of TILs (Asn-) harvested by Comparative Example 1; meanwhile, Figure 4 The results of B showed that the proportion of PD-1 and TCF-1 double-positive cells in the TIL(Asn-) harvested in Comparative Example 1 was significantly lower than that in the TIL(Asn-) harvested in Comparative Example 1. These results suggest that the method of the present invention can reduce the elevated expression of inhibitory checkpoints and functionally limited phenotypic features in amplified TILs, which is beneficial for improving the immune function status of the final product.

[0061] 5) Improve mitochondrial structure and mitochondrial-related metabolic characteristics, and reduce oxidative stress levels. Specific methods include: 5.1 Transmission electron microscopy: 5.1.1 Transfer an appropriate amount of the cell suspension harvested in Example 1 and Comparative Example 1 to a centrifuge tube, centrifuge at 2100 r for 5 min, and discard the supernatant (the cell pellet should be at least the size of a mung bean). 5.1.2 Add electron microscopy fixative until the precipitate is covered, and fix at 4 ℃ in the dark for 2 h; 5.1.3 The sample was transported to the Scientific Compass platform using ice packs at 4 °C for transmission electron microscopy observation.

[0062] 5.2 Mito-Tracker Green staining: 5.2.1 Transfer an appropriate amount of the cell suspension harvested from Example 1 and Comparative Example 1 into a centrifuge tube and count the cells; 5.2.2 Add 1 million TIL to a flow cytometer, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.2.3 Add PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.2.4 Resuspend the cells in 1 ml PBS, add MitoTracker Green working solution and mix well to a final concentration of 100 nM. Incubate at 37 °C in the dark for 30 min. 5.2.5 Add 1 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.2.6 Add 1 ml of PBS and rinse again, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.2.7 Resuspend in 200 μl PBS and perform flow cytometry analysis.

[0063] 5.3 Mito SOX Red staining: 5.3.1 Transfer an appropriate amount of the cell suspension harvested from Example 1 and Comparative Example 1 into a centrifuge tube and count the cells; 5.3.2 Add 1 million TIL to a flow cytometer, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.3.3 Add PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.3.4 Resuspend the cells in 1 ml PBS, add MitoSOX Red working solution and mix well to a final concentration of 5 μM. Incubate at 37 °C in the dark for 15 min. 5.3.5 Add 1 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.3.6 Add 1 ml of PBS and rinse again, centrifuge at 2100 r for 5 min, and discard the supernatant; 5.3.7 Resuspend in 200 μl PBS and perform flow cytometry analysis.

[0064] The results are as follows Figure 5 As shown. Figure 5 Transmission electron microscopy observation results of A and Figure 5 The comparison of the number of mitochondria and cristae per cell in TIL B showed that, compared with the TIL (Asn-) harvested in Comparative Example 1, the number of mitochondria per cell in TIL (Asn+) harvested in Example 1 was significantly increased, and the number of cristae per cell was also significantly increased, suggesting improved mitochondrial number, structure, and metabolic function. Meanwhile, Figure 5 Mito-Tracker Green staining results for C showed that the peak of TIL(Asn+) harvested by the method in Example 1 was skewed to the right. Figure 5 (Left figure of C), MFI is significantly elevated ( Figure 5 (C, right figure) Figure 5 The Mito SOX Red staining results of D showed that the peak of TIL(Asn+) harvested by the method in Example 1 was shifted to the left. Figure 5 (Left figure of D), MFI is significantly reduced ( Figure 5 (See right figure D), indicating increased mitochondrial quality indicators and decreased oxidative stress levels. These results demonstrate that the method of this invention can improve the number and structure of mitochondria in TILs, reduce oxidative stress levels, and enhance metabolic adaptability.

[0065] 6) Measurement of reducing lipid peroxidation-related damage and enhancing antioxidant stress capacity. Specific methods are as follows: 6.1 Body 581 / 591 staining: 6.1.1 Transfer an appropriate amount of the cell suspension harvested from Example 1 and Comparative Example 1 into a centrifuge tube and count the cells; 6.1.2 Add 1 million TIL to a flow cytometer, centrifuge at 2100 r for 5 min, and discard the supernatant; 6.1.3 Add PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 6.1.4 Resuspend the cells in 1 ml PBS, add BODIPY 581 / 591 working solution and mix well to a final concentration of 2 μM. Incubate at 37 °C in the dark for 20 min. 6.1.5 Add 1 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 6.1.6 Add 1 ml of PBS and rinse again, centrifuge at 2100 r for 5 min, and discard the supernatant; 6.1.7 Resuspend in 200 μl PBS and perform flow cytometry analysis.

[0066] 6.2 MDA Elisa Detection: 6.2.1 Transfer an appropriate amount of the cell suspension harvested from Example 1 and Comparative Example 1 into a centrifuge tube and count the cells; 6.2.2 Add 1 million TIL to a centrifuge tube, centrifuge at 2100 r for 5 min, and discard the supernatant; 6.2.3 Add 1 ml of PBS to rinse, centrifuge at 2100 r for 5 min, and discard the supernatant; 6.2.4 Add 100 μl of cell lysis buffer to lyse the cells, centrifuge at 10,000 g-14,000 g for 5 min, and collect the supernatant for assay. 6.2.5 Remove the pre-coated plate, set one blank well without adding any liquid; add 50 μl of supernatant and 50 μl of biotinylated antigen to each of the remaining test wells, mix well, attach the sealing film, and incubate at 37 ℃ for 60 min; 6.2.6 Manual washing of the plate: Discard the liquid in the hole, fill with washing solution, let stand for 10 seconds and then shake dry. Repeat this step 3 times and then pat dry. 6.2.7 Except for the blank wells, add 50 μl of enzyme-labeled avidin to each well, mix well, cover with sealing film, and incubate at 37 ℃ for 30 min; 6.2.8 Manual washing of the plate: Discard the liquid in the hole, fill with washing liquid, let stand for 10 seconds and then spin dry. Repeat this step 3 times and then pat dry. 6.2.9 Add 50 μl each of colorimetric reagent A and colorimetric reagent B to both the blank well and the test well, shake to mix, and incubate at 37 ℃ in the dark for 15 min. Add 50 μl of stop solution to each well. 6.2.10 Read the microplate reader at a wavelength of 450 nm. First, use a blank well to zero the instrument, then measure the optical density value of each well. Normalize the results to calculate the relative MDA using a 1=1 ratio.

[0067] The results are as follows Figure 6 As shown. Figure 6A and Figure 6 The BODIPY 581 / 591 staining results of B showed that the peak of TIL (Asn+) harvested by the method in Example 1 was skewed to the left and the MFI was significantly reduced; Figure 6 The MDA ELISA results of C showed that the intracellular MDA level of TILs (Asn+) harvested by the method in Example 1 was significantly reduced, suggesting that aspartic acid deprivation treatment can reduce lipid peroxidation and related damage, enhance the antioxidant stress capacity of TILs, and help TILs maintain their functional state at the end of culture and in the tumor microenvironment.

[0068] In summary, the method of this invention, through a simple and feasible process intervention of briefly depriving aspartic acid before harvest (during the initial amplification step and / or rapid amplification step), enhances the antitumor immune effect of the reinfused TIL preparation without altering the composition of the major TIL subsets. This results in amplified TILs exhibiting stronger proliferative activity, higher cytotoxic effects and cytokine secretion capacity, lower inhibitory checkpoint expression, and superior mitochondrial-related metabolic characteristics and lower levels of oxidative stress / lipid peroxidation-related damage. This is beneficial for maintaining an effective and durable antitumor response after reinfusion.

[0069] It should be noted that, during the TIL amplification process, this invention involves subjecting the TILs to aspartic acid deprivation / restriction treatment within a predetermined time window before the expected harvest date (e.g., replacing the medium with RPMI-1640 without L-aspartic acid and continuing cultivation for a period of time, as shown in the examples). This can improve the proliferative activity, immune effector function, and metabolic adaptability of the amplified products. Based on this, other aspartic acid deprivation / restriction treatment techniques, such as reducing the aspartic acid concentration to below the level of conventional culture conditions, or using a basal culture medium system from a different source / brand that also achieves "aspartic acid deficiency or significant reduction" to replace the treatment in the above examples, can also achieve the same or similar effects. This invention will not list them all.

[0070] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for improving TIL production quality by depriving aspartic acid to regulate immune metabolism, characterized in that, The method includes an initial amplification phase and a rapid amplification phase; wherein, in the later stages of the initial amplification phase and / or rapid amplification phase, the cells are cultured in an aspartic acid-deprived medium until the cells at each stage are harvested.

2. The method for improving TIL production quality by regulating immune metabolism through aspartic acid deprivation according to claim 1, characterized in that, The method involves changing the cell medium to aspartate-deprived medium during the later stages of the initial expansion phase. The initial expansion phase includes the following steps: A1. Take isolated tumor tissue, cut it into small pieces, and inoculate it into intact culture medium for culture; A2. Three days before the expected harvest date, change the medium to aspartic acid-deprived medium and continue culturing. A3. TIL cells were harvested after culturing in an aspartate-deprived medium for 3 days. The rapid expansion phase includes the following steps: B1. The TIL cells and feeder cells harvested in the initial expansion phase were added to a complete culture medium and stimulants were added. The culture was then recorded as day 0. B2. After culturing for 6 days, use complete culture medium to scale up the culture until TIL cells are harvested.

3. The method for improving TIL production quality by regulating immune metabolism through aspartic acid deprivation according to claim 1, characterized in that, The method, during the later stages of rapid amplification, involves changing the cell medium to an aspartate-deprived medium for further culture. The initial amplification stage includes the following steps: A11. Take isolated tumor tissue, cut it into small pieces, and inoculate it into intact culture medium for culture until the harvest day to harvest TIL cells; The rapid expansion phase includes the following steps: B11. The TIL cells and feeder cells harvested in the initial expansion phase were added to a complete culture medium and stimulants were added. The culture was then recorded as day 0. B12. After culturing to day 6, the cells were scaled up using complete culture medium. Three days before the expected harvest date, the medium was changed to a medium containing aspartic acid deprivation for continued scale-up culture. B13. After culturing in an aspartate-deprived medium for 3 days, TIL cells were harvested.

4. The method for improving TIL production quality by regulating immune metabolism through aspartate deprivation according to claim 1, characterized in that, The method involves changing the cell medium to aspartate-deprived medium during the initial amplification phase and the later stages of the rapid amplification phase. The initial amplification phase includes the following steps: A1. Take isolated tumor tissue, cut it into small pieces, and inoculate it into intact culture medium for culture; A2. Three days before the expected harvest date, change the medium to aspartic acid-deprived medium and continue culturing. A3. TIL cells were harvested after culturing in an aspartate-deprived medium for 3 days. The rapid expansion phase includes the following steps: B11. The TIL cells and feeder cells harvested in the initial expansion phase were added to a complete culture medium and stimulants were added. The culture was then recorded as day 0. B12. After culturing to day 6, the cells were scaled up using complete culture medium. Three days before the expected harvest date, the medium was changed to a medium containing aspartic acid deprivation for continued scale-up culture. B13. After culturing in an aspartate-deprived medium for 3 days, TIL cells were harvested.

5. The method for improving TIL production quality by regulating immune metabolism through aspartate deprivation according to any one of claims 1-4, characterized in that, During the initial expansion phase, the total cell culture time is 2 to 4 weeks; during the rapid expansion phase, the total cell culture time is 13 to 14 days.

6. The method for improving TIL production quality by regulating immune metabolism through aspartate deprivation according to any one of claims 2-4, characterized in that, The complete culture medium described in each step includes: basal culture medium, 6000 IU / ml IL-2, 10% human AB serum and 1% gentamicin; The aspartate-deprived culture medium described in each step includes: basal culture medium with L-aspartate content of 0-2 mg / L, 6000 IU / ml IL-2, 10% human AB serum and 1% gentamicin.

7. The method for improving TIL production quality by regulating immune metabolism through aspartic acid deprivation according to claim 6, characterized in that, The basal culture medium is RPMI-1640 medium.

8. The method for improving TIL production quality by regulating immune metabolism through aspartate deprivation according to any one of claims 2-4, characterized in that, In step B1 or B11, the feeder cells are irradiated peripheral blood mononuclear cells; The ratio of TIL cells to feeder cells is: 2-3 × 10⁻⁶ cells per 2 million TIL cells. 8 Each feeder cell. The stimulant is an anti-CD3 antibody; the final concentration of the stimulant added is 30 ng / ml.

9. The method for improving TIL production quality by regulating immune metabolism through aspartate deprivation according to any one of claims 2-4, characterized in that, In step A2 or B12, the expected harvest date is determined based on the time required for the TIL to fill a hole in a perforated plate; In step B12, the expected harvest date is determined based on the time required for the TIL quantity to meet the patient's infusion dose requirements.

10. A method for obtaining TIL according to any one of claims 1-9, characterized in that, The TIL CD8 + T cells accounted for more than 80%, CD4 + The percentage of T cells is over 14%, the percentage of GZMB+ expression is over 32%, the percentage of TNFα+ expression is over 610%, the percentage of IFNγ+ expression is over 50%, and the percentage of PD-1+ and TCF-1+ expression is over 310%.

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