A large-scale cell culture method of malignant pleural and peritoneal effusion tumor-associated lymphocytes

Abstract

The application provides a large-scale cell culture method of malignant pleural and peritoneal effusion tumor-associated lymphocytes, and products and applications thereof, and the method comprises the following steps: (1) separating mononuclear cells from the pleural effusion and / or peritoneal effusion of a tumor patient; (2) culturing the mononuclear cells in a culture medium containing IL-2, lymphoblast activation factor and inactivated fetal bovine serum; (3) performing expansion culture by using a culture medium containing IL-2, 0KT-3, IFNγ and inactivated fetal bovine serum; and (4) continuing culture in a WAVE bioreactor by using a cell bag. The method provided by the application has the advantages of fast proliferation, high cell survival rate, good composition of cultured TALs cells, greatly improved culture efficiency compared with conventional methods, and tumor-specific killing effect.

Description

Technical Field

[0001] This application pertains to the fields of tumor therapy and cell biology. Specifically, this application provides a method for large-scale cell culture of malignant pleural and peritoneal effusion tumor-associated lymphocytes, as well as the product and application thereof. Background Technology

[0002] Cancer immunotherapy utilizes the body's own immune system to specifically kill malignant tumor cells. It is one of the most promising cancer treatment methods after chemotherapy and targeted therapy, and it is also one of the fastest-growing medical technologies in recent years.

[0003] Tumor tissue is typically surrounded by a large population of tumor-infiltrating lymphocytes (TILs) highly enriched with tumor-specific T cells. Isolating and expanding TILs in vitro before reinfusing them into patients offers unique advantages, particularly for solid tumors. Studies have shown that they possess strong anti-tumor effects, effectively controlling the growth and metastasis of liver and lung cancer. Furthermore, clinical trials in melanoma showed a disease control rate as high as 80.3% and an objective response rate of 36.4%. Tumor-associated lymphocytes (TALs) in malignant pleural and peritoneal effusions, stimulated by tumor antigens in the fluid, are a special form of tumor-infiltrating lymphocytes (TILs), exhibiting similar tumor recognition capabilities and tumor-specific killing effects as TILs in solid tumors. After being induced and expanded in vitro by a series of cytokines, the cytokines are reinfused into the patient's body and can exert therapeutic effects through the following pathways: (1) They specifically recognize and kill autologous tumor cells, causing damage to the blood vessels supplying the tumor, causing them to close, reducing exudation and exerting anti-tumor effects, effectively inhibiting the normal growth of tumor cells; (2) They can inhibit the production of protein factors that can damage the peritoneal surface by tumor cells, inhibit the release of intracellular histamine components, so as to reduce the exudation on the peritoneal surface and promote the repair of the damaged peritoneal surface, so that the exudate can be absorbed as soon as possible, thereby achieving therapeutic effects; (3) They can improve the body's immune function.

[0004] Extracting tumor cells (TILs) from tumor tissue is technically challenging, the cell quantity is limited, and some advanced-stage patients cannot tolerate surgical resection of tumor tissue. Furthermore, TILs exhibit poor selectivity for expanding effector CD8+ lymphocytes, making it difficult to achieve clinically therapeutic cell numbers. Additionally, the expanded TILs suffer from insufficient chemotaxis towards tumor lesions and are prone to senescence, hindering the clinical application and development of this technology. Moreover, the use of large amounts of IL-2 during TIL culture can promote the expansion of exhausted T cells (Tregs), thereby affecting the anti-tumor activity of TILs and leading to tumor immune escape. Therefore, developing a functionally enhanced TIL cell line suitable for large-scale expansion and possessing highly efficient tumor-killing activity is a pressing issue that needs to be addressed for the clinical application of TILs cells.

[0005] Currently, relatively mature preparation processes include:

[0006] Using magnetic beads coated with anti-CD3 / CD28 to stimulate the expansion of naive T cells from peripheral blood mononuclear cells (PBMCs) and using polyclonal stimulants to directly expand T cells in PBMCs or TILs offers advantages such as simplicity, speed, and the ability to obtain a large number of T cells in a short time. For example, patent application CN202311280818.9 discloses a method for culturing malignant pleural / abdominal effusion TILs, which involves adding 10%-15% inactivated human AB serum, 100 IU / mL penicillin, 100 μg / mL streptomycin, 1000-2000 U / mL human IL-2, 10-30 ng / mL human IL-21, and 10-30 ng / mL human IL-33 to serum-free 1640 medium to stimulate and expand naive T cells.

[0007] Patent application CN202111144812.X discloses a method for culturing and isolating tumor-specific tumor-associated immune cells (TILs). Tumor organoids and tumor-associated immune cells are simultaneously isolated and cultured from malignant pleural and peritoneal fluid samples of the same source. The tumor organoids and immune cells are then co-cultured to obtain tumor-specific TILs.

[0008] Patent applications CN202211676281.3 and CN202210595591.6 disclose a method for preparing and applying T lymphocytes derived from pleural and peritoneal fluid. By isolating and screening tumor-specific T lymphocytes in pleural and peritoneal fluid, and directly stimulating them with factors such as CD3 / CD28 and IFN-γ, T lymphocytes with a high proportion of CD8+ cells and reaching clinical levels are obtained, which have tumor-specific killing effects.

[0009] However, the above-mentioned culture methods still need improvement in obtaining the number and / or proportion of tumor-specific lymphocytes (i.e., cytotoxic T lymphocytes, CD8+ T cells). They suffer from high cell exhaustion rates, low proliferation rates, and long processing times, exceeding 20 days. Summary of the Invention

[0010] The purpose of this invention is to design a highly efficient method for culturing tumor-associated lymphoid cells (TALs) based on metabolic reprogramming. This method isolates and cultured TALs from malignant pleural and peritoneal effusions in cancer patients, overcoming the difficulty of obtaining tumor tissue from patients with advanced cancer. The obtained TALs exhibit high tumor-killing activity. By initiating metabolic reprogramming, the mitochondrial biosynthetic capacity of lymphocytes increases, enhancing oxidative phosphorylation metabolism. This allows them to maintain sustained combat effectiveness in the hypoxic environment of tumor tissue, while also increasing tissue infiltration, thus providing clinical tumor-killing effects.

[0011] The TALs cells cultured using this protocol contain over 95% T lymphocytes, less than 1% B lymphocytes, less than 5% NK cells, and less than 2% Treg cells. The proportion of CD8+ T cells with tumor-specific killing activity can reach over 80%. The proliferation rate exceeds 1000-fold within 14-16 days, reaching 10^10. 10 -10 11 The invention produces a significant number of lymphocytes with a cell viability of over 90%. Compared to conventional methods, this invention greatly improves the culture efficiency of T cells while exhibiting tumor-specific killing effects.

[0012] This application provides a method for large-scale cell culture of lymphocytes associated with malignant pleural and peritoneal effusion tumors, the method comprising:

[0013] (1) Isolation of mononuclear cells from pleural effusion and / or ascites of cancer patients;

[0014] (2) Mononuclear cells were cultured in a culture medium containing IL-2, lymphoblast activation factor and inactivated fetal bovine serum;

[0015] (3) Amplification culture was performed using a medium containing IL-2, OKT-3, IFNγ and inactivated fetal bovine serum;

[0016] (4) Cells were cultured in WAVE bioreactors using cell bags.

[0017] Further, in step (1), pleural fluid and / or ascites fluid of the patient are collected using a collection bag containing heparin sodium anticoagulant, lymphocytes are separated using lymphocyte separation solution, and mononuclear cells are collected from the cloud-like white membrane layer cells on the interface.

[0018] Further, in step (2), a culture medium containing 500-1500 U / ml IL-2, 2-7 μg / ml or 1-3 ng / mL lymphoblast-stimulating factor, and 2-8% inactivated fetal bovine serum is added to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 5 -1x10 7 Cultured at 1 cell / ml.

[0019] Further, in step (2), X-Vivo15 medium containing 1000 U / ml IL-2, 2-5 μg / ml or 2 ng / mL lymphoblast-stimulating factor, and 5% inactivated fetal bovine serum was added to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Culture for 1-2 days.

[0020] Furthermore, the lymphoblastic stimulating factor is phytohemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS), pokeweed mitogen (PWM), staphylococcal protein A (SPA), polyflavin, thymosin, zoledronic acid, or one or more combinations thereof.

[0021] Furthermore, the lymphoblast-stimulating factor is PHA-L.

[0022] Further, in step (3), X-Vivo15 medium containing 500-1500 U / ml IL-2, 40-60 ng / ml 0KT-3, 40-60 ng / ml CD-28, 10-30 ng / ml IL-15, 10-30 ng / ml IL-21, 5-15 ng / ml IFNγ and 3-8% inactivated fetal bovine serum is added and cultured for 4-6 days;

[0023] Add X-Vivo15 medium containing 500-1500 U / ml IL-2, 10-30 ng / ml 0KT-3, 40-60 ng / ml IL-7, 5-15 ng / ml IFNγ and 3-8% inactivated fetal bovine serum, and culture for 4-6 days.

[0024] Further, in step (3), X-Vivo15 medium containing 1000 U / ml IL-2, 50 ng / ml OKT-3, 50 ng / ml CD-28, 20 ng / ml IL-15, 20 ng / ml IL-21, 10 ng / ml IFNγ, and 5% inactivated fetal bovine serum was added to the half-replacement medium to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml, cultured for 5 days;

[0025] Replace the medium with X-Vivo15 medium containing 1000 U / ml IL-2, 20 ng / ml OKT-3, 50 ng / ml IL-7, 10 ng / ml IFNγ, and 5% inactivated fetal bovine serum, adjusting the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml, cultured for 5 days, with fluid replenishment every 2 days.

[0026] Furthermore, in steps (2) and (3), the culture is carried out at 37°C in an incubator with 5% CO2.

[0027] On the other hand, this application provides tumor-infiltrating lymphocytes obtained by culturing using the above-described method.

[0028] Furthermore, this application provides the use of the above-described method or the above-described tumor-infiltrating lymphocytes in the preparation of drugs for treating tumors.

[0029] Beneficial effects:

[0030] Malignant pleural and peritoneal fluid from patients with solid tumors was used to prepare tumor-associated lymphocytes that can be used for the treatment of solid tumors, solving the problem of difficulty in obtaining tumor tissue for patients with advanced solid tumors.

[0031] The amplification time is short, and the number of cells amplified in vitro can reach 10 within 14-16 days. 10 -10 11 The quantity is sufficient for autologous infusion in patients with solid tumors. The expansion factor relative to the starting cell number can reach over 1000 times.

[0032] The method employs metabolic reprogramming technology and cell viability factors, rather than engineered cell technology, making it simple and easy to perform. The expanded lymphocytes are predominantly T cells, with fewer B lymphocytes and NK cells. CD8+ T lymphocytes are the most abundant, while Treg cells are few, exhibiting strong cell viability and cytotoxicity.

[0033] Tumor-associated lymphocytes derived from malignant pleural and peritoneal effusions, after in vitro expansion, exhibit strong tumor specificity and killing effects.

[0034] The materials are readily available, avoiding surgical sampling, and can also be used for patients with advanced solid tumors that are difficult to operate on. Attached Figure Description

[0035] Figure 1 Amplification curves of TALs from in vitro amplification culture of 3 patients. Detailed Implementation

[0036] Example 1. Isolation and culture of tumor-associated lymphocytes (TALs) from malignant pleural and peritoneal effusions in cancer patients.

[0037] (1) Collect malignant pleural and peritoneal fluid from cancer patients:

[0038] Pharyngeal and peritoneal fluid samples were collected from cancer patients who gave informed consent, using collection bags containing heparin sodium anticoagulant. Patient 1: Peritoneal mesothelioma, 200 mL malignant ascites. Patient 2: Renal carcinoma patient, 200 mL malignant ascites. Patient 3: Lung cancer patient, 200 mL malignant pleural effusion.

[0039] (2) Separation of mononuclear cells and tumor cells:

[0040] On day 0, lymphocytes were separated using Ficoll lymphocyte separation medium and centrifuged using density gradient under sterile conditions. The intermediate white membrane layer was then aspirated to obtain mononuclear cells, which were counted and cultured. Simultaneously, the bottom sediment was collected for primary culture of tumor cells for use in killing assays.

[0041] (3) Activation of lymphoblast-stimulating factors to stimulate metabolic reprogramming:

[0042] Collect the mononuclear cells after counting and add them to X-Vivo15 medium containing 1000 U / ml IL-2, 2 μg / ml lymphoblast-stimulating factor (phytohemagglutinin PHA-L), and 5% inactivated fetal bovine serum to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml were incubated at 37°C in a 5% CO2 incubator for 2 days.

[0043] During this process, we also tried different methods, such as replacing 2 μg / ml phytohemagglutinin with stimulating factors such as 2 μg / ml Phytolacca mitomycin (PWM), 2 ng / ml thymosin, and 2 ng / ml zoledronic acid for 2 days, and first culturing with 2 μg / ml phytohemagglutinin for 1 day, and then replacing 2 μg / ml phytohemagglutinin with 2 μg / ml Phytolacca mitomycin (PWM), 2 ng / ml thymosin, and 2 ng / ml zoledronic acid for 1 day (see Table 2 for details).

[0044] (4) Amplification culture:

[0045] From day 3 to 7, the medium was partially changed, and X-Vivo15 medium containing 1000 U / ml IL-2, 50 ng / ml OKT-3, 50 ng / ml CD-28, 20 ng / ml IL-15, 20 ng / ml IL-21, 10 ng / ml IFNγ, and 5% inactivated fetal bovine serum was added to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml were cultured in an incubator at 37°C and 5% CO2.

[0046] From days 7 to 11, the medium was changed by adding X-Vivo15 medium containing 1000 U / ml IL-2, 20 ng / ml OKT-3, 50 ng / ml IL-7, 10 ng / ml IFNγ, and 5% inactivated fetal bovine serum, adjusting the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml were cultured in an incubator at 37°C with 5% CO2; fluid was replenished every 2 days.

[0047] From days 12 to 15, cells were cultured in large-volume cell bags in a WAVE bioreactor until they expanded to 10⁶ cells / year. 10 -10 11 Order of magnitude.

[0048] (5) Observe cell proliferation activity, morphology, and cell count:

[0049] Every two days, the expanded cells are counted. The cell solution in the culture flask is mixed by pipetting, and 10 μL is taken for counting three times. The average value is taken, and the proliferation curve is plotted after converting the cell number into the proliferation factor.

[0050] (6) Live cell counting and calculation of cell viability:

[0051] Trypan blue assay: Mix the amplified cell suspension with 0.4% trypan blue solution at a ratio of 9:1 and mix thoroughly. Dead cells will be stained distinctly blue, while live cells will remain colorless and transparent. Count the live and dead cells separately within 3 minutes.

[0052] Cell viability (%) = (Total number of live cells / (Total number of live cells + Total number of dead cells)) × 100%

[0053] (7) Functional cell detection:

[0054] The TBNK flow cytometry assay kit was used to detect the virus. 20 μl of each fluorescently labeled monoclonal antibody was added to 100 μl of TALs cells cultured for 15 days and reacted at room temperature in the dark for 15 min.

[0055] Cells were washed once with PBS and analyzed by flow cytometry. The total number of lymphocytes, T cells, and CD4+ was counted separately. + CD8 + Functional cells such as NK cells.

[0056] (8) Aseptic endotoxin detection:

[0057] Use the horseshoe crab reagent method recommended by the pharmacopoeia.

[0058] Limulus amebocyte lysate (LAL) reagent sensitivity verification: This examines the accuracy of the LAL reagent's sensitivity, the correctness of the operator's procedures, and whether the test conditions meet regulations. Based on the LAL reagent's labeled sensitivity value (λ), the national standard or working standard for bacterial endotoxins is dissolved and mixed with water for bacterial endotoxin testing to prepare four concentrations of endotoxin standard solutions: 2λ, λ, 0.5λ, and 0.25λ. Each concentration of endotoxin standard solution is mixed with an equal volume of LAL reagent solution, with four parallel tubes prepared for each concentration. Two additional tubes are prepared with an equal volume of water for bacterial endotoxin testing as negative controls. The solutions in the tubes are gently mixed, the tubes are sealed, and the tubes are vertically placed in a thermostat at 37℃±1℃ for 60 minutes±2 minutes. The test is considered valid when all tubes at the highest concentration (2λ) are positive, all tubes at the lowest concentration (0.25λ) are negative, and the negative control tubes are negative. The geometric mean of the endpoint concentrations is calculated using the following formula, which is the measured value of the LAL reagent sensitivity (λc):

[0059] λc=antilg(∑X / n)

[0060] In the formula, X is the logarithm of the endpoint concentration (1g). The endpoint concentration refers to the concentration of the last positive result in a series of decreasing endotoxin concentrations; n is the number of parallel tubes for each concentration.

[0061] When λc is between 0.5 and 2λ (inclusive), it can be used for bacterial endotoxin testing, and the sensitivity λ is used as the sensitivity of that batch of horseshoe crab reagent.

[0062] Interference assays were conducted to determine the minimum non-interfering dilution factor for the tested cells.

[0063] Detection of bacterial endotoxins in cell solutions after amplification culture: Positive and negative controls and parallel tubes were set up for detection. The limit for bacterial endotoxins should generally not exceed 0.50 EU / ml.

[0064] (9) Mycoplasma detection:

[0065] Mycoplasma was detected using PCR.

[0066] Before performing PCR testing, thorough preparation is essential: ensure a clean and sterile laboratory environment to avoid cross-contamination. Prepare the necessary reagents, including PCR reaction buffer, primers, DNA polymerase, dNTPs, etc.

[0067] Test sample: Cell suspension after amplification culture.

[0068] To prepare the PCR reaction system, add the following ingredients sequentially to the reaction tubes of the PCR instrument:

[0069] DNA template: Extract DNA from the cell suspension after amplification and culture.

[0070] Primers: Specific primers are used to amplify the target DNA sequence.

[0071] dNTPs: Four types of deoxynucleotides, which serve as the basic materials for PCR reactions.

[0072] DNA polymerase: used for DNA synthesis.

[0073] PCR buffer: PCR reaction system.

[0074] Setting up the PCR instrument:

[0075] Initial denaturation: 94℃, 2-5 minutes, to ensure DNA denaturation.

[0076] Denaturation: 94℃, 30 seconds, to separate double-stranded DNA.

[0077] Annealing: Set the temperature according to the Tm value of the primers, 30 seconds, to allow the primers to bind.

[0078] Extension: 72℃, 1 minute, DNA polymerase synthesizes new DNA strands.

[0079] Number of loops: Usually set to 35 loops.

[0080] The final step: ensuring that all DNA strands are synthesized intact.

[0081] Electrophoresis detection: The PCR products are subjected to agarose gel electrophoresis, and the amplification results are observed. This determines whether mycoplasma DNA is present in the sample.

[0082] Experimental results:

[0083] Amplification curves of TALs in vitro amplification culture from 3 different patients ( Figure 1 The results are as follows: Within 15 days of culture, the number of lymphocytes can increase by 1000-fold.

[0084] Cell viability: The viability of TALs cells expanded and cultured in vitro from the three different patients was all above 95%. See Table 1 for details.

[0085] Detection of functional cells: The cells cultured from the in vitro expansion of TALs in the three different patients were predominantly CD8+ T cells, with the T cell proportion exceeding 95%. NK cell counts were low, all below 5%. See Table 1 for details.

[0086] Bacterial endotoxin detection: The bacterial endotoxin levels in the in vitro expanded cultures of TALs from three different patients were all below 0.5 EU / mL, indicating sterility. See Table 1 for details.

[0087] Mycoplasma detection: Mycoplasma detection was negative in in vitro amplified cells from TALs of three different patients, indicating the absence of mycoplasma. See Table 1 for details.

[0088] Table 1. Results of TALs in vitro expansion culture for three patients.

[0089] Patient 1 Patient 2 Patient 3 Increased days 15 16 15 Amplification factor 1071 1113 1161 Number of cells (individuals) 8.74 x 10 10 ]] 6.82 x 10 10 ]] 1.17 x 10 11 ]] Cell viability 97.6％ 98.4％ 99.2％ Number of functional cells (individuals) 8.3 x 10 10 ]]> 6.64 x 10 10 ]]> 1.14 x 10 11 ]]> T cell ratio 95.7％ 97.2％ 96.8％ CD8+ T cell ratio 85.1％ 84.2％ 86.5％ B cell proportion 0.61％ 0.56％ 0.63％ NK cell ratio 1.65％ 1.67％ 1.44％ Bacterial endotoxins <0.5 EU / mL <0.5 EU / mL <0.5 EU / mL Mycoplasma - - -

[0090] In the second step of activating lymphoblast metabolic reprogramming, different methods were tried, including replacing 2 μg / ml phytohemagglutinin with 2 μg / ml phosphatidylcholine (PWM), 2 ng / ml thymosin, and 2 ng / ml zoledronic acid for 2 days, and first culturing with 2 μg / ml phytohemagglutinin for 1 day, then replacing 2 μg / ml phytohemagglutinin with 2 μg / ml PWM, 2 ng / ml thymosin, and 2 ng / ml zoledronic acid for 1 day. The TALs amplification results of one patient after using different stimulating factors are shown in Table 2. Good amplification results were also obtained, with an amplification fold of 600-1000 times within 16 days, cell viability above 90%, T cell count above 80%, and NK cell count below 5%.

[0091] Table 2. Results of TALs in vitro expansion culture using different stimulating factors.

[0092]

[0093] Example 2. Flow cytometry phenotyping of cell subpopulations of TALs after in vitro expansion and culture.

[0094] (1) The proportion of T lymphocyte subsets and their subtypes was detected by flow cytometry.

[0095] (2) Collect cells cultured on days 0, 7, 15, or 16, and take 1×10⁻⁶ cells. 4 One cell was placed in a flow cytometry tube, centrifuged at 1500 r / min for 5 min, and the supernatant was discarded.

[0096] (3) Add 100 μL PBS to resuspend the cells, add fluorescently labeled antibodies: CD4, CD8, CD16, CD28, CD56, CD25, CD45RO, CD19, CD127, etc., and stain at room temperature in the dark for 30 min.

[0097] (4) After washing and centrifuging again, add PBS to each tube to resuspend.

[0098] (5) After testing with the FACSCaliber cytometer, perform data analysis.

[0099] (6) Results: See Table 3 for details.

[0100] The proportions of CD4+ T cells, CD8+ T cells, CD4+CD25+ regulatory T cells, and CD8+CD56+ natural killer T cells in TALs were detected by flow cytometry on days 0, 7, and 15. Compared with the initially isolated TALs, total T cells significantly increased during culture, reaching over 90%, CD8+ T cells significantly increased, reaching over 80% on day 15, and NKT cells gradually increased; while the proportion of CD4 T cells gradually decreased, and Tregs remained at a low level throughout in vitro culture. These results suggest that this culture system primarily selectively expands the CD8+ T cell subset, which is beneficial for exerting cytotoxic effects to specifically kill tumor cells, while maintaining a low level of regulatory T cells and good cell viability.

[0101] Table 3. Flow cytometry phenotypic results of cell subsets of TALs after in vitro expansion and culture (percentage)

[0102]

[0103] Example 3. Detection of the cytotoxic activity of TALs after in vitro expansion and culture against tumor cells.

[0104] (1) Take the TALs amplified and cultured in Example 1 for tumor cell killing activity detection.

[0105] (2) Take the tumor cells isolated from the patient in Example 1 and culture the primary tumor cells in DMEM complete medium containing 10% FBS until the logarithmic growth phase is reached.

[0106] (3) Primary tumor cells in the logarithmic growth phase were selected as the experimental target cells, and their density was adjusted to 1×10⁻⁶. 5 Cells / ml; human cervical cancer cells HeLa cells (purchased from ATCC) were used as positive target cells for control.

[0107] (4) Adjust the TALs cell density to 1×10⁻⁶ 5 cells / ml, 1×10 6 cells / ml and 2×10 6 Three groups were formed, with TALs mixed with target cells (100 μl each) at effector-to-target ratios of 1:1, 10:1, and 20:1. Corresponding negative controls were set up, including control wells with only tumor target cells, control wells with only TALs cells, and control wells with blank culture medium. Each group had 3 parallel wells.

[0108] (5) Incubate at 37℃ in a 5% CO2 incubator. Add 10 μl of CCK8 reagent at 12h and 24h of incubation and mix well. Continue incubating for another h, then measure the OD value at 450nm wavelength for each well. Calculation method for cell-killing activity:

[0109] Cell-killing activity = (1 - (target cell pore OD value - TALs cell pore OD value) / target cell control pore OD value) × 100%.

[0110] Experimental results:

[0111] As shown in Table 4, TALs expanded and cultured in vitro exhibited strong killing activity against tumor cells at different effector-target ratios (1:1, 10:1, 20:1), especially against tumor cells derived from the patient's own tumor cells.

[0112] Table 4. Killing effect of TALs after in vitro expansion and culture.

[0113]

[0114] Example 4. Tumor cell remnants in expanded cultured TALs

[0115] (1) Take the TALs amplified and cultured in Example 1 for tumor cell residue detection.

[0116] (2) Centrifuge the amplified TALs, and take the supernatant and cell pellet separately for smear preparation.

[0117] (3) After fixation, antibodies against tumor marker melanocyte differentiation protein-A (MART-1), tumor antigen gp-100 and neurogenic tumor antigen S100 were added for immunohistochemical staining.

[0118] (4) Observe under a microscope to determine whether there is positive staining and whether there are detectable residual tumor cells.

[0119] Experimental results:

[0120] As shown in Table 5, after in vitro expansion and culture of TALs, the detection results of tumor markers MART-1, gp-100 and S100 were all negative in both the supernatant and cell pellet, indicating that no tumor markers MART-1, gp-100 and S100 were detected, and no tumor cells remained in the in vitro expanded and cultured TALs.

[0121] Table 5. Detection of residual tumor cells in TALs after in vitro amplification and culture.

[0122]

[0123] Example 5. Metabolic reprogramming of high-concentration lymphoblast-stimulating factor

[0124] (1) Collect malignant pleural and peritoneal fluid from cancer patients

[0125] Pharyngeal and peritoneal fluid samples were collected from cancer patients who gave informed consent, using collection bags containing heparin sodium anticoagulant. Patient 4: Colonic adenoma, 200 mL malignant ascites. Patient 5: Renal carcinoma patient, 200 mL malignant ascites. Patient 6: Lung cancer patient, 200 mL malignant pleural effusion.

[0126] (2) Separate mononuclear cells and tumor cells, as in Example 1.

[0127] (3) Activate lymphoblast metabolic reprogramming

[0128] Add X-Vivo15 medium containing 1000 U / ml IL-2, 5 μg / ml lymphoblast-stimulating factor (phytohemagglutinin PHA-L), and 5% inactivated fetal bovine serum to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml were cultured in an incubator at 37°C and 5% CO2.

[0129] (4) The amplification and culture steps are the same as in Example 1.

[0130] (5) Observe cell proliferation activity and morphology, cell count, live cell count, calculate cell viability, and functional cell detection, etc., as in Example 1.

[0131] (6) Detection of bacterial endotoxins and mycoplasma: Same as in Example 1.

[0132] (7) Flow cytometry phenotyping of amplified TALs cell subpopulations, tumor killing assay, tumor cell residue, etc., are the same as in Examples 2-4.

[0133] Experimental results:

[0134] Amplification and cell viability of TALs in vitro culture from three different patients: Within 15 days of culture, the number of lymphocytes increased by approximately 1000-fold, and the cell viability was above 95%. See Table 6 for details.

[0135] Detection of functional cells: The cells cultured from the in vitro expansion of TALs in the three different patients were predominantly CD8+ T cells, with T cells accounting for over 95%. NK cells were present in smaller numbers, all below 5%. See Table 6 for details.

[0136] Bacterial endotoxin and endotoxin detection: Bacterial endotoxin levels in TALs amplified and cultured from three different patients were all below 0.5 EU / mL, indicating sterility. Mycoplasma detection was negative in all cases, indicating the absence of mycoplasma. See Table 6 for details.

[0137] Table 6. Results of TALs in vitro amplification culture for three patients.

[0138] Patient 4 Patient 5 Patient 6 Increased days 15 15 15 Amplification factor 1074 1035 1099 Number of cells (individuals) 7.73 x 10 10 ]] 9.22 x 10 10 ]]> 1.06 x 10 11 ]]> Cell viability 97.8％ 97.6％ 98.5％ Number of functional cells (individuals) 7.41 x 10 10 ]] 8.86 x 10 10 ]]> <![CDATA[1.03×10 11 ]]> T cell ratio 96.5％ 96.8％ 97.1％ CD8+ T cell ratio 88.2％ 87.0％ 86.7％ B cell proportion 0.63％ 0.68％ 0.62％ NK cell ratio 1.47％ 1.62％ 1.54％ Bacterial endotoxins <0.5 EU / mL <0.5 EU / mL <0.5 EU / mL Mycoplasma - - -

[0139] As shown in Table 7, the changes in the proportions of CD4+ T cells, CD8+ T cells, CD4+CD25+ regulatory T cells, and CD8+CD56+ natural killer T cells in TALs were detected by flow cytometry on days 0, 7, and 15. Compared with the initially isolated TALs, the total T cells increased significantly during culture, reaching over 90%, and the proportion of CD8+ T cells also increased significantly, reaching over 80% on day 15. NKT cells gradually increased, while the proportion of CD4 T cells gradually decreased, and Treg cells remained at a low level throughout in vitro culture. The results suggest that this culture system mainly selectively expands the CD8+ T cell subset, which is beneficial for exerting cytotoxic effects to specifically kill tumor cells, while maintaining a low level of regulatory T cells and good cell viability.

[0140] Table 7. Flow cytometry phenotypic results of cell subsets of TALs after in vitro expansion and culture (percentage)

[0141]

[0142] As shown in Table 8, TALs expanded and cultured in vitro exhibited strong killing activity against tumor cells at different effector-target ratios (1:1, 10:1, 20:1), especially against tumor cells derived from the patient's own tumor cells.

[0143] Table 8. Killing effect of TALs after in vitro expansion and culture on tumor cells.

[0144]

[0145] As shown in Table 9, after in vitro expansion and culture of TALs, the detection results of tumor markers MART-1, gp-100 and S100 were all negative in both the supernatant and cell pellet, indicating that no tumor markers MART-1, gp-100 and S100 were detected, and no tumor cells remained in the in vitro expanded and cultured TALs.

[0146] Table 9. Residual tumor cells in TALs after in vitro expansion culture.

[0147]

[0148] Example 6. Cell culture with low concentration of IL-2

[0149] (1) Collect malignant pleural and peritoneal fluid from cancer patients, using the same method as in Example 5.

[0150] (2) Separate mononuclear cells and tumor cells, using the same method as in Example 5.

[0151] (3) Activate lymphoblast metabolic reprogramming:

[0152] Cells were cultured in X-Vivo15 medium containing 200 U / ml IL-2, 5 μg / ml lymphoblast-stimulating factor (phytohemagglutinin PHA-L), and 5% inactivated fetal bovine serum.

[0153] (4) Amplification culture:

[0154] From day 3 to 7, X-Vivo15 medium containing 200 U / ml IL-2, 50 ng / ml OKT-3, 50 ng / ml CD-28, 20 ng / ml IL-15, 20 ng / ml IL-21, and 10 ng / ml IFNγ, along with 5% inactivated fetal bovine serum, was added to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml were cultured in an incubator at 37°C and 5% CO2.

[0155] From days 7 to 11, every two days, X-Vivo15 medium containing 200 U / ml IL-2, 20 ng / ml OKT-3, 50 ng / ml IL-7, 10 ng / ml IFNγ, and 0.5% inactivated fetal bovine serum was added to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml were cultured in an incubator at 37°C and 5% CO2.

[0156] From days 12 to 15, cells were cultured in large-volume cell bags in a WAVE bioreactor until they expanded to 10⁶ cells / year. 10 -10 11 Order of magnitude.

[0157] (5) Observe cell proliferation activity and morphology, and count cells. Perform live cell counting, calculate cell viability, functional cells, cell phenotype determination, cell killing test, sterility and mycoplasma detection, tumor cell residue, etc., as in Example 5.

[0158] Experimental results:

[0159] The in vitro expansion of TALs from three different patients showed that the number of lymphocytes increased by 1000-fold within 15 days of culture, with cell viability exceeding 95%. See Table 10 for details.

[0160] Detection of functional cells: The cells cultured from the in vitro expansion of TALs in the three different patients were predominantly CD8+ T cells, with T cells accounting for over 95%. NK cells were present in smaller numbers, all below 5%. See Table 10 for details.

[0161] Bacterial endotoxin and mycoplasma detection: Bacterial endotoxin levels in TALs amplified and cultured cells from three different patients were all below 0.5 EU / mL, indicating sterility. Mycoplasma detection was negative in all cases, indicating the absence of mycoplasma. See Table 10 for details.

[0162] Table 10. Results of TALs in vitro expansion culture for three patients.

[0163]

[0164] As shown in Table 11, the changes in the proportions of CD4+ T cells, CD8+ T cells, CD4+CD25+ regulatory T cells, and CD8+CD56+ natural killer T cells in TALs were detected by flow cytometry on days 0, 7, and 15. Compared with the initially isolated TALs, the total T cells increased significantly during culture, reaching over 90%, and the proportion of CD8+ T cells also increased significantly, reaching over 80% on day 15. NKT cells gradually increased, while the proportion of CD4 T cells gradually decreased, and Treg cells remained at a low level throughout in vitro culture. The results suggest that this culture system mainly has a selective expansion effect on the CD8+ T cell subset, which is beneficial for exerting cytotoxic effects to specifically kill tumor cells, while maintaining a low level of regulatory T cells and good cell viability.

[0165] Table 11. Flow cytometry phenotypic results of cell subsets of TALs after in vitro expansion and culture (percentage)

[0166]

[0167] As shown in Table 12, TALs expanded and cultured in vitro exhibited strong killing activity against tumor cells at different effector-target ratios (1:1, 10:1, 20:1), especially against tumor cells derived from the patient's own tumor cells.

[0168] Table 12 Killing effect of TALs after in vitro expansion and culture on tumor cells

[0169]

[0170] As shown in Table 13, after in vitro expansion and culture of TALs, the detection results of tumor markers MART-1, gp-100 and S100 were all negative in both the supernatant and cell pellet, indicating that no tumor markers MART-1, gp-100 and S100 were detected, and no tumor cells remained in the in vitro expanded and cultured TALs.

[0171] Table 13 Tumor cell remnants in TALs after in vitro expansion culture.

[0172]

Claims

1. A method for large-scale cell culture of malignant pleural and peritoneal effusion tumor-associated lymphocytes, characterized in that, The method includes: (1) Isolation of mononuclear cells from pleural effusion and / or ascites of cancer patients; (2) Add mononuclear cells to X-Vivo15 medium containing 1000 U / ml IL-2, lymphoblast-stimulating factor and 5% inactivated fetal bovine serum, and adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cultured for 2 days; the lymphoblast stimulation factors are 2 μg / ml phytohemagglutinin-L, 2 μg / ml pokeweed mitogen, 2 ng / ml thymosin, or 2 ng / ml zoledronic acid; (3) Add X-Vivo15 medium containing 1000 U / ml IL-2, 50 ng / ml OKT-3, 50 ng / ml CD-28, 20 ng / ml IL-15, 20 ng / ml IL-21, 10 ng / ml IFNγ and 5% inactivated fetal bovine serum to the half-replaced medium to adjust the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml, cultured for 5 days; Replace the medium with X-Vivo15 medium containing 1000 U / ml IL-2, 20 ng / ml OKT-3, 50 ng / ml IL-7, 10 ng / ml IFNγ, and 5% inactivated fetal bovine serum, adjusting the cell concentration to 1 x 10⁻⁶ cells / ml. 6 Cells / ml, cultured for 5 days, with fluid replenishment every 2 days; (4) Cells were cultured in WAVE bioreactors using cell bags.

2. The method according to claim 1, characterized in that, In step (1), pleural fluid and / or ascites are collected using a collection bag containing heparin sodium anticoagulant. Lymphocytes are separated using lymphocyte separation solution, and mononuclear cells are collected from the cloud-like white membrane layer cells at the interface.

3. The method according to claim 1, characterized in that, The lymphoblast stimulating factor mentioned in step (2) is 2 μg / ml phytohemagglutinin-L.

4. The method according to claim 1, characterized in that, In steps (2) and (3), the culture was carried out at 37°C in an incubator with 5% CO2.

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