TIL cell and isolated culture method thereof
Through low-temperature treatment and gradient centrifugation, TIL cells in pleural fluid are isolated and expanded, which solves the problem of poor isolation and culture effect of TIL cells in the prior art, and achieves high-purity and high-active TIL cells, with good therapeutic effects.
Patent Information
- Application Number
- CN202510249157.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-04
- Publication Date
- 2025-05-30
AI Technical Summary
In the prior art, the TIL cell isolation and culture effect is poor, affecting in vitro culture, proliferation fold and killing activity.
The tumor-infiltrating cells in the pleural fluid were isolated by low-temperature treatment and gradient centrifugation. The TIL cells were isolated and amplified through the steps of S1 to S8, including preserving the pleural fluid at 2 to 8°C, separating into clusters of tumor cells, performing centrifugation, precipitation and resuspension, Ficoll centrifugation and amplification culture.
High-purity TIL cell isolation and expansion were achieved, the activity and killing activity of lymphocytes were maintained, and the isolation and culture effect of TIL cells was significantly improved, and it had good effect on the treatment of ovarian cancer.
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Figure CN120060136A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a kind of TIL cells and a method for separating and culturing the same. Background Art
[0002] TIL cells (Tumor Infiltrating Lymphocytes) are infiltrating lymphocytes isolated from tumor tissues and have the ability to recognize and kill tumor cells. The main principle of TIL therapy is to utilize the patient's own TIL cells, which are activated and amplified in vitro under the action of cytokines such as IL-2, and then infused back into the patient's body. Since TIL cells are derived from tumor tissues, they have strong tumor homing characteristics and can accurately target tumor cells. Compared with CAR-T cell therapy, TIL therapy does not require genetic modification, thus avoiding related risks and complexities.
[0003] When using the patient's own TIL cells, the first thing to do is to isolate the TIL cells more thoroughly. The purity of TIL cells will affect in vitro culture, proliferation multiple, and killing activity. Therefore, how to further improve the effect of separating and culturing TIL cells is a problem that needs to be overcome in this field. Summary of the Invention
[0004] Aiming at the problem of how to further improve the effect of separating and culturing TIL cells in the prior art, the present invention provides a kind of TIL cells and a method for separating and culturing the same.
[0005] The technical solution adopted by the present invention is as follows:
[0006] A kind of TIL cells and a method for separating and culturing the same, comprising the following steps:
[0007] S1: Store the collected pleural effusion at 10 - 20 °C for a set time;
[0008] S2: Store the pleural effusion obtained in S1 at 2 - 8 °C for a set time;
[0009] S3: Separate the agglomerated tumor cells from the pleural effusion obtained in S2;
[0010] S4: Centrifuge the pleural effusion obtained in S3 to obtain the supernatant containing TIL cells;
[0011] S5: Precipitate the supernatant obtained in S4, resuspend the obtained precipitate, and then centrifuge again;
[0012] S6: Resuspend the precipitate obtained by centrifuging in S5 again;
[0013] S7: Centrifuge the cell resuspension obtained in S6 together with Ficoll, and then collect the precipitate after centrifugation.
[0014] S8: Perform amplification culture on the precipitate obtained in S7 to obtain amplified TIL cells.
[0015] Furthermore, the specific steps of S8 are as follows:
[0016] S801: Inoculate the tumor-infiltrating lymphocytes obtained in S7 at an inoculation density of 1 - 2×10 6 cells / mL.
[0017] S802: Supplement the culture medium on the 3rd day after inoculation to maintain the viable cell density at 1 - 2×10 6 cells / mL.
[0018] S803: On the 5th day after inoculation, evenly distribute the cells into a culture container with a larger volume and continue culturing. At the same time, continue to supplement the culture medium in the new culture container.
[0019] S804: On the 7th day after inoculation, continue to supplement the culture medium in the new culture container.
[0020] S805: Harvest the cells on the 9th day after inoculation.
[0021] After adopting this technical solution, in order to maintain the activity of lymphocytes, the present invention performs gradient cooling on the collected pleural effusion, which is beneficial to protecting the biological activity of lymphocytes in the pleural effusion and avoiding the influence of rapid cooling on the activation and proliferation of the separated lymphocytes during the later culture process.
[0022] Preferably, in S5, resuspension is performed using physiological saline, and after resuspension, centrifugation is carried out at 1500 - 2000 r / min for 5 - 10 min.
[0023] Furthermore, the culture medium in S8 is a T cell culture medium, including: basal medium RPMI1640, activator 1, activator 2, activator 3, and the supernatant (residual cells removed) separated by centrifugation. The addition amount of the supernatant accounts for 10 - 25% of the total volume of the culture medium. Among them, activator 1 is IFNγ, and the final concentration used is 500 - 1000 IU / mL; activator 2 is IL-15, and the final concentration used is 5 - 30 ng / mL; activator 3 is IL-2, and the final concentration used is 300 - 1000 IU / mL.
[0024] After adopting this technical solution, IL-15 and IL-2 share the γc receptor subunit, and both of these cytokines can induce the differentiation of T cells and natural killer cells (NK cells) and promote the proliferation of T cells and NK cells. Currently, IL-15 is often used in combination with IL-2 for in vitro amplification culture of T cells or NK cells.
[0025] Preferably, activator 1 is IFNγ (interferon - γ) with a final concentration of 1000 IU / mL; activator 2 is IL - 15 (interleukin - 15) with a final concentration of 15 ng / mL; activator 3 is IL - 2 (interleukin - 2) with a final concentration of 500 IU / mL.
[0026] Preferably, the addition amount of the supernatant accounts for 10 - 25% of the total culture medium volume.
[0027] Furthermore, the culture container used in S801 is a T75 culture flask with an inoculation amount of 30 - 50 mL; the added amount of the supplementary culture medium in S802 is 30 - 50 mL; the culture container used in S803 is a T225 culture flask, and the cells are evenly divided into two T225 culture flasks, and the added amount of the supplementary culture medium in each T225 culture flask is 50 - 80 mL. In S804, 120 - 150 mL of culture medium is added to each T225 culture flask.
[0028] Furthermore, in S6, RPMI1640 is used to resuspend the precipitate, and the concentration of Ficoll in S7 is 80 - 95%.
[0029] Preferably, the usage concentration of Ficoll is 95%.
[0030] After adopting this technical solution, RPMI1640 is used to resuspend the precipitate, and the usage concentration of Ficoll is 80 - 95%, thus fully ensuring the purity and viability of the cells.
[0031] After adopting this technical solution, during the low - temperature treatment process, more than 90% of the tumor cells have been removed, and the remaining trace - dispersed tumor cells. According to the separation principle of Ficoll, either too low or too high a concentration of Ficoll is not conducive to the separation of tumor cells and tumor - infiltrating lymphocytes. Therefore, the usage concentration of Ficoll is 80 - 95%.
[0032] Furthermore, the specific steps of centrifugation in S4 are as follows:
[0033] S401: First, centrifuge the pleural effusion at 2000 - 3000 r / min for 5 - 10 min and collect the supernatant;
[0034] S402: Centrifuge the supernatant obtained in S401 at 3500 - 4000 r / min for 5 - 10 min to obtain the supernatant.
[0035] Preferably, the preservation time in S1 is 2 h, and the preservation time in S2 is 4 h.
[0036] Preferably, the pleural effusion is derived from a large amount of fluid formed by the metastasis of invasive ovarian cancer tumors to the thoracic cavity.
[0037] A kind of TIL cells, obtained by using the method for isolating and culturing TIL cells described above.
[0038] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:
[0039] The present invention separates tumor-infiltrating cells in pleural effusion by means of low-temperature treatment and gradient centrifugation. In a low-temperature environment of 2-8 °C, the growth of tumor cells is inhibited, the viability is reduced, and a large number of tumor cells are contained in the pleural effusion of advanced cancer patients; the high-density and low-viability tumor cells aggregate into clusters; while tumor-infiltrating lymphocytes in the pleural effusion have been studied for 48 hours of stability, and their viability is not affected; after inoculation and culture, they can normally differentiate and proliferate. High-purity tumor-infiltrating lymphocytes (Tumor infiltrating lymphocytes, TILs) are obtained. After activating the tumor-infiltrating lymphocytes by a non-antibody activation method, autologous pleural effusion is used as a plasma substitute, combined with IL-2 stimulation, and billions of TIL cells with killing activity are obtained after 8 days of culture, which has a very good therapeutic effect on the treatment of ovarian cancer. Description of the Drawings
[0040] Figure 1 It is a microscopic examination result diagram of the sample taken when the pleural effusion is just taken;
[0041] Figure 2 It is a physical diagram of the drained pleural effusion after low-temperature placement;
[0042] Figure 3 It is a microscopic examination result diagram of the pleural effusion without tumor precipitate after low-temperature treatment;
[0043] Figure 4 It is a microscopic examination result diagram of the tumor-infiltrating lymphocytes separated by the separating liquid;
[0044] Figure 5 It is a microscopic examination result diagram of the pleural effusion of mice during animal experiments. Detailed Embodiments
[0045] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present application.
[0046] Example 1
[0047] A method for isolating and culturing TIL cells, where the pleural effusion is sourced from the pleural metastasis of ovarian cancer infiltrating tumors, forming a large amount of effusion. Under microscopic observation, it is confirmed to contain a large number of tumor cells and a small number of immune cells, as shown in Figure 1 (The blue-labeled ones are tumor cells. Malignant tumor cells usually increase in volume and contain some large "giant tumor cells". The red-labeled ones are lymphocytes with smaller volumes. Since there are too many tumor cells, only most of them are labeled), including the following steps:
[0048] S1: After the pleural effusion is collected, the collected pleural effusion together with the entire collection kit is stored at 20 °C for 2 h;
[0049] S2: Then the pleural effusion obtained in S1 together with the entire collection kit is placed in an incubator at 4 °C for 4 h. It can be observed that the tumor cells aggregate into clusters, as shown in Figure 2 ;
[0050] S3: After processing, the outer surface of the entire collection kit is sterilized and then transferred to a biosafety cabinet. The pleural effusion in the collection bag is slowly transferred to a 500 mL centrifuge tube, and a sample is taken for microscopic examination. The number of tumor cells is significantly reduced, as shown in Figure 3 (The blue label indicates tumor cells, the red label indicates tumor-infiltrating lymphocytes, and the black label indicates red blood cell clusters). Note: During the transfer process, large clumps of tumor cells are retained in the collection bag;
[0051] S4: The pleural effusion obtained in S3 is centrifuged to obtain the supernatant containing TIL cells, specifically as follows:
[0052] S401: First, centrifuge the pleural effusion at 2000 r / min for 10 min and collect the supernatant;
[0053] S402: Centrifuge the supernatant obtained in S401 at 3500 R for 10 min to obtain the supernatant;
[0054] S5: The supernatant obtained by centrifuging in S402 is precipitated, and the obtained precipitate is resuspended with physiological saline and then centrifuged at 2000 r / min for 10 minutes;
[0055] S6: Resuspend the precipitate obtained in S5 with RPMI1640;
[0056] S7: Take a new 50 mL centrifuge tube, add 15 mL of 95% Ficoll to each tube, then slowly add 25 mL of the cell suspension obtained in S6, centrifuge at 1200 g for 30 minutes (1 up and 1 down), and then collect the precipitate after centrifugation. Resuspend the precipitate with physiological saline and take a sample to detect the cell density and viability. The results are: cell density 1.68×10 6cells / mL, with a cell viability of 98%. At the same time, under microscopic observation, the cells were basically all tumor-infiltrating lymphocytes, see Figure 4 ;
[0057] S8: The tumor-infiltrating lymphocytes obtained in S7 were amplified and cultured to obtain amplified TIL cells, specifically as follows:
[0058] S801: The tumor-infiltrating lymphocytes obtained in S7 were inoculated into a T75 culture flask at an inoculation density of 2×10 6 cells / mL. In this example, the inoculation volume was 30 mL. The culture medium added to the T75 culture flask was a T cell culture medium, including: basal medium RPMI1640, activator 1, activator 2, activator 3, and the supernatant obtained by centrifugation (the supernatant separated in S5 needs to be centrifuged again at a high centrifugal force of 5000 g for 10 minutes to remove trace cells and cell debris remaining after the first centrifugation, etc.). The added volume ratio of the supernatant was 20% of the volume of the culture medium. Activator 1 was IFNγ, with a final usage concentration of 1000 IU / mL; activator 2 was IL-15, with a final usage concentration of 15 ng / mL; activator 3 was IL-2, with a final usage concentration of 500 IU / mL;
[0059] S802: On the 3rd day after inoculation, 30 mL of culture medium was supplemented to maintain the viable cell density at 2×10 6 cells / mL;
[0060] S803: On the 5th day after inoculation, the cells were evenly distributed into two T225 flasks and continued to be cultured. At the same time, 70 mL of culture medium was added to each of the two T225 flasks;
[0061] S804: On the 7th day after inoculation, 150 mL of culture medium was added to each of the two T225 flasks;
[0062] S805: Cell harvesting was performed on the 9th day after inoculation.
[0063] In order to explore the influence of the important parameters in the present invention on the results of cell separation and culture, the present invention conducted a single-variable experiment, specifically as follows. It should be noted that in order to highlight the differences between other examples (S) and comparative examples (D) and Example 1, the parts identical to Example 1 are not filled in:
[0064] Table 1
[0065]
[0066]
[0067] The process and results of the stability study of tumor-infiltrating lymphocytes in pleural effusion are as follows:
[0068] The experimental process was as follows: Pleural effusion was collected continuously for 2 days, with the amount ranging from 200 to 300 mL each day. The two portions of pleural effusion were separately stored at 2 - 8 °C for 24 h, and after 48 h, separation was carried out. After separation, the viability of lymphocytes was detected, as well as the cell proliferation curve and the flow cytometry results of the harvested cells. The specific results are shown in Table 1:
[0069] Table 2. Results of the stability study of tumor-infiltrating lymphocytes under low-temperature conditions
[0070]
[0071] As can be seen from Table 1, tumor-infiltrating lymphocytes can achieve a viability of over 98% under the low-temperature condition of 2 - 8 °C, and the killing activity can be maintained above 90%, proving that tumor-infiltrating lymphocytes have good stability under low-temperature conditions.
[0072] The TIL cells obtained in Example 1 were used to treat ovarian cancer. The specific process and results are as follows:
[0073] 1. Animal experiment
[0074] (1) Selection of animals: BALB / c mice (35 mice, 6 weeks old, 20 - 24 g, SPF level)
[0075] (2) Modeling method: 30 mice were selected for modeling. Tumor cells isolated from the pleural metastasis of ovarian cancer patients were transplanted by thoracic transplantation, 4000 tumor cells per mouse, thus generating tumors.
[0076] (3) Treatment method: Local thoracic injection. The treatment group was treated with a combination of 1×10 6 TIL cells + 3000 IU IL-2 (total volume 0.5 mL), once a day for 3 consecutive days. The positive control group was injected with cyclophosphamide at 0.1 mg / mouse (total volume 0.5 mL), and the negative control group was injected with 0.5 mL of normal saline. The normal control group was injected with 0.5 mL of normal saline.
[0077] (4) Two weeks after the treatment ended, mouse pleural effusion was collected for observation. The microscopic observation results are shown in Figure 5 , as Figure 5 shown. Compared with the state at the end of modeling, when pleural effusion was collected 14 days after treatment, in the TIL cell combined with IL-2 treatment group, the tumor cells in the pleural effusion disappeared, and the mouse hair and body weight were normal; in the cyclophosphamide treatment group, the tumor cells disappeared, but the hair became sparse and the body weight decreased, while in the normal saline treatment group, the tumor cells increased and tumor tissues were formed.
[0078] Safety data showed that, compared with the non-modeled normal mouse group, there were no gastrointestinal symptoms such as nausea and vomiting in the TIL cell combined with IL-2 treatment group, and the body weights of the mice were normal (as shown in Table 3); monitoring of blood biochemistry showed no electrolyte disorders or impaired liver and kidney functions, and there were no adverse drug reactions such as adrenal cortical function impairment, obesity, and diabetes.
[0079] Table 3. Changes in body weights of mice under different treatments
[0080]
[0081] 2. Clinical trials
[0082] (1) Inclusion criteria (case source: Ciai Cancer Hospital):
[0083] ① Through imaging examinations such as abdominal CT, chest CT, whole-body bone scan, or brain magnetic resonance imaging, it was determined that the ovarian cancer had spread to surrounding tissues or metastasized to distant organs, there was fluid in the chest or abdomen, and the depth was greater than 3 cm.
[0084] ② Hematological examination: By drawing blood for testing multiple tumor markers, such as carcinoembryonic antigen (CEA), carbohydrate antigen 12-5 (CA12-5), carbohydrate antigen 19-9 (CA19-9), etc., a significant increase in these markers may indicate a risk of metastasis.
[0085] ③ Clinical symptoms: The patient had symptoms caused by abdominal metastasis, such as abdominal pain, abdominal distension, nausea, vomiting, dyspnea, etc.
[0086] (2) Treatment method: Local thoracic perfusion. There were 20 cases in the treatment group, who were treated with the combined perfusion of 1 billion autologous TIL cells + 300,000 IU IL-2 (total volume of 20 mL), once a day for 3 consecutive days. There were 18 cases in the positive control group, who were perfused with cyclophosphamide at 50 mg per time (total volume of 0.5 mL), and 10 cases in the placebo group, who were perfused with 20 mL of normal saline.
[0087] (3) Evaluation criteria
[0088] 1. Imaging: According to the RECIST standard commonly used in Chinese clinical practice:
[0089] Complete remission (CR): All target lesions disappeared, and the short axis value of any pathological lymph node (regardless of whether it was a target lesion) must be < 10 mm and maintained for at least 4 weeks.
[0090] Partial remission (PR): Based on the total diameter at the baseline state, the sum of the diameters of all target lesions decreased by at least 30% and was maintained for at least 4 weeks.
[0091] Progressive disease (PD): With the smallest lesion diameter as a reference (including the sum of lesion diameters at baseline, if it is the minimum value), the sum of all target lesion diameters increases by at least 20%. In addition, in addition to the relative increase of 20% in the sum of diameters, the absolute value of the sum must also increase by at least 5 mm (Note: the appearance of one or more new lesions can also be considered as progressive disease). Stable disease (SD): With the sum of the smallest lesion diameters during the study as a reference, lesion reduction does not meet PR, and lesion increase does not meet PD.
[0092] 2. Serological evaluation method: The efficacy of chemotherapy is evaluated by detecting the levels of certain tumor markers related to certain tumors in the blood. If the level of related tumor markers increases by more than a certain percentage, or from normal to above normal, and repeated testing is performed to confirm, it may be considered as disease progression.
[0093] The results showed that 20 patients received a combination of 1 billion autologous TIL cells and 300,000 IU IL-2. (IL-2 can promote the proliferation and differentiation of T lymphocytes; maintain the long-term proliferation of the interleukin-2 cell line that the human body depends on. The TIL cells cultured for 9 days in this patent are in the exponential growth phase. After being reinfused or perfused into the body, the TIL cells will continue to proliferate in large quantities under the stimulation of IL-2, playing an immunomodulatory role in the body. There are two ways to use IL-2: 1. Intravenous drip, 200,000 to 300,000 IU are added to the TIL injection solution, with a volume of 50 to 100 mL; 2. Intrapleural administration, after the pleural effusion is drained, 200,000 to 300,000 IU are added to the TIL injection solution for local perfusion, with a volume of 10 to 30 mL.)
[0094] Compared with before and after treatment, 1 person achieved complete remission (CR), 12 people achieved partial remission (PR), the overall objective response rate (ORR) was 65%, and the disease control rate (DCR) was 85%. The median progression-free survival was 6.9 months, the half-year overall survival rate reached 83.1%, and the one-year overall survival rate (OS) was 69.3%. No complications or adverse events were reported in the study. Compared with the positive control cyclophosphamide group, which had chemotherapy side effects such as nausea, hair loss, and weight loss, the TIL cell combined with IL-2 treatment group had no side effects, while in the negative control group, more than 90% of patients had disease progression, and the treatment advantage of the TIL cell combined with IL-2 treatment group was significantly prominent.
[0095] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.
Claims
1. A method for isolating and culturing TIL cells, characterized in that: The following steps are involved: S1: Store the collected pleural effusion at 10-20°C for 2-3 hours; S2: Store the pleural effusion obtained in S1 at 2-8°C for 3-6 hours; S3: Separate the clustered tumor cells from the pleural effusion obtained in S2; S4: centrifuging the pleural effusion obtained in S3 to obtain a supernatant containing TIL cells; S5: precipitating the supernatant obtained in S4, resuspending the obtained precipitate and centrifuging it again; S6: resuspend the precipitate obtained by centrifugation in S5; S7: centrifuge the cell resuspension obtained in S6 together with Ficoll, and then collect the precipitate after centrifugation; S8: amplify and culture the precipitate obtained in S7 to obtain amplified TIL cells.
2. A TIL cell and a method for isolating and culturing the same according to claim 1, characterized in that: The specific steps of S8 are as follows: S801: The tumor infiltrating lymphocytes obtained in S7 were divided into 1-2×10 6 Inoculate at an inoculation density of pcs / mL; S802: Add medium on the third day after inoculation to maintain a viable cell density of 1-2×10 6 Pieces / mL; S803: On the 5th day after inoculation, the cells are evenly divided into culture containers with larger capacity for further culture, and the culture medium is continuously added to the new culture containers; S804: Continue to add culture medium to the new culture container on the 7th day after inoculation; S805: Cells were harvested on day 9 after seeding.
3. The method for isolating and culturing TIL cells according to claim 2, characterized in that: The culture medium in S8 is T cell culture medium, including: basic culture medium RPMI1640, activator 1, activator 2, activator 3 and supernatant separated by centrifugation, the amount of supernatant added accounts for 10-25% of the total culture medium volume, activator 1 is IFNγ, and the final concentration is 500-1000 IU / mL; activator 2 is IL-15, and the final concentration is 5-30 ng / mL; activator 3 is IL-2, and the final concentration is 300-1000 IU / mL.
4. The method for isolating and culturing TIL cells according to claim 2, characterized in that: The culture container used in S801 is a T75 culture flask, and the inoculation volume of the T75 culture flask is 30-50 mL; the amount of culture medium added in S802 is 30-50 mL; the culture container used in S803 is a T225 culture flask, the cells are evenly divided into two T225 culture flasks, and the amount of culture medium added to each T225 culture flask is 50-80 mL, and 120-150 mL of culture medium is added to each T225 culture flask in S804.
5. A method for isolating and culturing TIL cells according to any one of claims 1 to 4, characterized in that: In S6, RPMI1640 was used to resuspend the precipitate, and the concentration of Ficoll in S7 was 80-95%.
6. A method for isolating and culturing TIL cells according to any one of claims 1 to 4, characterized in that: The specific steps of centrifugation in S4 are as follows: S401: First, centrifuge the pleural effusion at 2000-3000 r / min for 5-10 min, and collect the supernatant; S402: Centrifuge the supernatant obtained in S401 at 3500-4000 r / min for 5-10 min to obtain a supernatant.
7. A TIL cell, characterized in that: The TIL cells are obtained by the isolation and culture method according to any one of claims 1 to 7.