Synergistic memory type NK cell (EM-NK) culture medium and application thereof

Abstract

The invention relates to a memory-enhancing NK cell (EM-NK) culture medium and application thereof. The synergistic memory type NK cell (EM-NK) culture medium comprises a cell factor and antibody combination and mobilized peripheral blood plasma. According to the invention, the plasma component of the mobilized PBMC is added into a culture system and contains bioactive substances such as various cytokines and exosomes, so that the proliferation and activity enabling of cells are facilitated, and the stability and effectiveness in the preparation process are kept. Meanwhile, due to the fact that a specific cell factor and antibody combination is added into the culture medium, the purity of the obtained synergistic memory type NK cells is obviously improved, and meanwhile the killing rate is also obviously improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an enhanced memory NK cell (EM-NK) culture medium and its uses. Background Technology

[0002] Cells, as an important component of the innate immune system, play a crucial role in defending against infection and monitoring cancer. In recent years, with in-depth research, the memory-like function of NK cells has been gradually discovered and has attracted widespread attention. Memory-like NK cells possess enhanced responses and long-term protective capabilities against specific antigens, providing new strategies for immunotherapy and vaccine development. However, current methods for preparing memory-like NK cells still have many shortcomings. Traditional NK cell preparation methods mainly rely on in vitro culture and gene modification, which suffer from low efficiency, complex operations, and high costs. The purity of the prepared NK cells is generally low, containing impurities from other immune cells (such as T cells and B cells), which affects the purity and therapeutic efficacy of memory-like NK cells. During the preparation process, the activity of memory-like NK cells may be damaged, leading to weakened cytotoxicity and killing power, thereby reducing the therapeutic effect.

[0003] Therefore, developing an efficient, stable, and reproducible preparation method to effectively induce NK cell memory-like properties and significantly enhance cell activity is of great significance for promoting the application of NK cells in the field of immunotherapy. Summary of the Invention

[0004] This application provides an enhanced memory NK cell (EM-NK) culture medium and its application. The EM-NK culture medium of this invention includes a specific combination of cytokines and antibodies (i.e., 50 ng / mL IL-2, 50 ng / mL IL-7, 10 ng / mL IL-12, 50 ng / mL IL-15, 50 ng / mL IL-18, 50 ng / mL anti-CD3 antibody, and 50 ng / mL anti-CD16 antibody) and 10% (volume) of mobilized peripheral blood plasma. Enhanced memory NK immune cells (EM-NK) cultured in the above culture medium exhibit stronger tumor-killing effects and proliferative capacity. Simultaneously, these NK cells also possess the ability to recognize antigens a second time, thus demonstrating higher efficacy and durability in tumor immunotherapy.

[0005] The present invention is achieved using the following technical solution.

[0006] On one hand, the present invention provides an enhanced memory NK cell (EM-NK) culture medium comprising a combination of cytokines and antibodies.

[0007] Preferably, the cytokine and antibody combination consists of 50 ng / mL IL-2, 50 ng / mL IL-7, 10 ng / mL IL-12, 50 ng / mL IL-15, 50 ng / mL IL-18, 50 ng / mL anti-CD3 antibody, and 50 ng / mL anti-CD16 antibody.

[0008] Preferably, the culture medium further comprises mobilized peripheral blood plasma.

[0009] Preferably, the volume concentration of the mobilized peripheral blood plasma is 10%.

[0010] Preferably, the culture medium further comprises a basal culture medium.

[0011] Preferably, the basal culture medium is RPMI 1640 medium or RPMI-1640 medium containing 10% FBS.

[0012] On the other hand, the present invention provides the application of the above-mentioned culture medium in the preparation of enhanced memory NK cells (EM-NK).

[0013] Compared with the prior art, this application has at least the following beneficial technical effects:

[0014] The enhanced memory NK cell (EM-NK) culture medium of the present invention includes CD3 antibody, CD16 antibody, recombinant human interleukin-2 (rhIL-2), recombinant human interleukin-12 (rhIL-12), recombinant human interleukin-15 (rhIL-15), recombinant human interleukin-18 (rhIL-18), and recombinant human interleukin-7 (rhIL-7). This culture medium optimizes the ratio, concentration, and addition time of each factor, thereby improving the quality of cell culture.

[0015] The enhanced memory NK cells (EM-NK) prepared in this invention benefit from the addition of plasma components from mobilized PBMCs to the culture system. These plasma components contain various cytokines and exosomes, which promote cell proliferation and activity, maintaining stability and effectiveness during the preparation process. Furthermore, the addition of the aforementioned cytokine and antibody combination to the culture medium significantly improves the purity and killing rate of the resulting enhanced memory NK cells. Attached Figure Description

[0016] Figure 1The cell proliferation (multiplication factor) under three different culture systems (a, b, and c) was shown. Group a: final concentrations of 5 ng / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-12, 5 ng / mL IL-15, 5 ng / mL IL-18, 5 ng / mL anti-CD3 antibody, and 5 ng / mL anti-CD16 antibody; Group b: final concentrations of 50 ng / mL IL-2, 50 ng / mL IL-7, 10 ng / mL IL-12, 50 ng / mL IL-15, and 50 ng / mL IL-18.

[0017] Group C: 50 ng / mL anti-CD3 antibody, 50 ng / mL anti-CD16 antibody; Group C: final concentration of 100 ng / mL IL-2.

[0018] 100ng / mL IL-7, 100ng / mL IL-12, 100ng / mL IL-15, 100ng / mL IL-18, 100ng / mL

[0019] Anti-CD3 antibody, 100 ng / mL anti-CD16 antibody; in the figure, blue represents group a, orange represents group b, and gray represents group c;

[0020] Figure 2 To illustrate cell proliferation (multiplication factor) under four different plasma concentration systems: Group A: no G-PBMC plasma added; Group B: 5% (volume) G-PBMC plasma added; Group C: 10% (volume) G-PBMC plasma added; Group D: 20% (volume) G-PBMC plasma added. In the figure, blue represents Group A, orange represents Group B, gray represents Group C, and yellow represents Group D. Figure 3 The phenotypes of NK cells obtained by conventional culture methods and the enhanced memory NK cells of this invention are as follows: Figure 3 A represents the traditional cultured NK cell phenotype; Figure 3 B represents the phenotype of the enhanced memory NK cells of the present invention;

[0021] Figure 4 Enhanced CT images of the abdominal and pelvic cavities before and after treatment with the enhanced memory NK cell therapy of the present invention for patients with malignant peritoneal mesothelioma, wherein (A) is a CT image before NK cell infusion; and (B) is a CT image after NK cell infusion. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0023] Example 1: Traditional method for culturing memory-like NK cells

[0024] (1) Peripheral blood was drawn from healthy volunteers and placed in a blood collection tube containing heparin anticoagulant. Ficoll separation solution was poured into a 50 mL centrifuge tube, and peripheral blood diluted twice with PBS solution was slowly added along the tube wall. The tube was centrifuged at 3000 rpm for 10 min at 4°C. After centrifugation, the intermediate white membrane layer was aspirated to obtain mononuclear cells, which were then placed in a new 15 mL centrifuge tube. 5 mL of PBS was added to resuspend the mononuclear cell layer, and the tube was centrifuged at 3000 rpm for 5 min. The supernatant was discarded to obtain the cell pellet. 5 mL of erythrocyte lysis buffer was added to the pellet, and after resuspending, the tube was centrifuged at 3000 rpm for 5 min. After removing the supernatant, the cells were washed once with PBS to obtain the cells, which were NK cells.

[0025] (2) The NK cells were seeded in RPMI-1640 medium containing 10% FBS, and IL-12, IL-15 and IL-18 were added at a final concentration of 10 ng / mL, 10 ng / mL and 50 ng / mL. The cells were cultured in a 37°C, 5% CO2 incubator. The cell status and density were observed. The medium and cytokines were added every 2-3 days. The cells were harvested after 14 days. These were memory-like NK cells.

[0026] Example 2: Collection of peripheral blood mononuclear cells and isolation of NK precursor cells after mobilization

[0027] (1) Peripheral blood mononuclear cells were collected from healthy donors after subcutaneous injection of granulocyte colony-stimulating factor (rhG-CSF). The mobilization method was as follows: human granulocyte colony-stimulating factor (rhG-CSF) 5 μg / (kg.d) q 12h was administered subcutaneously for 4.5 days. The collection method was to use a blood cell separator to collect 200-300 mL of peripheral blood hematopoietic stem cell collection material (G-PBMCs) from 10000 mL of circulating whole blood.

[0028] (2) Transfer 5 mL of LG-PBMC into a 15 mL centrifuge tube, add an equal volume of lymphocyte separation medium, and ensure a clear interface between the blood and the separation medium. Centrifuge at 2000 rpm for 20 min, then transfer the intermediate white membrane layer to a new 15 mL centrifuge tube.

[0029] (3) Add about 10 mL of PBS, mix well, centrifuge (1500 rpm, 10 min), wash, discard the supernatant, and repeat the washing once.

[0030] (4) Resuspend G-PBMCs in 1 mL of MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) to achieve an appropriate cell concentration.

[0031] (5) Add an appropriate amount of NK cell Biotin-Antibody Cocktail to the cell suspension (50 μL per 10^7 cells) and incubate on ice for 10 min.

[0032] (6) Add NK cell MicroBead Cocktail (per 10) 7 Add 50 μL of the solution to each cell and incubate on ice for 15 min.

[0033] (7) Add about 10 mL of MACS buffer, mix well, and centrifuge to wash (1500 rpm, 10 min). Discard the supernatant and resuspend the cells in 500 μL of MACS buffer.

[0034] (8) Place the MS separation column in the magnetic field of the MACS sorter and wet the separation column with 500 μL of MACS buffer. Add the cell suspension to the separation column to allow NK cells to adhere to the column.

[0035] (9) Wash the separation column three times with 500 μL of MACS buffer to remove unadsorbed non-NK cells.

[0036] (10) Remove the separation column from the magnetic field and place it on a clean collection tube. Add 1 mL of MACS buffer to wash off the NK cells adsorbed on the column.

[0037] (11) Take a portion of the sorted NK cell suspension and use flow cytometry to detect the expression of CD3, CD56 and CD16, and calculate the purity of the sorted NK cells.

[0038] Example 3 Enhancement Activation

[0039] The NK cells prepared in Example 2 were irradiated with light at a wavelength of 635 nm and a power density of 50 mW / cm². 2 Irradiate for 20 minutes.

[0040] Example 4: Determination of the cytokines and antibodies added to the culture system:

[0041] (1) Add the following three groups of cytokines and antibodies to RPMI-1640 medium containing 10% FBS:

[0042] Group A: Final concentrations of 5 ng / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-12, 5 ng / mL IL-15, 5 ng / mL IL-18, 5 ng / mL anti-CD3 antibody, and 5 ng / mL anti-CD16 antibody;

[0043] Group b: Final concentrations of 50 ng / mL IL-2, 50 ng / mL IL-7, 10 ng / mL IL-12, 50 ng / mL IL-15, 50 ng / mL IL-18, 50 ng / mL anti-CD3 antibody, and 50 ng / mL anti-CD16 antibody;

[0044] Group C: Final concentrations of 100 ng / mL IL-2, 100 ng / mL IL-7, 100 ng / mL IL-12, 100 ng / mL IL-15, 100 ng / mL IL-18, 100 ng / mL anti-CD3 antibody, and 100 ng / mL anti-CD16 antibody;

[0045] (2) The NK cells activated in Example 3 were cultured at 37°C in a 5% CO2 incubator in RPMI-1640 medium containing 10% FBS, supplemented with the aforementioned three groups of cytokines and antibodies. The culture was continued for 7 days, with the NK cell seeding density being 1 x 103. 6 cells / ml.

[0046] (3) The proliferation of the obtained cells is as follows Figure 1 As shown, from Figure 1 It is evident that adding group B cytokines and antibodies yields the best results.

[0047] Example 5: Preparation and Addition of G-PBMC Plasma

[0048] 1. Preparation of G-PBMC plasma:

[0049] a. Collected G-PBMCs (collected as described in step (1) of Example 2) were added to 50 mL of 6% hydroxyethyl starch, and red blood cells were separated by natural sedimentation. After 30 min of suspension sedimentation, the settled red blood cells in the cell bag were removed.

[0050] b. Centrifuge the sample bag using a large-capacity, low-speed blood bag centrifuge at 800g for 10 minutes. Collect the upper plasma layer after centrifugation. Store at -20 degrees Celsius for later use. Allow the sample to return to room temperature before use.

[0051] 2. Addition of G-PBMC plasma:

[0052] The NK cells prepared in Example 2 were cultured in RPMI-1640 medium containing 10% FBS, with the addition of the group b cytokines and antibodies described in Example 4 and the G-PBMC plasma prepared in step 1. The culture was carried out at 37°C in a 5% CO2 incubator for 7 days. Every two days, the same concentration of plasma, RPMI-1640 medium containing 10% FBS, and the group b cytokines and antibodies described in Example 4 were added. The seeding density of NK cells was 1 x 102. 6 cells / ml, harvest memory-like NK cells; the following shows the results when adding the plasma prepared in step 1:

[0053] Group A: No G-PBMC plasma added; Group B: 5% (by volume) G-PBMC plasma added;

[0054] Group C: Added 10% (by volume) of G-PBMC plasma;

[0055] Group D: Added 20% (by volume) of G-PBMC plasma;

[0056] As a result, Figure 2 As shown in the figure. The results showed that group C: the addition of 10% (by volume) of G-PBMC plasma had the best effect.

[0057] Therefore, the culture system for EM-NK was ultimately determined to include a combination of group b cytokines and antibodies, as well as 10% (volume) G-PBMC plasma from group C.

[0058] Example 6: Preparation and Quality Control of Enhanced Memory NK Cells (EM-NK Cells) of the Present Invention

[0059] The NK cells prepared in Example 2 were irradiated with laser as described in Example 3. Then, they were cultured, expanded, and quality-controlled in an EM-NK culture system containing 10% FBS in RPMI-1640 medium supplemented with the previously determined group b cytokine and antibody combination and group C 10% (volume) G-PBMC plasma to ensure its safety and efficacy. The NK cell seeding density was 1 x 102 6 The enhanced memory NK cells of this invention are obtained by measuring cells / ml.

[0060] Example 7: Phenotypic identification of the enhanced memory NK cells of the present invention and NK cells cultured using conventional methods.

[0061] (1) A blank control group, a conventional culture group (culture scheme of Example 1), and an experimental group (scheme of Example 6 of this invention) were set up, and 1×10⁻⁶ samples were collected from each group. 6 Centrifuge 1500 rpm for 3 minutes to obtain NK cells. After centrifugation, discard the supernatant. Wash once with 1 mL of PBS by centrifugation and discard the supernatant.

[0062] (2) No cytokines or antibodies were added to the blank control group. 5 μL of PEMouseAnti-Human CD3 and 2.5 μL of FITC MouseAnti-Human CD56 were taken from the conventional culture group and the experimental group, respectively, and mixed by vortexing. The reaction was carried out in the dark for 15 min, and mixed again after 7 min.

[0063] (3) Take 1 mL of PBS, centrifuge and wash, and discard the supernatant. Add 500 μL of PBS to resuspend, and then use the instrument to detect the NK cell phenotype of each group.

[0064] (4) Cell phenotype detection was performed on the conventional culture group NK cells and the enhanced memory NK cells prepared by the method of the present invention using the above method. The results are shown in Figure (). Figure 3 As shown in the figure, the enhanced memory NK cells prepared by the method of the present invention have a purity far greater than that of NK cells in the traditional culture group.

[0065] Example 8: Evaluation of the cytotoxic function of the enhanced memory NK cells of the present invention and NK cells cultured using conventional methods.

[0066] (1) NK cells from the conventional culture group (culture scheme of Example 1) and the experimental group (scheme of Example 6 of this invention) were used as effector cells, and the cell concentration was adjusted to 1×10⁻⁶ using RPMI 1640. 7 Units / mL are available for use.

[0067] (2) K562 leukemia cells were used as target cells. Target cells in good growth condition were collected, washed once with 1×PBS, and resuspended at 5×10⁻⁶. 6 Units / mL are available for use.

[0068] (3) Add 5 μL Annexin V to the target cell suspension, mix once during the process, add 5 times the volume of complete culture medium, wash once at 1200 rpm for 3 min, discard the supernatant, and resuspend the cells in culture medium to 1×10⁻⁶. 6 per mL.

[0069] (4) Add the treated effector cells and target cells to 5 mL flow cytometry tubes at different effector-to-target ratios (1:1, 5:1, 10:1, 20:1). Add 100 μL of target cells and the corresponding volume of effector cells to each tube, and keep two separate tubes containing target cells and effector cells as controls. After mixing the effector cells and target cells, centrifuge at 300 g for 1 min to ensure close contact between the cells, and incubate in a 5% CO2, 37°C incubator for 4 h.

[0070] (5) Wash the cells by centrifugation with pre-cooled PBS, dilute 5×Binding Buffer with double-distilled water to make 1× working solution, take 500μL of 1×Binding Buffer to resuspend the cells, add 10μL of 7-AAD to each tube, gently vortex to mix, and incubate at room temperature in the dark for 5min.

[0071] (6) Calculate the killing rate of NK cells against K562 tumor cells in the traditional culture group and the experimental group respectively.

[0072] Table 1 Comparison of NK kill rates between the control group and the experimental group (EM-NK)

[0073] Effectiveness-to-target ratio NK kill rate (%) in the control group EM-NK kill rate (%) in the experimental group 1:1 10.4 16.7 5:1 15.8 26.4 10:1 34.1 45.2 20:1 57.5 66.7

[0074] Example 9: Application of the present invention in enhancing memory-type NK cell tumor immunotherapy:

[0075] Research Protocol: Patients with stage IV cancer who have not responded well to surgery, radiotherapy, or chemotherapy, or whose life expectancy is less than 6 months, will be screened. They will be treated with the enhanced memory NK cells (obtained from Example 6) of this invention, administered intravenously to exert their anti-tumor effect. The single infusion dose is 50 × 10⁻⁶ cells / day. 8 The medication is administered once every 1-2 weeks, with at least 4 infusions per course of treatment. Patient immune function and antitumor efficacy are evaluated. CR indicates complete remission, PR indicates partial remission, and SD indicates stable disease.

[0076] Study Results: Between 2023 and 2024, a total of 8 patients aged 45-84 years were enrolled and received 4-17 EM-NK infusions, with a single dose of 50×10⁻⁶. 8 The total dose is 200-850 billion.

[0077] Efficacy: All enrolled patients had stage IV peritoneal metastases or combined extraperitoneal metastases. After surgery, chemotherapy, targeted therapy, etc., the OS of patients treated with the enhanced memory NK cell therapy of this invention reached up to 81 months, all of which were higher than the expected lifespan. Four patients were in complete remission, two patients were in partial remission, and two patients died of systemic organ failure due to disease-related reasons despite stable tumors.

[0078] Safety evaluation: A total of 62 infusions were administered to 8 patients. Some patients experienced cytokine release reactions such as fever and chills, which were relieved by oral ibuprofen or intravenous dexamethasone. No other adverse events occurred. Two patients died due to multiple organ failure caused by long-term tumor consumption, which was unrelated to the enhanced memory NK cell infusion of this invention. The patient conditions and treatment results of the enhanced memory NK cell infusion of this invention are shown in Table 2.

[0079] Table 2

[0080]

[0081]

[0082] Among them, the enhanced CT results of the abdominal and pelvic cavities of patient No. 2 with malignant peritoneal mesothelioma before and after enhanced memory NK cell therapy are as follows: Figure 4 As shown in the CT images after NK infusion, the amount of ascites in (B) is reduced compared to before.

[0083] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and they should not be used to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An enhanced memory NK cell (EM-NK) culture medium comprising a combination of cytokines and antibodies, and mobilized peripheral blood plasma.

2. The enhanced memory NK cell (EM-NK) culture medium according to claim 1, wherein, The cytokine and antibody combination consists of 50 ng / mL IL-2, 50 ng / mL IL-7, 10 ng / mL IL-12, 50 ng / mL IL-15, 50 ng / mL IL-18, 50 ng / mL anti-CD3 antibody, and 50 ng / mL anti-CD16 antibody.

3. The enhanced memory NK cell (EM-NK) culture medium according to claim 1 or 2, wherein, The culture medium also contains mobilized peripheral blood plasma.

4. The enhanced memory NK cell (EM-NK) culture medium according to claim 3, wherein, The volume concentration of the mobilized peripheral blood plasma was 10% (volume concentration).

5. The enhanced memory NK cell (EM-NK) culture medium according to any one of claims 1 to 4, wherein, The culture medium also includes a basal culture medium.

6. The enhanced memory NK cell (EM-NK) culture medium according to claim 5, wherein, The basal culture medium is RPMI-1640 medium or RPMI-1640 medium containing 10% FBS.

7. The use of the enhanced memory NK cell (EM-NK) culture medium according to any one of claims 1 to 6 in the preparation of enhanced memory NK cells (EM-NK).

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