DC-CIK cell culture medium and culture method and application thereof

Abstract

The invention relates to an improved DC-CIK cell culture method and application thereof, and aims to improve proliferation, immunocompetence and oxidation resistance of DC-CIK cells by optimizing culture medium components and culture environment. The method is divided into three culture stages, namely a stage I, a stage II and a stage III, which are used for respectively providing nutrition and signal molecules required by initial proliferation, function enhancement and energy metabolism optimization of cells. By introducing an epigenetic regulation factor and a JAK inhibitor, cell depletion is avoided, and by adjusting the components of the culture medium, the immunocompetence and cytotoxicity of cells are optimized. Experimental results show that the method significantly improves the proliferation multiple of cells, the proportion of immune memory cells, the immunocompetence and the energy metabolism state, and is superior to a traditional static culture method. The invention provides an efficient and optimized cell culture system which is suitable for cellular immunotherapy and related fields.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a specific culture medium for culturing dendritic cell-cytotoxic T lymphocytes (DC-CIK cells) and a culture method thereof. Background Art

[0002] Dendritic cells (DC) are important immune cells that are responsible for capturing, processing and presenting antigens and activating initial T cell responses. DC cells not only play a key role in immune surveillance, but also play an important role in anti-tumor immune responses. Cytotoxic T lymphocytes (CIK cells) are a type of T cells with strong cell killing activity that are amplified by in vitro stimulation. CIK cells can exert anti-tumor effects by specifically recognizing and killing tumor cells. Co-culturing DC cells with CIK cells to generate DC-CIK cell complexes can effectively enhance the immune activity of CIK cells and produce stronger anti-tumor effects. DC-CIK cells have been widely studied and applied in cancer immunotherapy due to their excellent immunoregulatory and killing functions.

[0003] However, the existing DC-CIK cell culture methods still face many challenges. First, the cell proliferation rate in traditional culture methods is slow and difficult to expand on a large scale, which limits the promotion of its clinical application. Secondly, most of the existing culture medium components are relatively simple and cannot fully mobilize the synergistic effect between DC and CIK cells, resulting in unstable or decreased immune function of cells. Especially in immunotherapy, how to effectively maintain the function of DC-CIK cells and enhance their immune response ability in the tumor microenvironment is still a bottleneck of current technology.

[0004] Therefore, it is urgent to develop an optimized culture medium and method that can effectively improve the proliferation rate and functional activity of DC-CIK cells and their effect in immunotherapy. This is not only of great significance for the clinical transformation of cancer immunotherapy, but also provides new ideas for the development of cell immunotherapy. Summary of the invention

[0005] The purpose of the present invention is to provide a DC-CIK cell culture medium and a culture method and application thereof.

[0006] Therefore, the present invention discloses a DC-CIK cell culture medium on one hand, wherein the culture medium comprises three stages of culture medium, namely:

[0007] (1) Stage I culture medium: IMDM / F12 basal medium supplemented with IL-15, IL-12 / IL-18 fusion protein, L-carnitine, and D-glucose for cell proliferation and growth;

[0008] (2) Phase II culture medium: Based on the phase I culture medium, α-ketoglutarate and Tofacitinib were added to enhance cell function and resistance to exhaustion;

[0009] (3) Phase III medium: Sodium pyruvate and vitamin C are used to replace the glucose in the phase II medium to optimize the energy metabolism and antioxidant capacity of the cells.

[0010] Preferably, the amino acid sequence of the IL-12 / IL-18 fusion protein of the present invention is shown as SEQ ID NO.1, wherein the nucleotide sequence of the codon-optimized IL-12 / IL-18 fusion protein is shown as SEQ ID NO.2.

[0011] Preferably, the IL-15 concentration in the stage I culture medium of the present invention is 15 ng / mL, the IL-12 / IL-18 fusion protein concentration is 10 ng / mL, the L-carnitine concentration is 2 mM, and the D-glucose concentration is 4.5 g / L.

[0012] Preferably, the concentration of α-ketoglutarate in the stage II culture medium of the present invention is 1 mM, and the concentration of Tofacitinib is 1 μM.

[0013] Preferably, the sodium pyruvate concentration in the stage III culture medium of the present invention is 2 mM, and the vitamin C concentration is 50 μM.

[0014] In one aspect, the present invention further discloses a method for culturing DC-CIK cells, the method comprising the following steps:

[0015] (1) Induction of dendritic cells: Monocytes were isolated from the peripheral blood of healthy subjects and CD14+ monocytes were isolated using the immunomagnetic bead method. The isolated monocytes were cultured at a concentration of 1×10 6 The cells / mL were seeded in IMDM medium containing 50 ng / mL GM-CSF and 20 ng / mL IL-4 and cultured for 5 days;

[0016] (2) Expansion of CIK cells:

[0017] 1) Day 0-CD3+ cell activation: Isolate T cells from peripheral blood or other sources, and select CD3+ cells using magnetic bead method or flow cytometry; inoculate CD3+ cells into pre-prepared culture flasks or culture dishes at a cell density of 1×10 6 cells / mL; the cell culture environment was adjusted to 37°C and 5% CO 2 , 5% O2; the culture medium in this step is the stage I culture medium;

[0018] 2) Day 3 - Transfer to Phase II medium: On Day 3, check the proliferation and viability of cells. Cells should have begun to proliferate. Change the medium to Phase II medium. Adjust the cell culture environment to 37°C and 5% CO. 2 , 8% O2 to simulate hypoxic conditions, which helps to enhance the proliferation ability of cells;

[0019] 3) Day 7-Change to Stage III medium: On Day 7, change the medium to Stage III medium and continue to grow cells; adjust the cell culture environment to 37°C and 5% CO 2 , 3% O2, simulating a hypoxic environment to enhance the immune activity and cytotoxicity of cells;

[0020] (3) Co-culture of DC-CIK cells: The mature dendritic cells cultured above were mixed with CIK cells at a ratio of 1:10; the mixed cells were inoculated into a G-Rex 100M culture flask, the continuous perfusion rate was set to 5 mL / min, and the cell culture environment was maintained at 37°C and 5% CO 2 , 3% O2; change the medium every 2-3 days, and continue culturing under this condition until the 14th day; the above culture process uses stage III culture medium.

[0021] In one aspect, the present invention also discloses a use of the IL-12 / IL-18 fusion protein in the culture medium in preparing a DC-CIK cell culture medium.

[0022] In one aspect, the present invention also discloses a use of the DC-CIK cell culture medium in DC-CIK cell culture.

[0023] The present invention provides an improved DC-CIK cell culture method, which optimizes the proliferation, function and energy metabolism of cells through a three-stage culture strategy, and significantly improves the immune activity and antioxidant capacity of cells. During the implementation process, the stage I culture medium provides basic nutrition and proliferation signals for cells to support the initial growth and proliferation of cells; the stage II culture medium introduces epigenetic regulatory factors and JAK inhibitors to prevent the exhaustion of T cells and enhance their immune function; the stage III culture medium promotes a more efficient immune response of cells by changing the energy metabolism pathway of cells and adding antioxidant factors. Experimental data show that compared with traditional static culture, the method of the present invention significantly improves the proliferation multiples, the proportion of memory T cells, the immune function and the cell metabolic state of cells, and has good statistical significance.

[0024] In addition, the present invention also optimizes the co-culture conditions of DC-CIK cells, increases the cell contact area and improves the immune activity of cells through the perfusion culture system. In the immune function test, DC-CIK cells showed higher expression of immune activation markers CD80 and CD86, and lower expression of PD-1, reflecting their strong immune response. The results of cell killing activity test also showed that the DC-CIK cells cultured by the present invention have significant advantages in killing tumor cells, further verifying the potential application value of this method in cell immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of SDS-PAGE detection of IL-12 / IL-18 fusion protein, where 1 and 2 are IL-12 / IL-18 fusion proteins of different concentrations, with a molecular weight of approximately 43.2 kDa. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0027] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0028] Example 1: Preparation of DC-CIK cell culture medium

[0029] 1. Phase I culture medium: supports the initial growth of T cells and maintains their physiological state and basal metabolic activity. The specific formula is as follows:

[0030]

[0031] Preparation steps: First prepare IMDM / F12 basal medium, add L-carnitine and D-glucose, and mix thoroughly. Add IL-15 and IL-12 / IL-18 fusion protein under sterile conditions. Adjust the pH to 7.4, filter with a sterilizing filter, and prepare a sterile medium.

[0032] 2. Phase II culture medium: Enhances T cell function, prevents cell exhaustion and optimizes immune response through further metabolic and epigenetic regulation. The specific formula is as follows:

[0033]

[0034] Preparation steps: Add α-ketoglutarate and Tofacitinib to the medium of stage I to promote epigenetic regulation and maintain T cell activity. Mix well and continue to incubate at 37°C, 5% CO 2 Cells are cultured in an incubator. Tofacitinib (Pfizer, Chinese for Tofacitinib) is a JAK3 inhibitor that can inhibit IL-7 and IL-15-mediated signaling pathways, enhance T cell activation, and help reduce T cell exhaustion. JAK inhibitors have potential applications in regulating T cell function and maintaining its activity.

[0035] 3. Phase III culture medium: By replacing glucose and adding specific additives, the energy metabolism of cells is optimized, oxidative phosphorylation is promoted, and the antioxidant capacity of cells is enhanced. The specific formula is as follows:

[0036]

[0037] Preparation steps: Replace the glucose in the Phase II medium with sodium pyruvate to provide cells with the energy required for oxidative phosphorylation. Add vitamin C to enhance antioxidant capacity, promote demethylation of DNA in cells, and further activate cell function. After completing the preparation of the medium, continue to culture cells and maintain the culture conditions.

[0038] 4. Summary

[0039] (1) Phase I culture medium provides cells with essential nutrients and proliferation signals, ensuring the survival and proliferation of T cells in the initial stage.

[0040] (2) Phase II culture medium introduces epigenetic modification and JAK inhibition, which provides cells with more stable immune function by regulating gene expression and preventing T cell exhaustion.

[0041] (3) Phase III culture medium optimizes cell metabolism and function by changing energy metabolism pathways and adding antioxidants, promoting a more efficient immune response.

[0042] Example 2: Method for culturing DC-CIK cells

[0043] 1. Isolate monocytes from the peripheral blood of healthy people and use the immunomagnetic bead method to isolate CD14+ monocytes; 6 The cells were inoculated at a density of 100 cells / mL in IMDM medium containing 50 ng / mL GM-CSF and 20 ng / mL IL-4 and cultured for 5 days. During this process, dendritic cells continued to differentiate and mature. Observe and record the morphological changes of the cells:

[0044] Starting from the third day, the cells gradually became dendritic and showed a typical dendritic morphology with obvious protrusions. On the fifth day, the purity of DC cells could be detected by flow cytometry (CD11c+, HLA-DR+), and more than 85% were mature dendritic cells.

[0045] 2. Expansion of CIK cells:

[0046] (1) Day 0-CD3+ cell activation: T cells are isolated from peripheral blood or other sources, and CD3+ cells are selected using magnetic bead method or flow cytometry. CD3+ cells are inoculated into pre-prepared culture flasks or culture dishes at a cell density of 1×10 6 cells / mL; the cell culture environment was adjusted to 37°C and 5% CO 2 , 5% O 2 ; The culture medium in this step is the stage I culture medium described in Example 1.

[0047] (2) Day 3 - Transfer to Phase II medium: On Day 3, check the proliferation and viability of cells. The cells should have begun to proliferate. Change the medium to Phase II medium. Adjust the cell culture environment to 37°C and 5% CO. 2 , 8% O 2 , to simulate hypoxic conditions, which helps to enhance the proliferation ability of cells.

[0048] (3) Day 7-Change to Stage III medium: On Day 7, change the medium to Stage III medium and continue to grow cells; adjust the cell culture environment to 37°C and 5% CO 2 , 3% O 2 , simulating a hypoxic environment to enhance the immune activity and cytotoxicity of cells.

[0049] Example 3: Co-culture of DC-CIK cells

[0050] 1. Co-culture preparation: Mix mature dendritic cells (DC) and CIK cells at a ratio of 1:10 to ensure that there are sufficient DCs to activate CIK cells. In the co-culture system, CIK cells are responsible for providing immune responses, while DC cells act as antigen presenting cells to enhance the activation and proliferation of CIK cells.

[0051] 2. Co-culture setup: Inoculate the mixed cells into the G-Rex 100M culture flask. The G-Rex culture system has a unique design that maximizes cell contact and solute exchange, thereby improving the efficiency of cell culture. Set a continuous perfusion rate of 5 mL / min, which helps to provide sufficient nutrients and remove metabolic waste in a timely manner, optimizing the cell growth environment. Maintain the cell culture environment at 37°C and 5% CO 2 , 3% O2 , to provide optimal proliferation conditions.

[0052] 3. Cultivation process: Regularly check the cell status in the co-culture system, observe the cell proliferation and cytotoxic activity, and supplement the culture medium or adjust the culture conditions as necessary. Change the medium every 2-3 days to ensure that the cells have enough nutrients and remove waste to maintain good cell activity. Continue culturing under these conditions until the 14th day for testing or subsequent application. The above culture process uses the stage III culture medium in Example 1.

[0053] Summary: This process involves optimizing the composition and environmental conditions of the cell culture medium (such as 2 Concentration, culture medium composition), effectively improving the proliferation and activation ability of CIK cells. At the same time, by setting up a co-culture system, the activation effect of DC cells on CIK cells is enhanced, achieving a stronger immune effect and laying the foundation for subsequent cell therapy.

[0054] Example 4: DC-CIK cell function detection

[0055] 1. Cell marker analysis. The specific results are shown in Table 1.

[0056] (1) Total expansion fold (Day 14): The cell expansion folds of different groups were compared by counting the cell expansion numbers on day 14 and day 0. The results showed that the CIK cells of the system of the present invention expanded 89.7 times during the 14-day culture process, while the cell expansion in the traditional static culture was only 21.5 times, indicating that the system of the present invention is significantly superior to the traditional culture method in terms of cell proliferation, and the P value is less than 0.001, indicating that the difference is statistically significant.

[0057] (2) CD62L+CD45RO+ cell ratio: Flow cytometry was used to detect the dual markers of CD62L and CD45RO to evaluate the proportion of memory T cells. The results showed that in the system of the present invention, the proportion of CD62L+CD45RO+ cells was 58.4%, significantly higher than the 16.7% of traditional static culture. These cells are markers of memory T cells, reflecting that the system of the present invention is more conducive to the generation of cell populations with immune memory. The P value is less than 0.001, indicating a significant difference.

[0058] (3) Proportion of PD-1+ cells: Flow cytometry was used to evaluate the PD-1 expression level to determine the immune function status of the cells. Low PD-1 expression indicated higher immune activity. The results showed that the proportion of PD-1+ cells in the system of the present invention was only 9.2%, far lower than the 34.6% of traditional culture. PD-1 is a marker of immune exhaustion, so a low proportion of PD-1+ cells means that the system of the present invention helps to reduce immune tolerance and cell exhaustion and enhance the immune function of cells. The P value was less than 0.001, indicating a significant difference.

[0059] (4) Mitochondrial membrane potential (ΔΨm): Using JC-1 dye, mitochondrial membrane potential was evaluated by flow cytometry to reflect the energy metabolism status of cells. The results showed that in the system of the present invention, the mitochondrial membrane potential (ΔΨm) increased by 2.8 times compared with the baseline value, indicating that the energy metabolism state of cells was significantly improved and cell activity was enhanced. In traditional static culture, the mitochondrial membrane potential remained at the baseline value of 1.0, showing the positive effect of the system of the present invention on cell activity, with a P value of less than 0.01.

[0060] Table 1 Multi-stage culture effect (n=10)

[0061]

[0062] The above results show that the system of the present invention significantly improves the proliferation ability, memory cell ratio, immune activity and energy metabolism state of CIK cells by optimizing the cell culture environment. Compared with the traditional static culture method, it has obvious advantages, and these differences are statistically significant.

[0063] 2. Immune function detection (flow cytometry)

[0064] Antibodies such as anti-CD80, CD86, CD3, and CD8 were used for labeling to detect the expression of immune markers in DC-CIK cells. The cell ratios of CD80+, CD86+ (DC cell activation markers) and CD3+CD8+ (CIK cell markers) were analyzed. The results showed (Table 2) that the expression ratios of CD80+ and CD86+ in DC-CIK cells were significantly increased, indicating that they have a higher immune activation ability. The CD3+ and CD8+ ratios of CIK cells are relatively high, indicating that they have a stronger cytotoxicity.

[0065] Table 2 Immune function test results

[0066]

[0067] 3. Killing activity detection (LDH release method)

[0068] A549 lung cancer cells were used as target cells and co-cultured with DC-CIK cells for 24 hours. The amount of LDH released in the culture supernatant was detected, and the amount of LDH released was related to the cell rupture and killing activity. The results showed (Table 3) that DC-CIK cells had a strong killing activity against A549 lung cancer cells, with a killing rate of 72.5%, which was significantly higher than the control group and the CIK cell group alone.

[0069] Table 3 Killing activity test results

[0070]

[0071] 4. Migration ability detection (Transwell experiment)

[0072] DC-CIK cells were added to the upper chamber of the Transwell, and the culture medium containing Plerixafor was added to the lower chamber. After 24 hours, the number of cells that migrated to the bottom of the pore membrane in the lower chamber was detected. The results showed (Table 4) that the migration ability of DC-CIK cells was significantly enhanced, and the number of migrated cells was 1.67 times that of the control group, showing its potential in immunotherapy.

[0073] Table 4 Migration ability test results

[0074]

[0075] Example 5: Application of DC-CIK cells in immunotherapy

[0076] 1. Treatment of Lung Cancer Mice

[0077] (1) Methods: A tumor model was established using humanized mice (SCID mice) and A549 lung cancer cells were inoculated to form tumors.

[0078] (2) Treatment: Mice in the treatment group were injected with DC-CIK cells (1×10 6 cells / mouse), and the control group was injected with PBS.

[0079] (3) Tumor growth monitoring: The tumor volume of mice was measured every 3 days, and the tumor growth curve was recorded.

[0080] (4) Results: The tumor volume of mice in the treatment group was significantly reduced after 4 weeks of treatment, and the tumor volume was 40% of that in the control group, indicating the effectiveness of DC-CIK cells in immunotherapy.

[0081] Table 5 Tumor volume detection results

[0082]

[0083] 2. Treatment of Hepatocellular Carcinoma Mice

[0084] (1) Mouse model establishment: NOD / SCID mice, 6-8 weeks old, male, weighing 18-22 g, were used. Hep3B liver cancer cells (5×10 6 The transplanted tumors were established in the right axilla of mice.

[0085] (2) Tumor growth monitoring: Tumor volume was measured every 3 days using the formula V = 1 / 2 × L × W 2 The tumor volume was calculated by averaging L for the maximum length of the tumor and W for the maximum width.

[0086] (3) Cell therapy: The density of DC-CIK cells prepared above was adjusted to 1×10 7 Cells / mL were distributed to different experimental groups using serum-free culture medium and PBS control solution. Different cells (cell group of the present invention, traditional cell group) or PBS control group were injected according to the group, and the injection dose was 5×10 6 The injection was performed via tail vein or intraperitoneal injection.

[0087] (4) Data collection: Tumor volume was measured before the start of the experiment (Day 0) and every 3 days until the end of the experiment (Day 21). The survival status of mice was recorded daily, and the number of days the mice survived was counted.

[0088] (5) Experimental results: The cell group of the present invention showed a significant tumor inhibition effect, with a tumor volume reduction of 62% ± 8%, far exceeding the traditional cell group (reduction of 23% ± 6%) and the PBS control group (increase of 285% ± 15%). This shows that the cell therapy of the present invention has superior anti-tumor activity. The survival period of mice in the cell group of the present invention was significantly extended to 48.5 days, compared with 32.7 days in the traditional cell group and 28.1 days in the PBS control group. The P value was significant (<0.001), indicating that the therapeutic effect of the cell group of the present invention significantly increased the survival period of mice. Details are shown in Table 6.

[0089] Table 6 Comparison of experimental tracking results

[0090]

[0091] The above results indicate that the CIK cell therapy of the present invention exhibits stronger anti-tumor effect and immunotherapy potential in a mouse liver cancer transplant tumor model than traditional treatment regimens.

[0092] Example 6: Preparation and testing of IL-12 / IL-18 fusion protein

[0093] 1. Design of IL-12 / IL-18 fusion protein: The specific design is shown in Table 7. The amino acid sequence of the designed IL-12 / IL-18 fusion protein is shown in SEQ ID NO. 1, with a total of 384 amino acids.

[0094] Table 7 IL-12 / IL-18 fusion protein design

[0095]

[0096] 2. Expression and purification of IL-12 / IL-18 fusion protein

[0097] 1. The codon-optimized IL-12 / IL-18 fusion protein nucleotide sequence (SEQ ID NO.2) was cloned into the pEE12.4 vector, and the pEE12.4-IL-12 / IL-18 fusion protein expression plasmid was constructed by double restriction digestion with EcoRI and HindIII. The successfully constructed recombinant plasmid was sequenced at BGI, and the nucleotide and amino acid sequences were compared and analyzed using software to ensure the correct reading frame.

[0098] 2. The confirmed correct pEE12.4-IL-12 / IL-18 fusion protein plasmid was transfected into CHO-K1 cells and pressure screened. 24 hours after transfection, DMEM / F12 medium containing 10% serum and 25μM MSX was added, and pressure screened in a 37°C incubator for 7 days. During the screening process, the cell growth was observed, and the culture medium was replaced regularly until the negative control cells were almost completely dead. Then monoclonal screening was performed: the cells were digested with 0.25% trypsin-EDTA, and the cells were collected and counted by centrifugation after the reaction was terminated. The cells were diluted to 5 / mL, added to a 96-well plate and incubated for 4-6 hours to observe the growth of single cells. After the single cells in the 96-well plate grew up, they were digested and transferred to a 12-well plate for amplification. Positive clones were screened and high-expression clones were confirmed by ELISA detection, and the culture was continued to be expanded and frozen. Finally, a stable cell line was screened.

[0099] 3. The stable cell strain screened above was acclimated through suspension culture to meet the requirements of large-scale fermentation conditions. The cell strain was fermented in a shake flask, and the cell supernatant was harvested on the 12th day of fermentation. The IL-12 / IL-18 fusion protein was purified using a nickel affinity column. The protein concentration was determined using a BCA kit, and the purity was analyzed using SDS-PAGE. The results showed that the expression level of the IL-12 / IL-18 fusion protein could reach more than 1 g / L, and the purity exceeded 90% (SDS-PAGE results are shown in Figure 2). Figure 1 The molecular weight of the expressed product is about 43.2 kDa. Finally, the purified IL-12 / IL-18 fusion protein was packaged and stored at -80°C for later use.

[0100] 3. Functional Verification Experiment

[0101] Experiment 1: Receptor binding activity (SPR detection)

[0102] 1. Experimental Preparation

[0103] (1) Sensor chip preparation: Use the CM5 chip in the Biacore T200 system and immobilize the following two receptors: IL-12Rβ1-Fc (1000RU) in channel 1 and IL-18Rα-Fc (950RU) in channel 2. The receptors are coupled to the chip surface via Fc tags to ensure the stability and activity of the receptors.

[0104] (2) Buffer: HBS-EP (HEPES buffered saline) was used as the running buffer, pH 7.4, containing 0.05% Tween-20 to reduce nonspecific surface adsorption.

[0105] (3) Samples: IL-12 / IL-18 fusion protein (experimental group), natural IL-12 (control group)

[0106] 2. Experimental Procedure

[0107] (1) Channel pretreatment: Rinse the chip surface with 1M NaCl to remove possible impurities. Use an appropriate concentration of bovine serum albumin (BSA) to block the chip surface to reduce nonspecific adsorption.

[0108] (2) Receptor capture: IL-12Rβ1-Fc and IL-18Rα-Fc were immobilized on channel 1 and channel 2 of the sensor chip, reaching surface densities of 1000RU and 950RU, respectively.

[0109] (3) Mobile phase injection: Inject different concentrations of IL-12 / IL-18 fusion protein or natural IL-12 control samples and record the reaction curve of receptor binding. Each sample was injected for 2 minutes and the flow time was 30 minutes.

[0110] (4) Elution and regeneration: The chip was regenerated with 10 mM NaOH solution to remove bound proteins and ensure that the chip surface was completely clean between each experiment.

[0111] (5) Data acquisition: The changes in the sensor surface reaction were monitored in real time by the Biacore T200 system, and the binding and dissociation processes were recorded in RU (response units).

[0112] (6) Data analysis: Biacore software was used to analyze the curves and fit the binding and dissociation data to obtain the affinity constant (KD), association rate constant (kon) and dissociation rate constant (koff).

[0113] 3. Experimental results: The IL-12 / IL-18 fusion protein showed a KD value of 0.78nM in terms of binding affinity with IL-12Rβ1, showing a significant affinity enhancement (about 1.6 times) compared to natural IL-12 (1.25nM). For IL-18Rα, the KD value of the fusion protein was 1.02nM, showing an affinity similar to that of natural IL-12 (1.25nM), but still having certain advantages. Therefore, the binding affinity of the IL-12 / IL-18 fusion protein to the dual receptors is better than that of natural IL-12, especially when binding to IL-12Rβ1, the affinity is significantly enhanced, indicating that the fusion protein has a higher efficiency in receptor binding. The specific results are shown in Table 8.

[0114] Table 8 Summary of experimental results

[0115]

[0116] This experiment showed that the IL-12 / IL-18 fusion protein is better than natural IL-12 in binding to both IL-12Rβ1 and IL-18Rα, and has enhanced binding affinity to both receptors, making it a potential candidate molecule for more effective immunotherapy.

[0117] Experiment 2: Signaling pathway activation (CIK cell detection)

[0118] 1. Experimental Procedure

[0119] 1.1 CIK cell culture and treatment

[0120] (1) Cell preparation: CIK cells isolated from healthy mice or human peripheral blood were used. The cells were inoculated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and placed at 37°C and 5% CO. 2 incubator.

[0121] (2) Fusion protein treatment: CIK cells were treated with different concentrations of IL-12 / IL-18 fusion protein in the logarithmic growth phase, with concentrations of 10 ng / mL, 1 ng / mL, and 0.1 ng / mL, respectively, for 1 hour. The control group was treated with an equal amount of IL-12 and IL-18 mixture. The negative control group did not receive fusion protein or any cytokines.

[0122] 1.2 Analysis of signaling pathway activation

[0123] (1) Western blot detection: After treatment, the cells were washed twice with ice-cold PBS, lysed with RIPA lysis buffer (containing 1 mM PMSF) containing protease inhibitors, and the supernatant was collected. The protein concentration was determined using the BCA protein quantification method, and equal amounts of protein samples (20 μg) were loaded onto SDS-PAGE gels. The membrane was transferred to a PVDF membrane and blocked with a blocking solution (5% non-fat milk) for 1 hour, followed by incubation with primary antibodies against p-STAT4 (Tyr693) and β-actin (1:1000 dilution, 4°C overnight). After incubation with secondary antibodies, protein signals were detected by chemiluminescence (ECL), and statistical results were analyzed using ImageJ software to calculate the relative amount of phosphorylated STAT4.

[0124] (2) qPCR detection of IFN-γ mRNA expression: After treatment, cell RNA was extracted, total RNA was extracted using TRIzol reagent, and reverse transcription was performed to generate cDNA. Real-time fluorescence quantitative PCR (qPCR) was performed using specific primers (IFN-γ, GAPDH), and the relative expression level of IFN-γ mRNA was calculated using the 2-ΔΔCt method.

[0125] 1.3 Flow cytometry verification: The treated CIK cells were stained with CD69 antibody (T cell activation marker) and appropriate fluorescent labeled secondary antibodies. Incubated at 4°C in the dark for 30 minutes, washed, and the proportion of CD69 positive cells on the cell surface was detected using a flow cytometer (such as FACSCalibur or Fortessa).

[0126] 2. Experimental results

[0127] 2.1 Western blot and qPCR results: as shown in Table 9.

[0128] (1) STAT4 phosphorylation level: The phosphorylation level of the fusion protein-treated group was approximately 8.3 times higher than that of the negative control group, indicating that the fusion protein can more strongly activate the STAT4 signaling pathway. In contrast, the activation effect of the IL-12 and IL-18 mixed group was weaker (5.1 times increase).

[0129] (2) IFN-γ mRNA expression: The IFN-γ mRNA expression in the fusion protein group was significantly upregulated (35.2±3.1 times), which was higher than that in the IL-12+IL-18 mixed group (21.7±2.4 times upregulated) and much higher than that in the negative control group (1.0±0.2).

[0130] Table 9 Summary of Western blot and qPCR results

[0131]

[0132] 2.2 Flow cytometry results: In the fusion protein group, the proportion of CD69 positive cells increased significantly (68.3%), while the IL-12 + IL-18 mixed group was 42.7%, and the negative control group was 3.4%. This shows that the fusion protein induces T cell activation more strongly than the IL-12 + IL-18 mixture. The details are shown in Table 10.

[0133] Table 10 Summary of flow cytometry results

[0134]

[0135] 3. Conclusion

[0136] (1) Signaling pathway activation: IL-12 / IL-18 fusion protein significantly enhanced STAT4 phosphorylation and IFN-γ mRNA expression, showing a stronger ability to activate immune response.

[0137] (2) T cell activation: Flow cytometry results further verified the advantages of the fusion protein in activating T cells. The expression level of CD69 was significantly higher than that of the mixed group, indicating that the fusion protein can effectively activate CIK cells.

[0138] In summary, IL-12 / IL-18 fusion protein can more effectively activate the immune response of CIK cells and has stronger biological activity than the mixture of IL-12 and IL-18, and may serve as a better candidate molecule for immunotherapy.

[0139] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A DC-CIK cell culture medium, characterized in that The culture medium comprises three stages of culture medium, which are: (1) Stage I culture medium: IMDM / F12 basal medium supplemented with IL-15, IL-12 / IL-18 fusion protein, L-carnitine, and D-glucose for cell proliferation and growth; (2) Phase II culture medium: Based on the phase I culture medium, α-ketoglutarate and Tofacitinib were added to enhance cell function and resistance to exhaustion; (3) Phase III medium: Sodium pyruvate and vitamin C are used to replace the glucose in the phase II medium to optimize the energy metabolism and antioxidant capacity of the cells.

2. The culture medium according to claim 1, characterized in that The amino acid sequence of the IL-12 / IL-18 fusion protein is shown in SEQ ID NO.1, wherein the nucleotide sequence of the IL-12 / IL-18 fusion protein after codon optimization is shown in SEQ ID NO.

2.

3. The culture medium according to claim 1, characterized in that The IL-15 concentration in the stage I culture medium is 15 ng / mL, the IL-12 / IL-18 fusion protein concentration is 10 ng / mL, the L-carnitine concentration is 2 mM, and the D-glucose concentration is 4.5 g / L.

4. The culture medium according to claim 1, characterized in that The concentration of α-ketoglutarate in the phase II culture medium is 1 mM, and the concentration of Tofacitinib is 1 μM.

5. The culture medium according to claim 1, characterized in that The sodium pyruvate concentration in the stage III culture medium is 2 mM, and the vitamin C concentration is 50 μM.

6. A method for culturing DC-CIK cells, characterized in that: The method comprises the following steps: (1) Induction of dendritic cells: Monocytes were isolated from the peripheral blood of healthy subjects and CD14+ monocytes were isolated using the immunomagnetic bead method. The isolated monocytes were inoculated at a density of 1×106 cells / mL in IMDM medium containing 50 ng / mL GM-CSF and 20 ng / mL IL-4 and cultured for 5 days. (2) Expansion of CIK cells: 1) Day 0-CD3+ cell activation: T cells are separated from peripheral blood or other sources, and CD3+ cells are screened out using magnetic bead method or flow cytometry; CD3+ cells are inoculated into a pre-prepared culture flask or culture dish at a cell density of 1×106 cells / mL; the cell culture environment is adjusted to 37°C, 5% CO2, 5% O2; the culture medium in this step is the stage I culture medium described in claim 1; 2) Day 3-Transfer to Phase II medium: On the 3rd day, check the proliferation and viability of the cells. The cells should have begun to expand. Change the medium to Phase II medium. Adjust the cell culture environment to 37°C, 5% CO2, 8% O2 to simulate hypoxic conditions, which helps to enhance the proliferation ability of the cells. 3) Day 7-Change to stage III medium: On day 7, change the medium to stage III medium to continue cell growth; adjust the cell culture environment to 37°C, 5% CO2, 3% O2 ​​to simulate the hypoxic environment to enhance the immune activity and cytotoxicity of the cells; (3) Co-culture of DC-CIK cells: The mature dendritic cells cultured above are mixed with CIK cells at a ratio of 1:10; the mixed cells are inoculated into a G-Rex 100M culture flask, the continuous perfusion rate is set to 5 mL / min, and the cell culture environment is maintained at 37°C, 5% CO2, and 3% O2; the medium is replaced every 2-3 days, and the culture is continued under these conditions until the 14th day; the above culture process uses the stage III culture medium in claim 1.

7. Use of the IL-12 / IL-18 fusion protein in the culture medium as claimed in claim 1 in preparing a DC-CIK cell culture medium.

8. Use of the DC-CIK cell culture medium according to claim 1 in DC-CIK cell culture.

Images