NK cell induction expansion culture additive, culture medium and application thereof

Abstract

The present application relates to a kind of NK cell induction amplification culture additive, culture medium and its application.The described one additive includes tanshinone I and berberine.Certainly, PBMC is isolated from human peripheral blood, tanshinone I (final concentration 0.1~10 µM) is added in culture system on the 3rd day of NK activation culture, and berberine (final concentration 10~100 µM) is cultured, tanshinone I and berberine are both natural products and widely available, combined use can significantly improve the proportion of memory NK cell, simultaneously with the characteristics of high activity, high purity, high amplification multiple and high killing activity.Using flow cytometry, the expression positive rate of some markers CD62L, CD127, Eomes of NK cell is significantly increased.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and immunotherapy, specifically relating to an NK cell induction and expansion culture additive, culture medium, and their applications. Background Technology

[0002] Natural killer (NK) cells are core effector cells of the innate immune system. Because they can directly recognize and kill malignant cells and are not restricted by the major histocompatibility complex (MHC), they have shown great potential in adoptive immunotherapy for tumors. However, conventional NK cells expanded using conventional methods (such as high-dose interleukin-2) have inherent limitations in vivo, including short survival time, limited homing ability, and inability to form long-term immune memory. These limitations lead to less than satisfactory clinical efficacy, especially in preventing tumor recurrence.

[0003] In recent years, studies have found that NK cells, similar to T and B lymphocytes, also possess the ability to form "memory" subsets. After being stimulated by primary antigens or cytokines, memory NK cells can survive in vivo for a long time and generate a faster, stronger, and more durable immune response when encountering the same or similar threats again. Compared with conventional NK cells, memory NK cells have the following significant advantages: (1) Durable in vivo survival and long-term protection: After reinfusion, memory NK cells can survive in the host for a long time, forming long-term immune surveillance and effectively preventing tumor recurrence. (2) Enhanced restimulation response: When encountering tumor antigens again, memory NK cells can rapidly expand and produce a large number of effector molecules (such as IFN-γ, perforin, and granzyme), and their response speed and intensity far exceed those of primary NK cells. (3) Superior homing and tissue residency characteristics: Some memory NK cell subsets (such as tissue-resident memory NK cells) highly express specific homing receptors, enabling them to migrate and reside more effectively in specific tissues, enhancing their ability to clear local lesions. (4) Potential antigen specificity: Adaptive / memory NK cells (such as the NKG2C+CD57+ subset) can generate memory responses to specific antigens, providing the possibility for developing more targeted NK therapies. Given the above advantages, inducing and enriching memory NK cells has become a recognized strategy to improve the efficacy of NK cell therapy. Currently, the mainstream induction protocol uses a combination of cytokines interleukin-12, interleukin-15, and interleukin-18 to perform a short-term "pre-stimulation" of quiescent NK cells for 12-24 hours at the beginning of culture. However, in translational applications, especially in the large-scale, standardized preparation of NK cells derived from peripheral blood of healthy donors, the cells induced by this classic protocol are difficult to fully utilize the aforementioned memory advantages, and interleukin-12 has significant systemic inflammatory toxicity, the residual risk of which restricts its widespread acceptance in clinical applications. At the same time, interleukin-12 and interleukin-18 are both expensive GMP-grade recombinant cytokines, which are the main factors leading to high treatment costs and are not conducive to the popularization of "universal" drugs.

[0004] In summary, although memory NK cells theoretically possess significant advantages, existing induction techniques suffer from low efficiency, high cost, and functional deficiencies, severely hindering the full realization of these advantages and their clinical translation. Therefore, there is an urgent need in this field to develop a novel induction strategy that can reprogram conventionally expanded NK cells into memory NK cells on a large scale, efficiently, and with high quality in a more economical and safer manner. This would truly realize the theoretical advantages of durable in vivo protection and robust secondary response, providing core technology for the development of next-generation "universal" NK cell therapy products. Summary of the Invention

[0005] Based on the above background, the present invention provides an NK cell induction and expansion culture additive, which is composed of tanshinone I with a final concentration of 0.1~10µM and berberine with a final concentration of 10~100µM.

[0006] Preferably, the NK cell induction and expansion culture additive is composed of tanshinone I at a final concentration of 5 µM and berberine at a final concentration of 50 µM.

[0007] An NK cell induction and expansion culture medium includes a basal culture medium, IL-2 at a final concentration of 500 IU / mL, IL-21 at a final concentration of 20 ng / mL, IL-15 at a final concentration of 20 ng / mL, 10% heat-inactivated autologous plasma, and the aforementioned NK cell induction and expansion culture additive.

[0008] Preferably, the basal culture medium is either SCGM NK medium or MACS NK medium.

[0009] Preferably, the NK cell induction and expansion culture additive is added starting on the third day of NK cell culture.

[0010] An application of an NK cell induction and expansion culture medium for in vitro induction, expansion, and enhancement of NK cell memory phenotype and function.

[0011] Memory NK cells are a subset of NK cells that possess long-lasting immune memory function, formed through clonal expansion and functional differentiation in response to specific antigen stimulation. They promote NK cell proliferation and epigenetic remodeling via downstream signaling pathways such as STAT4 and STAT5, thereby driving the stable expression of memory-related genes and enabling cells to achieve long-term survival and rapid re-response. During this process, the phenotype of memory NK cells undergoes characteristic reprogramming. On the one hand, enhanced expression of CD62L, a biomarker related to survival and homing, facilitates cell homing, thus participating in the establishment and maintenance of the memory pool; high-level expression of CD127 provides crucial signaling support for long-term cell survival; and high expression of the transcription factor Eomes is closely related to the terminal differentiation and functional persistence of memory NK cells, promoting cytotoxicity and stabilizing memory-related gene programming. Therefore, those skilled in the art can effectively assess the formation level and functional status of memory NK cells by analyzing the significant upregulation of CD62L, CD127, and Eomes expression. Therefore, it can be explained that the specific embodiment effectively promotes the formation of memory NK cells with homing and long-term survival capabilities by adding a composition, thereby enhancing the long-term immune memory function of NK cells as a whole.

[0012] Beneficial Effects: This invention, by adding a well-defined composition to the NK culture medium and combining it with commercially available NK culture media, ensures high NK cell viability while enabling rapid and large-scale proliferation of NK cells, resulting in NK cells with high purity, high cytotoxic activity, and a high memory phenotype. The tanshinone I contained in this invention promotes memory metabolic characteristics by altering glucose and lipid metabolism. Berberine contained in this invention activates the AMPK pathway to enhance memory formation and improves the quality of memory NK cells by regulating cell metabolism and homing ability.

[0013] The NK cells cultured with the composition tanshinone I and berberine of this invention exhibit high NK cell viability, high expansion rate, high purity, and high cytotoxicity. Compared with the classic interleukin-12 / 15 / 18 induction system, it can increase the positive rates of CD62L, CD127, and Eomes in memory NK cells cultured for 14 days by 33.8, 29.4, and 37.4 percentage points, respectively. Attached Figure Description

[0014] Figure 1 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M1 experimental group of Example 1, obtained by flow cytometry. Figure 2 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M2 experimental group of Example 1, obtained by flow cytometry. Figure 3 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M3 experimental group of Example 1, obtained by flow cytometry. Figure 4 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M4 experimental group of Example 1, obtained by flow cytometry. Figure 5 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M5 experimental group of Example 1, measured by flow cytometry. Figure 6 This is a characterization graph of the memory phenotype positivity rate of NK cells in the first experimental group of Example 2, measured by flow cytometry. Figure 7 This is a characterization graph of the memory phenotype positivity rate of NK cells in the second experimental group of Example 2, obtained by flow cytometry. Figure 8 This is a characterization graph of the memory phenotype positivity rate of NK cells in the third experimental group of Example 2, measured by flow cytometry. Figure 9 This is a characterization graph of the memory phenotype positivity rate of NK cells in the fourth experimental group of Example 2, measured by flow cytometry. Figure 10This is a characterization graph of the memory phenotype positivity rate of NK cells in the fifth experimental group of Example 2, measured by flow cytometry. Figure 11 This is a characterization graph of the memory phenotype positivity rate of NK cells in the sixth experimental group of Example 2, measured by flow cytometry. Figure 12 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M1 experimental group of Example 3, obtained by flow cytometry. Figure 13 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M2 experimental group of Example 3, obtained by flow cytometry. Figure 14 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M3 experimental group of Example 3, obtained by flow cytometry. Figure 15 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M4 experimental group of Example 3, obtained by flow cytometry. Figure 16 This is a characterization graph of the memory phenotype positivity rate of NK cells in the M5 experimental group of Example 3, obtained by flow cytometry. Figure 17 This is a characterization graph of the memory phenotype positivity rate of NK cells in the first experimental group of Example 4, measured by flow cytometry. Figure 18 This is a characterization graph of the memory phenotype positivity rate of NK cells in the second experimental group of Example 4, measured by flow cytometry. Figure 19 This is a characterization graph of the memory phenotype positivity rate of NK cells in the third experimental group of Example 4, measured by flow cytometry. Figure 20 This is a characterization graph of the memory phenotype positivity rate of NK cells in the fourth experimental group of Example 4, measured by flow cytometry. Figure 21 This is a characterization graph of the positive rate of memory phenotype of NK cells in the fifth experimental group of Example 4, measured by flow cytometry. Figure 22 This is a characterization graph of the memory phenotype positivity rate of NK cells in the sixth experimental group of Example 4, obtained by flow cytometry. Detailed Implementation

[0015] The technical solutions of the present invention will be further described below with reference to specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] To more clearly illustrate the purpose, technical solution, and advantages of this invention, the following detailed description is provided in conjunction with the accompanying drawings.

[0017] The experimental method used in this invention for culturing peripheral blood NK cells is as follows.

[0018] I. Experimental Materials: SCGM NK medium (Sartorius, catalog number 20802-0500), MACS NK medium (Mitteni, catalog number 130-114-429), rhIL-2 (nearshore protein, catalog number GMP-CD66), rhIL-15 (nearshore protein, catalog number GMP-C016), rhIL-21 (nearshore protein, catalog number GMP-CC45), rhIL-12 (nearshore protein, catalog number GMP-CI58), rhIL-18 (nearshore protein, catalog number GMP-CH29), Danshen Berberine I (Aladdin, T407817), Berberine (Aladdin, catalog number B422528), CD3 antibody (Baijin Biotechnology, catalog number 300439), CD56 antibody (Baijin Biotechnology, catalog number 362508), CD62L antibody (Baijin Biotechnology, catalog number 362508), CD127 antibody (Baijin Biotechnology, catalog number 362508), Eomes antibody (Thermo Fisher Scientific, catalog number 25-4877-41).

[0019] II. Experimental Methods: (1) NK cell culture: The method used in this invention involves isolating fresh PBMCs from human whole blood using density centrifugation for NK cell activation and expansion. On day 0, the isolated PBMC cell suspension is transferred to a prepared T75 culture flask coated with monoclonal CD16 antibody, and the cell density is adjusted to 2 × 10⁶ cells / mL using NK medium. 6 Cells were cultured at a concentration of 500 IU / mL for rhIL-2, 20 ng / mL for rhIL-21, 20 ng / mL for rhIL-15, and 10% heat-inactivated autologous plasma. The cells were then incubated at 37 °C in a 5% CO2 incubator for cell activation. On day 3 after activation, the cells were transferred to new T75 culture flasks containing NK medium supplemented with the aforementioned rhIL-2, rhIL-15, rhIL-21, and 10% heat-inactivated autologous plasma. Cell viability and density were measured every two days, and fluid was replenished to maintain a cell density of 1–1.5 × 10⁶ cells / mL. 6 Cells / mL, the composition of the invention is added starting on day 3 and culture continues until day 14 to obtain the cells to be treated.

[0020] The classic interleukin-12 / 15 / 18 induction system culture method: Fresh PBMCs were isolated from human whole blood using density centrifugation for NK cell activation and expansion. On day 0, the isolated PBMC cell suspension was transferred to a T75 culture flask, and the cell density was adjusted to 2 × 10⁶ cells / mL using NK medium. 6 Cells were cultured at a concentration of 10 ng / mL for rhIL-12, 10 ng / mL for rhIL-15, 50 ng / mL for rhIL-18, and 10% heat-inactivated autologous plasma. The cells were then incubated at 37°C in a 5% CO2 incubator for 16–18 h for activation. After 16–18 h of pulsed activation, cells were collected, washed once with pre-warmed medium, and resuspended in complete medium containing 30 ng / mL rhIL-15. The cells were then transferred to new culture flasks and cultured at 37°C in a 5% CO2 incubator. Cell viability and density were measured every two days, and fluid was replenished to maintain a cell density of 1–1.5 × 10⁶ cells / mL. 6 Cells were cultured at a density of 10 cells / mL. The replenishment medium was a complete medium containing 30 ng / mL rhIL-15 and 10% heat-inactivated autologous plasma. The cells were cultured until day 14 to obtain the cells to be treated.

[0021] (2) Detection of NK cell viability and proliferation efficiency: Samples were taken on days 0, 3, 5, 7, 9, 11, and 14 of culture (corresponding to D0, D3, D5, D7, D9, D11, and D14) for counting to obtain data on cell proliferation and cell viability.

[0022] (3) Identification of NK cell surface markers: On day 14 of cell culture, NK cell surface markers were measured and analyzed. Approximately 1.0 × 10⁶ cells were collected. 6 Centrifuge cells at 2000 rpm for 5 min, discard the supernatant, and resuspend in PBS. Incubate the cells with fluorescently labeled CD3, CD56, CD62L, and CD127 antibodies at 4 °C for 15 min. Wash twice with PBS, discard the supernatant, and resuspend in 0.5 mL PBS. Analyze the processed cells using flow cytometry.

[0023] (4) Identification of intracellular markers in NK cells: On day 14 of cell culture, intracellular markers of NK cells were measured and analyzed. Approximately 1.0 × 10⁶ cells were collected. 6Centrifuge cells at 2000 rpm for 5 min, discard the supernatant, and resuspend in PBS. Incubate the cells with fluorescently labeled CD3 and CD56 antibodies at 4 °C for 15 min. Wash twice with PBS, discard the supernatant, and fix and permeate the cells at 4 °C in the dark for 30–60 min. After incubation, wash twice with 2 mL of pre-chilled permeabilization buffer, discard the supernatant, and resuspend the permeabilized cells with 100 µL of pre-chilled permeabilization buffer diluted with Eomes antibody. Incubate at 4 °C in the dark for 30–60 min. After incubation, wash twice with 2 mL of permeabilization buffer, discard the supernatant, and resuspend in 0.5 mL of PBS. Analyze the processed cells using flow cytometry.

[0024] (5) NK cell killing activity assay (CCK8 assay): Vigorously growing K562 cell lines were used as target cells. After washing twice with RPMI-1640 medium, the cell density was adjusted to 1×10⁻⁶ cells / year. 6 / mL. 50 µL was seeded into each well of a 96-well plate. NK cells cultured to day 14 were harvested and the cell density adjusted to 1×10⁹ / mL. 6 / mL, 2×10 6 / mL, 5×10 6 / mL, 1×10 7 / mL, 50 µL was added to a 96-well plate to achieve target-effect ratios of 1:1, 2.5:1, 5:1, 10:1, and 20:1, mainly divided into four groups: experimental group (K562+NK), control group (NK+RPMI-1640), maximum release group (K562+RPMI-1640), and blank group (RPMI-1640), with 3 replicates per group. After co-culturing the seeded cells in an incubator for 12 h, 10 μL of CCK-8 reagent (MCE, catalog number HY-K0301) was added to each well, shaken well, and cultured for another 3 h. The absorbance at 450 nm was measured using a microplate reader, and the killing rate was calculated as follows: 1 - [experimental group (OD value) - control group (OD value) - blank group (OD value)] / [maximum release group (OD value) - blank group (OD value)] × 100.

[0025] The present invention will now be described in detail with reference to specific embodiments.

[0026] Example 1: NK cells were cultured and identified strictly according to the above experimental method. Starting from day 3 (D3), different additives were added to SCGM NK medium. The experimental groups were set up as follows: M1 (tanshinone I): Tanshinone I was added to SCGM NK medium containing rhIL-2 at a final concentration of 500 IU / mL, rhIL-21 at a final concentration of 20 ng / mL, rhIL-15 at a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma at a concentration of 5 µM. M2 (berberine): Berberine was added to SCGM NK medium containing rhIL-2 at a final concentration of 500 IU / mL, rhIL-21 at a final concentration of 20 ng / mL, rhIL-15 at a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma at a concentration of 50 µM. M3 (Heat-inactivated autologous plasma): Only heat-inactivated autologous plasma was added to SCGM NK medium, with the addition ratio controlled at 10%, as a blank control; M4 (complex combination): Add tanshinone I to SCGM NK medium containing rhIL-2 at a final concentration of 500 IU / mL, rhIL-21 at a final concentration of 20 ng / mL, rhIL-15 at a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma at a final concentration of 5 µM, and berberine at a final concentration of 50 µM. M5 (classic interleukin-12 / 15 / 18 induction system): rhIL-12, rhIL-15, and rhIL-18 were added sequentially to SCGM NK medium to achieve final concentrations of 10 ng / mL, 10 ng / mL, and 50 ng / mL, respectively. Pulsed induction was performed for 16±2 hours. After the pulse induction ended, rhIL-12 / rhIL-18 were washed away, and the medium was replaced with SCGM NK medium containing rhIL-15 (30 ng / mL) and cultured until day 14.

[0027] The test results are as follows: 1. Cell viability of NK cells: The experimental results are shown in Table 1. Table 1

[0028] The addition of the composition of this invention maintained high cell viability. On day 14, the viability of experimental group M1 was 96.53%, experimental group M2 was 95.19%, experimental group M3 was 94.22%, experimental group M4 was 97.31%, and experimental group M5 was 95.67%. The addition of the composition maintained the high viability level of NK cells at harvest.

[0029] 2. The fold increase of NK cells, the experimental results are shown in Table 2: Table 2

[0030] The addition of the composition of this invention maintained the high cell amplification capacity. On day 14, the NK cell amplification in the M1 experimental group was 1215.04 times, the M2 experimental group was 1158.4 times, the M3 experimental group was 1052.45 times, the M4 experimental group was 1315.63 times, and the M5 experimental group was 1178.56 times. Compared with the blank control group (M3) without the addition of active ingredients and the classic interleukin-12 / 15 / 18 induction system group (M5), the addition of the composition did not reduce the amplification fold, but rather slightly increased it.

[0031] 3. Purity of NK cells; experimental results are shown in Table 3: Table 3

[0032] The addition of the composition of this invention maintained high NK cell purity. On day 14, the purity of experimental group M1 was 96.6%, experimental group M2 was 95.1%, experimental group M3 was 94.2%, experimental group M4 was 98.6%, and experimental group M5 was 91.3%. The addition of the composition maintained high NK cell purity at harvest.

[0033] 4. The killing efficiency of NK cells is shown in Table 4: Table 4

[0034] The addition of the composition of this invention maintains high NK cell killing activity. At an effector-to-target ratio of 5:1, the NK cell killing rate on day 14 was 69.2% in the M1 experimental group, 71.2% in the M2 experimental group, 64.8% in the M3 experimental group, 76.6% in the M4 experimental group, and 62.1% in the M5 experimental group. Compared with the blank control group (M3) without the addition of active ingredients and the classic interleukin-12 / 15 / 18 induction system group (M5), the composition of this invention (M4) can improve the NK cell killing efficiency by about 10%.

[0035] 5. The memory phenotype of NK cells, the experimental results are shown in Table 5: Table 5

[0036] Adding the composition of this invention significantly enhances the memory phenotype of harvested NK cells. On day 14, the positive rates of CD62L, CD127, and Eomes in NK cells of the M1 experimental group were 15.3%, 13.5%, and 18%, respectively; in the M2 experimental group, they were 15.8%, 16.4%, and 19.8%; in the M3 experimental group, they were 9.2%, 3.8%, and 9.5%; and in the M4 experimental group, they were 45.1%. The positive rates of CD62L, CD127, and Eomes in NK cells of the M5 experimental group were 11.3%, 5.9%, and 12.8%, respectively. Compared with the blank control group (M3) without added active ingredients, the composition of the present invention (M4) can increase the positive rates of CD62L, CD127, and Eomes by 35.9, 31.5, and 40.7 percentage points, respectively. Compared with the classic interleukin-12 / 15 / 18 induction system (M5), the composition of the present invention (M4) can increase the positive rates of CD62L, CD127, and Eomes by 33.8, 29.4, and 37.4 percentage points, respectively.

[0037] Example 2: NK cells were cultured and identified strictly according to the above experimental method. Starting from day 3 (D3), different additives were added to SCGM NK medium. The experimental groups were set up as follows: The first group is the low-concentration additive composition combination of the present invention: adding tanshinone I with a final concentration of 0.1 µM and berberine with a final concentration of 1 µM to SCGMNK medium containing rhIL-2 with a final concentration of 500 IU / mL, rhIL-21 with a final concentration of 20 ng / mL, rhIL-15 with a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma. The second group is the medium-concentration additive composition combination of the present invention: adding tanshinone I with a final concentration of 5 µM and berberine with a final concentration of 50 µM to SCGMNK medium containing rhIL-2 with a final concentration of 500 IU / mL, rhIL-21 with a final concentration of 20 ng / mL, rhIL-15 with a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma. The third group is a high-concentration additive composition combination of the present invention: adding tanshinone I with a final concentration of 10 µM and berberine with a final concentration of 100 µM to SCGMNK medium containing rhIL-2 with a final concentration of 500 IU / mL, rhIL-21 with a final concentration of 20 ng / mL, rhIL-15 with a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma. The fourth group was a control group without the added composition and heat-inactivated autologous plasma, containing only SCGM NK medium; The fifth group was a control group that only underwent heat inactivation of autologous plasma, with 10% heat-inactivated autologous plasma added to SCGM NK medium; The sixth group is a control of the classic interleukin-12 / 15 / 18 induction system.

[0038] The test results are as follows: 1. Cell viability of NK cells, the experimental results are shown in Table 6: Table 6

[0039] At day 14, the cell viability was 93.23% in group 1, 97.31% in group 2, 88.77% in group 3, 91.23% in group 4, 94.22% in group 5, and 95.67% in group 6. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 performed best in maintaining high cell viability.

[0040] 2. The fold increase of NK cells, the experimental results are shown in Table 7: Table 7

[0041] At day 14, the cell proliferation rate was 1018.15 in group 1, 1315.63 in group 2, 957.13 in group 3, 1023.59 in group 4, 1052.45 in group 5, and 1178.56 in group 6. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 performed best in maintaining a high cell proliferation rate.

[0042] 3. The purity of NK cells, the experimental results are shown in Table 8: Table 8

[0043] At day 14, the proportion of NK cells was 95.4% in group 1, 98.6% in group 2, 93.2% in group 3, 91% in group 4, and 94.2% in group 5. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 performed best in maintaining a high proportion of NK cells.

[0044] 4. The killing efficiency of NK cells, the experimental results are shown in Table 9: Table 9

[0045] At an effector-to-target ratio of 5:1, the NK cell killing rate on day 14 was 65.3% in group 1, 76.6% in group 2, 63.2% in group 3, 65.7% in group 4, 64.8% in group 5, and 62.1% in group 6. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 showed the most significant effect in improving NK cell killing efficiency.

[0046] 5. The memory phenotype of NK cells, the experimental results are shown in Table 10: Table 10

[0047] Adding the composition of this invention can significantly enhance the memory phenotype of harvested NK cells. On day 14, the positive rates of CD62L, CD127, and Eomes in the first group of NK cells were 15.1%, 14%, and 17.2%, respectively; in the second group, they were 45.1%, 35.3%, and 50.2%; in the third group, they were 17.5%, 16.8%, and 11.4%; in the fourth group, they were 6.2%, 3.4%, and 6.1%; and in the fifth group, they were 6.2%, 3.4%, and 6.1%. The positive rates of 27 and Eomes were 9.2%, 3.8%, and 9.5%, respectively. The positive rates of CD62L, CD127, and Eomes in NK cells of group 6 were 11.3%, 5.9%, and 12.8%, respectively. Therefore, compared with the low-concentration group, high-concentration group, and various control groups, the medium-concentration combination of group 2 was the most effective in enhancing the memory phenotype. Compared with group 5, it could increase the positive rates of CD62L, CD127, and Eomes by 35.9, 31.5, and 40.7 percentage points, respectively. Compared with the classic induction system (group 6), it could increase the positive rates of CD62L, CD127, and Eomes by 33.8, 29.4, and 37.4 percentage points, respectively.

[0048] Example 3: NK cells were cultured and identified strictly according to the above experimental method. Starting from day 3 (D3), different additives were added to the MACS NK medium. The experimental groups were set up as follows: M1 (tanshinone I): Tanshinone I was added to MACS NK medium containing rhIL-2 at a final concentration of 500 IU / mL, rhIL-21 at a final concentration of 20 ng / mL, rhIL-15 at a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma at a concentration of 5 µM. M2 (berberine): Berberine was added to MACS NK medium containing rhIL-2 at a final concentration of 500 IU / mL, rhIL-21 at a final concentration of 20 ng / mL, rhIL-15 at a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma at a concentration of 50 µM. M3 (Heat-inactivated autologous plasma): Only heat-inactivated autologous plasma was added to MACS NK medium at a ratio of 10% as a blank control. M4 (composition combination): Add tanshinone I to MACS NK medium containing rhIL-2 at a final concentration of 500 IU / mL, rhIL-21 at a final concentration of 20 ng / mL, rhIL-15 at a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma at a final concentration of 5 µM, and berberine at a final concentration of 50 µM. M5 (classic interleukin-12 / 15 / 18 induction system): Recombinant human IL-12, IL-15, and IL-18 were added sequentially to MACS NK medium to achieve final concentrations of 10 ng / mL, 10 ng / mL, and 50 ng / mL, respectively. Pulsed induction was performed for 16±2 hours. After the pulse induction ended, rhIL-12 / rhIL-18 was washed away, and the medium was replaced with MACS NK medium containing rhIL-15 (30 ng / mL) and cultured until day 14.

[0049] 1. Cell viability of NK cells: The experimental results are shown in Table 11. Table 11

[0050] The addition of the composition of this invention maintained high cell viability. On day 14, the viability of experimental group M1 was 93.54%, experimental group M2 was 93.76%, experimental group M3 was 91.69%, experimental group M4 was 96.59%, and experimental group M5 was 93.97%. The addition of the composition maintained a high level of NK cell viability at harvest.

[0051] 2. The fold increase of NK cells, the experimental results are shown in Table 12: Table 12

[0052] The addition of the composition of this invention maintained the high cell amplification capacity. On day 14, the NK cell amplification rate was 1694.8-fold in the M1 experimental group, 1611.24-fold in the M2 experimental group, 1598.07-fold in the M3 experimental group, 1724.06-fold in the M4 experimental group, and 1637.38-fold in the M5 experimental group. Compared with the blank control group (M3) without the addition of active ingredients and the classic interleukin-12 / 15 / 18 induction system group (M5), the addition of the composition did not reduce the amplification rate, but rather slightly increased it.

[0053] 3. Purity of NK cells; experimental results are shown in Table 13: Table 13

[0054] The addition of the composition of this invention maintained high NK cell purity. On day 14, the purity of experimental group M1 was 91.4%, experimental group M2 was 92.6%, experimental group M3 was 89.1%, experimental group M4 was 93.2%, and experimental group M5 was 87.7%. The addition of the composition maintained high NK cell purity at harvest.

[0055] 4. The killing efficiency of NK cells, the experimental results are shown in Table 14: Table 14

[0056] The addition of the composition of this invention maintains high NK cell killing activity. At an effector-to-target ratio of 5:1, the NK cell killing rate on day 14 was 70.5% in the M1 experimental group, 69.8% in the M2 experimental group, 63.2% in the M3 experimental group, 74% in the M4 experimental group, and 61.9% in the M5 experimental group. Compared with the blank control group (M3) without the addition of active ingredients and the classic interleukin-12 / 15 / 18 induction system group (M5), the composition of this invention (M4) can improve the NK cell killing efficiency by about 10%.

[0057] 5. The memory phenotype of NK cells, the experimental results are shown in Table 15: Table 15

[0058] Adding the composition of this invention significantly enhances the memory phenotype of harvested NK cells. On day 14, the positive rates of CD62L, CD127, and Eomes in NK cells of the M1 experimental group were 18.7%, 19.9%, and 17%, respectively; those of the M2 experimental group were 21.4%, 19.2%, and 19.2%; those of the M3 experimental group were 7.3%, 5.9%, and 9.1%; those of the M4 experimental group were 42.5%, 36.1%, and 51.4%; and those of the M5 experimental group were 8.1%, 6.2%, and 12.4%. These results were significantly improved compared to the blank control group (M3) without the active ingredient and the classic interleukin-12 / 15 / 18 groups. Compared to the induction system (M5), the composition (M4) of the present invention can increase the positive rates of CD62L, CD127, and Eomes by more than 34, 29, and 37 percentage points, respectively. Compared to the blank control group (M3) without the addition of active ingredients, the composition (M4) of the present invention can increase the positive rates of CD62L, CD127, and Eomes by 35.2, 30.2, and 42.3 percentage points, respectively; compared to the classic interleukin-12 / 15 / 18 induction system (M5), the composition (M4) of the present invention can increase the positive rates of CD62L, CD127, and Eomes by 34.4, 29.9, and 39.0 percentage points, respectively.

[0059] Example 4: NK cells were cultured and identified strictly according to the above experimental method. Starting from day 3 (D3), different additives were added to the MACS NK medium. The experimental groups were set up as follows: The first group is the low-concentration additive composition combination of the present invention: adding tanshinone I with a final concentration of 0.1 µM and berberine with a final concentration of 1 µM to MACSNK medium containing rhIL-2 with a final concentration of 500 IU / mL, rhIL-21 with a final concentration of 20 ng / mL, rhIL-15 with a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma. The second group is the medium-concentration additive composition combination of the present invention: adding tanshinone I with a final concentration of 5 µM and berberine with a final concentration of 50 µM to MACSNK medium containing rhIL-2 with a final concentration of 500 IU / mL, rhIL-21 with a final concentration of 20 ng / mL, rhIL-15 with a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma. The third group is a high-concentration additive composition combination of the present invention: adding tanshinone I with a final concentration of 10 µM and berberine with a final concentration of 100 µM to MACSNK medium containing rhIL-2 with a final concentration of 500 IU / mL, rhIL-21 with a final concentration of 20 ng / mL, rhIL-15 with a final concentration of 20 ng / mL, and 10% heat-inactivated autologous plasma. The fourth group was a control group without the added composition and heat-inactivated autologous plasma, containing only MACS NK medium; The fifth group was a control group that only underwent heat inactivation of autologous plasma, with 10% heat-inactivated autologous plasma added to MACS NK medium; The sixth group is a control of the classic interleukin-12 / 15 / 18 induction system.

[0060] 1. Cell viability of NK cells: The experimental results are shown in Table 16. Table 16

[0061] At day 14, the cell viability was 93.78% in group 1, 96.59% in group 2, 90.39% in group 3, 91.14% in group 4, 91.69% in group 5, and 93.9% in group 6. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 performed best in maintaining high cell viability.

[0062] 2. The fold increase of NK cells, the experimental results are shown in Table 17: Table 17

[0063] At day 14, the cell proliferation rate was 1647.16 in group 1, 1724.06 in group 2, 1418.55 in group 3, 1540.44 in group 4, 1598.07 in group 5, and 1637.38 in group 6. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 performed best in maintaining a high cell proliferation rate.

[0064] 3. Purity of NK cells; experimental results are shown in Table 18: Table 18

[0065] At day 14, the proportion of NK cells was 91.6% in group 1, 93.2% in group 2, 90.1% in group 3, 88.4% in group 4, 89.1% in group 5, and 87.7% in group 6. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 performed best in maintaining a high proportion of NK cells.

[0066] 4. The killing efficiency of NK cells, the experimental results are shown in Table 19: Table 19

[0067] At an effector-to-target ratio of 5:1, the NK cell killing rate on day 14 was 68.5% in group 1, 74% in group 2, 61.8% in group 3, 61.4% in group 4, 63.2% in group 5, and 61.9% in group 6. Therefore, compared with the low-concentration group, the high-concentration group, and various control groups, the medium-concentration combination in group 2 showed the most significant effect in improving NK cell killing efficiency.

[0068] 5. The memory phenotype of NK cells, the experimental results are shown in Table 20: Table 20

[0069] Adding the composition of this invention can significantly enhance the memory phenotype of harvested NK cells. On day 14, the positive rates of CD62L, CD127, and Eomes in the first group of NK cells were 18.8%, 16.5%, and 19.5%, respectively; in the second group, they were 42.5%, 36.1%, and 51.4%; in the third group, they were 19.5%, 14.8%, and 15.4%; in the fourth group, they were 5.2%, 6.1%, and 7.9%; in the fifth group, they were 7.3%, 5.9%, and 9.1%; and in the sixth group, they were 8.1%, 6.2%, and 12.4%. Therefore, compared with the low concentration group, high concentration group and various control groups, the medium concentration combination in the second group was the most effective in improving the memory phenotype. Compared with the fifth group, it increased the positive rates of CD62L, CD127 and Eomes by 35.2, 30.2 and 42.3 percentage points, respectively; compared with the classic induction system (the sixth group), it increased the positive rates of CD62L, CD127 and Eomes by 34.4, 29.9 and 39.0 percentage points, respectively.

[0070] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Based on the above background, the present invention provides an NK cell induction and expansion culture additive, composed of tanshinone I at a final concentration of 0.1~10 µM and berberine at a final concentration of 10~100 µM.

Claims

1. A culture medium for inducing and expanding NK cells, characterized in that, It consists of basal culture medium, IL-2 with a final concentration of 500 IU / mL, IL-21 with a final concentration of 20 ng / mL, IL-15 with a final concentration of 20 ng / mL, 10% heat-inactivated autologous plasma, tanshinone I with a final concentration of 5 µM, and berberine with a final concentration of 50 µM. The basal culture medium is either SCGM NK medium or MACS NK medium.

2. The application of the NK cell induction and expansion culture medium as described in claim 1, characterized in that, Used for the in vitro induction, expansion, and enhancement of NK cell memory phenotypes and functions.

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