A composition for enhancing NK cells and a method for culturing the same in vitro

Abstract

The application discloses a kind of for NK cell synergistic composition and its in-vitro culture method, it is related to cell culture technical field.The composition for the activation of NK cell includes buffer, and only the following final concentration of cytokine combination is contained in buffer: 100-500U / ml IL-2, 20-50ng / ml IL-7, 5-20ng / ml IL-12, 5-20ng / ml IL-15, 10-50ng / ml IL-18 and 10-30ng / ml IL-21.The composition for the activation of NK cell provided by the application is added to culture medium, the expansion fold and purity of peripheral blood autologous NK cell can be improved, and the killing activity of NK cell is enhanced.It is favorable to improve the curative effect of cell immunotherapy to patient.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically, to a composition for enhancing NK cells and a method for in vitro culture thereof. Background Technology

[0002] Natural killer cells (NK cells) are an important component of the immune system and play a key role in tumor immune surveillance and antiviral infection. Due to their ability to directly kill tumor cells without prior sensitization, they have become highly promising immunotherapy cells.

[0003] Many current cell immunotherapies involve extracting NK cells from peripheral blood mononuclear cells (PBMCs), activating and expanding them in vitro before reinfusion into patients. There are two main activation methods for NK cells: factor-based and feeder cell-based. Feeder cell-based methods typically use genetically modified K562 cells, which can improve the purity and expansion rate of NK cell culture. However, after infusion, the cells are removed from the optimal culture environment provided by feeder cells, leading to a shortened in vivo survival time and reduced cytotoxicity. More importantly, feeder cells are often selected from tumor cell lines (such as K562), whose potential tumorigenicity may pose safety risks, requiring rigorous safety assessment, especially in clinical applications. Using tumor cells as a feeder layer may raise ethical concerns, and patients and their families have low psychological acceptance of co-culturing tumor cells. Therefore, a pure factor-based NK cell culture method has been developed, where cytokines are used in conjunction to activate and expand feeder cells, eliminating the need for feeder cells.

[0004] Currently, the pure factor NK cell culture method still has problems such as low NK cell purity, low proliferation rate, and low NK cell count. This is because PBMCs contain 5-10% NK cells, but mainly T cells and B cells. These factors stimulate the proliferation of NK cells, and also stimulate the proliferation of T cells and even B cells.

[0005] In addition, existing NK cell expression or amplification kits suffer from poor uniformity and stability of cell samples obtained under the same culture conditions, poor stability of total cell number and NK cell number, and poor NK cell killing ability.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a composition for enhancing NK cells and a method for in vitro culture thereof to solve the above-mentioned technical problems.

[0008] This invention is implemented as follows:

[0009] In a first aspect, the present invention provides a composition for NK cell activation, comprising a buffer solution containing only a combination of cytokines at a final concentration of:

[0010] IL-2 at 100-500 U / ml, IL-7 at 20-50 ng / ml, IL-12 at 5-20 ng / ml, IL-15 at 5-20 ng / ml, IL-18 at 10-50 ng / ml, and IL-21 at 10-30 ng / ml.

[0011] In a second aspect, the present invention provides a culture medium comprising a basal culture medium and a composition added to the basal culture medium, the basal culture medium containing a combination of cytokines at final concentrations of: 100-500 U / ml IL-2, 20-50 ng / ml IL-7, 5-20 ng / ml IL-12, 5-20 ng / ml IL-15, 10-50 ng / ml IL-18 and 10-30 ng / ml IL-21.

[0012] Thirdly, the present invention provides a kit comprising the above-described composition or the above-described culture medium.

[0013] Fourthly, the present invention provides a method for expanding NK cells, which includes the following steps: adding the above-mentioned culture medium and PBMC cells to a culture container and culturing them.

[0014] The present invention has the following beneficial effects:

[0015] This invention provides a composition for NK cell activation, comprising specific concentrations of IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. The combined use of IL-7 and IL-12 increases NK cell expression levels and reduces T cell activation. Adding the NK cell activation composition provided by this invention to a culture medium can increase the expansion fold and purity of peripheral blood autologous NK cells, thereby enhancing NK cell cytotoxic activity.

[0016] Compared to feeder cell culture systems and existing commercially available pure factor NK cell culture systems, the NK cell culture medium and method provided in this invention produce safer NK cells with higher purity, greater yield, better activity, stronger cytotoxic activity, and better uniformity and stability of the expanded NK cell samples, as well as higher stability in total cell count and NK cell count. High-purity, high-activity, and high-cytotoxic NK cells are beneficial for improving the efficacy of cell immunotherapy for patients. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 To culture cell samples using the composition provided in Example 1 of this invention, the cell samples were cultured using the feeder cell method, and the statistical results of NK cell purity of the cell samples cultured using the factor method commercial kit are shown in the figure.

[0019] Figure 2 Flow cytometry results of three NK cell cultures obtained using the NK cell expansion method provided in Example 4;

[0020] Figure 3 The flow cytometry results are shown for cell samples 4, 5, and 6 cultured using the feeder cell method in Comparative Example 5.

[0021] Figure 4 The flow cytometry results for cell samples 7, 8, and 9, cultured using the commercial kit of the factor method, are shown in Comparative Example 6.

[0022] Figure 5 The figure shows the statistical results of the number of NK cells, NK cell activity, and NK cell killing activity of NK cell samples cultured according to the NK cell expansion methods provided in Example 4 and Comparative Examples 5-6.

[0023] Figure 6 The figure shows the detection results of NK cell amplification purity and NK cell amplification number in Experiment Example 3.

[0024] Figure 7 The figure shows the detection results of CD107a positive expression rate and NK cell activity in NK cells in Experiment Example 4.

[0025] Figure 8 The figure shows the statistical results of IFN-γ expression in NK cells in each treatment group in Experiment Example 5.

[0026] Figure 9 Flow cytometry images of IFN-γ factor in NK cells and K652 target cells obtained from each treatment group after incubation.

[0027] Figure 10 Flow cytometry images of IFN-γ factor in NK cells cultured from each treatment group after incubation with MM1.S and HepG2 target cells;

[0028] Figure 11 The graph shows the results of the three-luciferase activity assay in Experiment Example 6.

[0029] Figure 12 The results of the three-luciferase activity assay after NK cells targeted and killed HepG2-GFP-Luc cells in Experiment Example 6;

[0030] Figure 13 A graph showing the statistical results of NK cell expression levels;

[0031] Figure 14 Flow cytometry results of amplifying the same target cell samples using the same NK cell reagent kit;

[0032] Figure 15 The graph shows the percentage of NK cells, the number of NK cells, and the total number of cells amplified after amplifying the same target cell sample using the same NK cell reagent kit.

[0033] Figure 16 The flow cytometry results are shown below after amplification of PBMC samples from the above six different donors using the method provided in Example 4.

[0034] Figure 17 This is a graph showing the statistical results of NK cell expression levels on day 15 of the above 6 samples cultured using the method provided in Example 4.

[0035] Figure 18 The figure shows the statistical results of the number of NK cells and the total number of cells amplified after using the method provided in Example 4 to expand PBMCs from the above six different donor sources. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0037] In a first aspect, the present invention provides a composition for NK cell activation, comprising a buffer solution containing only a combination of cytokines at a final concentration of:

[0038] IL-2 at 100-500 U / ml, IL-7 at 20-50 ng / ml, IL-12 at 5-20 ng / ml, IL-15 at 5-20 ng / ml, IL-18 at 10-50 ng / ml, and IL-21 at 10-30 ng / ml.

[0039] Through screening, the inventors discovered that a combination of cytokines containing IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21 in the culture medium exhibited higher NK cell activation, higher NK cell purity, and a higher number of NK cells expanded compared to compositions without IL-7 or IL-12 alone. IL-7 and IL-12 also showed a synergistic effect in promoting NK cell proliferation.

[0040] Cytokine combinations include, for example, IL-2 at levels of 100-200 U / ml, 150-250 U / ml, 200-300 U / ml, 300-400 U / ml, and 400-500 U / ml; IL-7 at levels of 20-30 ng / ml, 30-40 ng / ml, and 40-50 ng / ml; IL-12 at levels of 5-10 ng / ml, 10-15 ng / ml, and 15-20 ng / ml; IL-15 at levels of 5-10 ng / ml, 10-15 ng / ml, and 15-20 ng / ml; IL-18 at levels of 10-20 ng / ml, 20-30 ng / ml, 30-40 ng / ml, and 40-50 ng / ml; and IL-21 at levels of 10-20 ng / ml and 20-30 ng / ml.

[0041] The buffer solution is selected from PBS or DPBS buffer.

[0042] In a second aspect, the present invention provides a culture medium comprising a basal culture medium and a composition added to the basal culture medium, the basal culture medium containing a combination of cytokines at final concentrations of: 100-500 U / ml IL-2, 20-50 ng / ml IL-7, 5-20 ng / ml IL-12, 5-20 ng / ml IL-15, 10-50 ng / ml IL-18 and 10-30 ng / ml IL-21.

[0043] Compared to feeder cell culture systems and existing commercially available pure factor NK cell culture systems, the NK cell culture medium and method provided in this invention produce safer NK cells with higher purity, greater yield, better activity, stronger cytotoxic activity, and better uniformity and stability of the expanded NK cell samples, as well as higher stability in total cell count and NK cell count. High-purity, high-activity, and high-cytotoxic NK cells are beneficial for improving the efficacy of cell immunotherapy for patients.

[0044] In a preferred embodiment of the present invention, the basal culture medium is selected from any one of DMEM, RPMI 1640, Corning 581 and X-VIVO.

[0045] In a preferred embodiment of the present invention, the basal culture medium is further supplemented with the following additives: insulin-transferrin-selenium-sodium pyruvate additive and autologous plasma. The volume percentage of insulin-transferrin-selenium-sodium pyruvate additive in the culture medium is 0%-1%, and the volume percentage of autologous plasma in the culture medium is 1-10%.

[0046] The volume percentage of autologous plasma in the culture medium is, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0047] Thirdly, the present invention provides a kit comprising the above-described composition or the above-described culture medium.

[0048] In a preferred embodiment of the present invention, the kit further includes: an antibody combination and a proliferation culture medium, wherein the antibody combination contains only antibodies in the following mass ratio: CD3 antibody, CD16 antibody and NKG2D antibody in a ratio of 0.01-1:2-8:2-8.

[0049] In one embodiment, the antibody combination contains only CD3 antibody, CD16 antibody and NKG2D antibody in the following mass ratios: 0.01-0.8:2-8:2-8, 0.05-0.5:2-8:2-8, 0.05-0.1:2-8:2-8, 0.1-0.5:2-6:2-6.

[0050] In one embodiment, the antibody combination is present in a buffer solution, for example, the buffer solution contains only the following final concentrations of antibody combination: 0.05-0.5 μg / mL of CD3 antibody, 3-8 μg / mL of CD16 antibody and 3-8 μg / mL of NKG2D antibody.

[0051] The invention reveals that coating cell culture containers with the antibody combination described above significantly improves the purity of the obtained NK cells; and compared to not adding CD3 antibodies, it significantly improves both the purity and quantity of the harvested NK cells. Increasing the amount of CD3 antibodies leads to T cells dominating the cell activation population, resulting in the majority of expanded cells being T cells, thus causing activation failure.

[0052] Furthermore, compared to compositions with added 2B4 antibody, compositions with added RetroNectin, or compositions with reduced CD16 antibody levels, the compositions provided by this invention, after coating cell culture containers, result in higher purity and greater number of NK cells obtained through culture.

[0053] Coating cell culture containers with the composition provided in this invention can significantly improve the purity (over 85%) and number of harvested NK cells, as well as enhance their activity, achieving a viability rate of over 90%. Therefore, this invention solves the problems of low purity, low fold increase, and low activity in NK cell amplification.

[0054] The proliferation medium includes a basal medium and additives added to the basal medium at the following final concentrations:

[0055] IL-2 at 500-1000 U / ml, IL-15 at 5-20 ng / ml, and IL-18 at 10-50 ng / ml.

[0056] The inventors discovered that introducing a proliferation medium (containing IL-2, IL-15, and IL-18) during proliferation culture resulted in NK cells cultured in this medium, with over 60% expressing CD107a. This indicates that the NK cells possess extremely high detoxification granule strength and thus exhibit extremely high cytotoxicity. Therefore, using the aforementioned proliferation medium can enhance the cytotoxic effect of the expanded NK cells.

[0057] Compared to proliferation media without the addition of IL-15 or IL-18, the proliferation media provided by this invention can further enhance the activity of NK cells.

[0058] In one embodiment, the proliferation medium includes a basal medium and an additive added to the basal medium at a final concentration as follows:

[0059] IL-2 at concentrations of 500-600 U / ml, 550-660 U / ml, 600-700 U / ml, 700-800 U / ml or 800-1000 U / ml; IL-15 at concentrations of 5-10 ng / ml, 10-15 ng / ml or 15-20 ng / ml; and IL-18 at concentrations of 10-20 ng / ml, 15-30 ng / ml, 20-40 ng / ml or 30-50 ng / ml.

[0060] Fourthly, the present invention provides a method for expanding NK cells, which includes the following steps: adding the above-mentioned culture medium and PBMC cells to a culture container and culturing them.

[0061] In a preferred embodiment of the present invention, the activation method further includes pre-coating the culture vessel, which includes: coating the culture vessel with an antibody combination containing only the following mass ratio of CD3 antibody, CD16 antibody and NKG2D antibody: 0.01-1:2-8:2-8; after coating, washing is performed, and then the above-mentioned culture medium and PBMC cells are added to the culture vessel.

[0062] The purpose of pre-coating is not only to significantly improve the purity (over 85%) and number of harvested NK cells, but also to enhance the activity of the obtained NK cells, with a viability rate of over 90%.

[0063] In a preferred embodiment of the present invention, the cultivation includes the following steps:

[0064] On day 3, supplement with culture medium containing 5-10% autologous plasma; on day 5, supplement with culture medium containing 3-5% autologous plasma; on day 7, supplement with culture medium containing 0-2% autologous plasma.

[0065] In a preferred embodiment of the present invention, cells are cultured until day 9, and then supplemented with proliferation medium every other day, and harvested on days 14-16; the proliferation medium includes basal medium and additives added to the basal medium at the following final concentrations:

[0066] IL-2 at 500-1000 U / ml, IL-15 at 5-20 ng / ml, and IL-18 at 10-50 ng / ml.

[0067] The basal culture medium was selected from any one of DMEM, RPMI 1640, Corning 581 and X-VIVO.

[0068] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0069] Example 1

[0070] This embodiment provides a composition for NK cell activation, comprising a combination of cytokines in DPBS (Guangdong Beso, CS001) at the following final concentrations: 100 U / ml IL-2, 20 ng / ml IL-7, 5 ng / ml IL-12, 5 ng / ml IL-15, 10 ng / ml IL-18, and 10 ng / ml IL-21.

[0071] Example 2

[0072] This embodiment provides an NK cell activation culture medium, comprising: basal culture medium, the cytokine composition of Example 1 added to basal RPMI 1640 culture medium, an insulin-transferrin-selenium-sodium pyruvate additive, and autologous plasma. The basal culture medium contains the following final concentrations of the cytokine combination: 500 U / ml IL-2, 20 ng / ml IL-7, 5 ng / ml IL-12, 10 ng / ml IL-15, 20 ng / ml IL-18, and 10 ng / ml IL-21.

[0073] The volume percentage of insulin-transferrin-selenium-sodium pyruvate additive (ITS-A) in the NK cell activation medium was 1%, the volume percentage of autologous plasma in the NK cell activation medium was 10%, and the volume percentage of basal medium in the NK cell activation medium was 89%.

[0074] Example 3

[0075] This embodiment provides an NK cell proliferation culture medium, which includes: basal RPMI 1640 medium, and IL-2 at a final concentration of 500 U / ml, IL-15 at a final concentration of 10 ng / ml, and IL-18 at a final concentration of 20 ng / ml added to the basal RPMI 1640 medium.

[0076] Example 4

[0077] This embodiment provides a method for in vitro expansion of high-purity peripheral blood autologous NK cells, comprising the following steps:

[0078] 1. NK cell activation antibody coating.

[0079] Add the antibody combination (10 mL) to a TC 75 culture flask, spread it evenly on the bottom surface, and let it stand at room temperature for more than 1 hour before use.

[0080] The antibody combination consisted of DPBS (Guangdong Beso, CS001) supplemented with 0.5 ug / ml CD3 antibody, 8 ug / ml CD16 antibody, and 8 ug / ml NKG2D antibody. The CD3 antibody was purchased from Tongli Haiyuan (catalog number GMP-TL101-0500); the CD16 antibody was purchased from Tongli Haiyuan (catalog number GMP-TL201-0500); and the NKG2D antibody was purchased from ACRO (NKD-H5245).

[0081] 2. Mononuclear cells were isolated from peripheral blood using density gradient centrifugation.

[0082] (1) Transfer peripheral blood from healthy donors into centrifuge tubes, centrifuge at 800g for 10 minutes, collect the upper layer of autologous plasma, inactivate it and set it aside for later use;

[0083] Plasma inactivation: Plasma is placed in a preheated water bath at 56°C for 30 minutes and then placed in a refrigerator at 4°C. After the plasma has cooled, it is centrifuged at 1200g for 10 minutes. The supernatant is then transferred to a new centrifuge tube to obtain inactivated autologous plasma.

[0084] (2) Dilute the lower layer of blood cells to the original volume with DPBS, mix well, and slowly add peripheral blood to the Ficoll layer at a ratio of peripheral blood: Ficoll = 2:1. Centrifuge at 700g for 20min. Ficoll was purchased from Dayou, catalog number 7912011.

[0085] (3) After centrifugation, the white membrane layer was aspirated, resuspended in DPBS buffer, and centrifuged at 800g for 10min at room temperature. This process was repeated twice to obtain peripheral blood mononuclear cells (PBMCs).

[0086] 3. NK cell activation

[0087] (1) Aspirate the liquid from the culture flask coated with antibody in step 1 above and wash it with 10ml DPBS.

[0088] (2) Resuspend the cells in the NK cell activation culture medium of Example 2 above, take samples for counting, and then add 4×10 7 Mononuclear cells were added to the T75 culture flask pre-coated in step (1), and NK cell activation medium from Example 2 (containing 17.8 ml of basic RPMI 1640 medium (with cytokine combination), 2 mL of plasma and 0.2 mL of ITS-A) was added to make a final volume of 20 ml. The flask was then placed in an incubator for culture.

[0089] (3) On day 3 of culture, supplement the culture medium with 10% (v / v) autologous plasma and 90% (v / v) NK cell activation medium;

[0090] (4) On day 5 of culture, supplement with culture medium containing 5% (v / v) autologous plasma and 95% (v / v) NK cell activation medium;

[0091] (5) On day 7, supplement the culture medium with 1% (v / v) autologous plasma and 99% (v / v) NK cell activation medium.

[0092] 4. NK cell proliferation

[0093] 1) After culturing to day 9, supplement with the NK cell proliferation medium from Example 3 every other day;

[0094] 2) Harvest cells on days 14-16.

[0095] Comparative Example 1

[0096] Compared with Example 2, the NK cell activation culture medium provided in this comparative example does not contain IL-12, while the contents of other components are the same as in Example 2.

[0097] Comparative Example 2

[0098] Compared with Example 2, the NK cell activation culture medium provided in this comparative example does not contain IL-7, while the contents of other components are the same as in Example 2.

[0099] Comparative Example 3

[0100] Compared with Example 3, the NK cell proliferation culture medium provided in this comparative example does not contain IL-15, while the contents of other components are the same as in Example 3.

[0101] Comparative Example 4

[0102] Compared with Example 3, the NK cell proliferation culture medium provided in this comparative example does not contain IL-18, while the contents of other components are the same as in Example 3.

[0103] Comparative Example 5

[0104] NK cells were cultured using feeder cells.

[0105] Resuscitate feeder cells and add feeder cells and PBMCs at a 1:1 volume ratio. The number of PBMCs is the same as the number added in the cytokine method in Example 4, which is 4 × 10⁻⁶. 7 In Example 4, step 1, the antibody package is replaced with the addition of feeder cells; the rest of the operation remains the same.

[0106] Comparative Example 6

[0107] NK cells were cultured using a commercial NK cell culture kit, specifically the NK induction kit from Guangdong Beso Pharmaceutical Co., Ltd., catalog number: T20202N2.

[0108] Comparative Example 7

[0109] Compared with Example 2, the NK cell activation culture medium provided in this comparative example does not contain IL-7 and IL-12, while the contents of other components are the same as in Example 2.

[0110] Comparative Example 8

[0111] Compared with Example 3, the NK cell proliferation culture medium provided in this comparative example does not contain IL-15 and IL-18, while the contents of other components are the same as in Example 3.

[0112] Experimental Example 1

[0113] This experiment was conducted to detect the purity of NK cells. The reagents used were as follows: APC-labeled anti-CD3 and PE-Cy7-labeled anti-CD56 were purchased from Biolegend, with the catalog number of CD3 being 981012 and the catalog number of CD56 being 985912.

[0114] Phenotypic analysis of NK cells was performed using flow cytometry. APC-labeled anti-CD3 and PE-Cy7-labeled anti-CD56 antibodies were incubated with NK cells (cell samples 1, 2, and 3 amplified using the NK cell amplification method provided in Example 4; cell samples 4, 5, and 6 cultured using the feeder cell method in Comparative Example 5; and cell samples 7, 8, and 9 cultured using the commercial factor method kit in Comparative Example 6) at room temperature in the dark for 10 minutes before analysis by flow cytometry. Experiments 1-3 were replicates of each other; experiments 4-6 were replicates of each other; and experiments 7-9 were replicates of each other.

[0115] Figure 1 In the above, samples 1, 2, and 3 are samples cultured using the composition provided in Example 1 of the present invention; samples 4, 5, and 6 are samples cultured using the feeder cell method; and samples 7, 8, and 9 are samples cultured using a commercial kit for factor culture.

[0116] Figure 1 The results showed that the average purity (CD3-CD56+ index) of the three NK cells cultured using the NK cell expansion method provided in Example 4 of the present invention reached 93.6%; the average purity of the three NK cells cultured using feeder cells in Comparative Example 5 was 87.7%; while the average purity of the three NK cells cultured using a commercial NK cell culture kit in Comparative Example 6 was only 45.9%.

[0117] Figure 2 These are flow cytometry results of three NK cell cultures cultured using the NK cell expansion method provided in Example 4. Figure 3 The images show the flow cytometry results of cell samples 4, 5, and 6 cultured using the feeder cell method in Comparative Example 5. Figure 4 The flow cytometry results for cell samples 7, 8, and 9, cultured using the factor method commercial kit, are shown in Comparative Example 6.

[0118] The results show that the NK cells amplified using the NK cell amplification method provided in Example 4 of this invention have extremely high NK cell purity.

[0119] Experimental Example 2

[0120] This experiment was conducted to detect the number of NK cells harvested, cell viability, and cytotoxic activity.

[0121] (1) The method for detecting NK cell count is as follows:

[0122] After NK cells were cultured to day 15 using the NK cell expansion methods provided in Example 4 and Comparative Examples 5-6, samples of the cultured cells were taken, and the cells were counted using a cell counter. The final harvested cell quantity was then calculated based on the culture volume.

[0123] (2) NK cell activity detection:

[0124] After culturing to day 15, cells in culture were sampled and trypan blue staining was used to detect NK cell activity.

[0125] (3) NK cell killing activity assay:

[0126] The expression level of CD107a was detected by flow cytometry to assess the killing effect of NK cells on target K562 tumor cells. The ratio of NK cells to target cells was 1:1.

[0127] The specific testing steps are as follows:

[0128] Target cells K562 (cell density 2×10⁻⁶) 6 / ml) and NK cells (cell density 2×10⁹ / ml) 6 Add 100 μL each of the two (b / ml) to a 96-well U-plate, for a total volume of 200 μL, with an effector-target ratio of 1:1. Then add 1 μL of CD107a (biolegend:328620) antibody to each well and incubate at 37°C for 4 hours. (After 1 hour of incubation, add 1 μL of GolgiStop (BD:554724) to 50 μL of complete culture medium, mix well, and then add 5 μL to each well and mix thoroughly.) After 4 hours, stain with CD3 (biolegend:981012) and CD56 (biolegend:985912) antibodies, incubate at room temperature in the dark for 10 minutes, and then perform detection.

[0129] Figure 5 Samples 1-3 are NK cell samples cultured according to the NK cell amplification method provided in Example 4; Samples 4-6 are NK cell samples cultured according to the NK cell amplification method provided in Comparative Example 5; Samples 7-9 are NK cell samples cultured according to the NK cell amplification method provided in Comparative Example 6.

[0130] Figure 5 The results show that the NK cell expansion method provided in Example 4 of this invention can obtain an extremely high total number of cells.

[0131] The NK cells cultured using the NK cell expansion method provided in Example 4 of this invention have higher activity than the commercial factor culture system of Comparative Example 6, and are far superior to the feeder cell culture system provided in Comparative Example 5.

[0132] The NK cells cultured using the NK cell expansion method provided in Example 4 of this invention exhibit significantly stronger target cell killing activity than the NK cells expanded using the NK cell expansion method provided in Comparative Examples 5-6.

[0133] In summary, compared with feeder cell culture systems and existing commercially available pure factor NK cell culture systems, the NK cell activation medium and culture method provided by this invention produce safer NK cells with higher cell purity, greater yield, better activity, and stronger killing activity, thus having better application value.

[0134] Experimental Example 3

[0135] In this experiment, the purity and number of NK cells amplified were detected using the NK cell activation culture media provided in Example 2, Comparative Examples 1-2, and Comparative Example 7, following the NK cell amplification method provided in Example 4. The methods for detecting the purity and number of NK cells amplified were the same as in Examples 1 and 2.

[0136] The results of NK cell expansion purity and NK cell expansion number detection were referenced. Figure 6 As shown, Figure 6 The "none" in the figure refers to the absence of IL7 and IL-12, i.e., Comparative Example 7. The results show that the combined use of IL-7 and IL-12 resulted in higher NK cell purity and greater NK cell proliferation compared to the use of IL-7 alone. The addition of IL-7 alone enhanced T cell activation, leading to a decrease in NK expression.

[0137] Experiment Example 4

[0138] In this experiment, the purity and number of NK cells amplified were determined using the NK cell proliferation culture media provided in Example 3, Comparative Examples 3-4, and Comparative Example 8, following the NK cell amplification method provided in Example 4. The methods for detecting NK cell cytotoxic activity and NK cell activity were the same as in Example 2.

[0139] The results of NK cell CD107a positive expression rate and NK cell activity were obtained by referring to... Figure 7 As shown, Figure 7 The “none” in the text refers to the absence of IL-15 and IL-18, i.e., Comparative Example 8.

[0140] The results showed that the combined use of IL-15 and IL-18 had higher NK cell killing activity than the use of IL-18 alone, and the NK cell activity remained at a higher level for 25 consecutive days.

[0141] Experimental Example 5

[0142] In this experimental example, the NK cell proliferation culture medium provided in Example 3, Comparative Examples 3-4, and Comparative Example 8 was used to detect IFN-γ after NK cell amplification according to the NK cell amplification method provided in Example 4.

[0143] The reagents involved are as follows: IFN-γ antibody was purchased from Biolegend, APC anti-human IFN-γ Antibody, catalog number: 502512.

[0144] The ability of D15 NK cells to release cytotoxic factors to target cells was detected by flow cytometry. Target cells included K652, MM1.S, and HepG2, with a NK cell to target cell ratio of 1:1. Proliferation medium was used as a blank control. The inhibitor BFA was added to block the release of IFN-γ factor into the extracellular space, which facilitated subsequent detection.

[0145] Test results refer to Figure 8 As shown, the flow cytometry plots of IFN-γ factor in NK cells and K652 target cells obtained from each treatment group after incubation are referenced. Figure 9 As shown, Figure 9 In the text, "Med" refers to the blank control (culture medium with the same volume as tumor cells). Flow cytometry images of IFN-γ factor in NK cells obtained from each treatment group after incubation with MM1.S and HepG2 target cells are shown below. Figure 10 As shown.

[0146] Figures 8-10 The results showed that the NK cells obtained by proliferation culture medium provided in Example 3 had higher IFN-γ levels than the NK cells obtained by proliferation culture medium provided in Comparative Examples 3-4 and 8.

[0147] Experimental Example 6

[0148] In this experimental example, the NK cell proliferation culture medium provided in Example 3, Comparative Examples 3-4, and Comparative Example 8 was used to perform a 3-Luciferase Activity Assay on the NK cell amplification after NK cell expansion according to the NK cell amplification method provided in Example 4.

[0149] NK cells were mixed with K562-GFP-Luc, MM1.S-GFP-Luc, and HepG2-GFP-Luc cells at effector-target ratios of 9:1, 3:1, 1:1, and 1:3, respectively. The corresponding number of NK cells and target cells were used. 5 Add the sample to a 96-well U-shaped plate and mix thoroughly to a final volume of 200 μL. Set up 2 × 10⁻⁶ samples respectively. 5 One target cell / well was used as a control. The cells were incubated in a 5% CO2 incubator at 37°C for 4 hours, and the NK cell killing activity was calculated by detecting the fluorescence intensity using a microplate reader.

[0150] Figure 11 and Figure 12The results of the three-luciferase activity assay showed that the NK cells obtained by proliferation medium provided in Example 3 had higher killing activity than the NK cells obtained by proliferation medium provided in Comparative Examples 3-4 and Comparative Example 8.

[0151] Experimental Example 7

[0152] This experiment tested the stability of NK cell expansion.

[0153] D15 NK expression and cell count were measured by flow cytometry using the NK cell reagent kit from Comparative Example 6. The assay method was the same as in Experiments 1-2.

[0154] The same NK cell reagent kit was used to amplify PBMC cell samples (6 PBMCs from different donors, referred to as sample 1, sample 2, sample 3, sample 4, sample 5 and sample 6) to test NK cell expression levels, NK cell percentage, NK cell number and total number of amplified cells.

[0155] Figure 13 The graph shows the statistical results of NK cell expression levels on day 15 of culture. Figure 14 Flow cytometry results of amplification of PBMCs from six different donors using the same NK cell reagent kit. Figure 15 The percentage of NK cells, the number of NK cells, and the total number of cells amplified after using the same NK cell reagent kit to amplify PBMCs from the above six different donor sources were recorded.

[0156] Figures 13-15 The results showed that there were significant differences in NK cell expression among the six samples. Samples 1, 4, and 5 had low NK cell expression, while sample 4 had a higher proportion of NK cells and sample 5 had a higher proportion of T cells. The data varied among the samples, indicating poor cell homogeneity and stability.

[0157] Figures 13-15 The results show that there are significant differences in the total number of cells and the number of NK cells among the six samples corresponding to NK expression, indicating that the stability of the kit is poor for different samples.

[0158] In contrast. Figure 16 The image shows the flow cytometry results after amplification of PBMC samples from the six different donors using the method provided in Example 4. Figure 17 The graph shows the statistical results of NK cell expression levels on day D15 of the above 6 samples cultured using the method provided in Example 4. Figure 18 The figure shows the statistical results of the number of NK cells and the total number of cells amplified after using the method provided in Example 4 to expand PBMCs from the above six different donor sources.

[0159] The results showed that the method provided in Example 4 had a more stable NK cell amplification effect on the same PBMC sample, and the number of amplified NK cells and the total number of amplified cells were higher.

[0160] In summary, the present invention has the following advantages:

[0161] 1) No trophoblast cells are added, eliminating the risk of tumorigenesis and making cell application safer;

[0162] 2) NK cells have high purity, reaching over 85%, with minimal sample variability;

[0163] 3) A greater number of NK cells are obtained; after 15 days of culture, the total number of cells can exceed 10 billion.

[0164] 4) The obtained NK cells have higher activity, with a survival rate of over 90%;

[0165] 5) The obtained NK cells have stronger killing ability.

[0166] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A reagent kit, characterized in that, It includes a culture medium, an antibody combination, and a proliferation medium. The culture medium includes a basal medium and a composition for NK cell activation. The basal medium contains the following final concentrations of the cytokine composition for NK cell activation: 100-500 U / ml IL-2, 20-50 ng / ml IL-7, 5-20 ng / ml IL-12, 5-20 ng / ml IL-15, 10-50 ng / ml IL-18, and 10-30 ng / ml IL-21. The basal medium also contains the following additives: insulin-transferrin-selenium-sodium pyruvate additive and autologous plasma. The volume percentage of the insulin-transferrin-selenium-sodium pyruvate additive in the culture medium is 0%-1%, and the volume percentage of the autologous plasma in the culture medium is 1-10%. The antibody combination contains only antibodies in the following mass ratio: CD3 antibody, CD16 antibody and NKG2D antibody in a ratio of 0.01-1:2-8:2-8. The proliferation medium includes a basal medium and additives added to the basal medium at the following final concentrations: 500-1000 U / ml of IL-2, 5-20 ng / ml of IL-15 and 10-50 ng / ml of IL-18. The basal culture medium is selected from any one of DMEM, RPMI 1640, Corning 581 and X-VIVO.

2. A method for expanding NK cells, characterized in that, It includes the following steps: Add the culture medium and PBMC cells as described in claim 1 to the culture vessel and culture.

3. The method for expanding NK cells according to claim 2, characterized in that, The amplification method further includes pre-coating the culture vessel, which includes: coating the culture vessel with an antibody combination containing only antibodies in the following mass ratio: 0.01-1:2-8:2-8 CD3 antibody, CD16 antibody and NKG2D antibody; after coating, washing is performed, and then the culture medium and PBMC cells as described in claim 1 are added to the culture vessel.

4. The method for amplifying NK cells according to claim 2, characterized in that, The cultivation process includes the following steps: On day 3, supplement the culture medium with 5-10% autologous plasma; on day 5, supplement the culture medium with 3-5% autologous plasma; on day 7, supplement the culture medium with 0-2% autologous plasma.

5. The method for amplifying NK cells according to claim 2, characterized in that, Cells were cultured until day 9, and then supplemented with proliferation medium every other day. Cells were harvested on days 14-16. The proliferation medium consisted of basal medium and additives added to the basal medium at the following final concentrations: IL-2 at 500-1000 U / ml, IL-15 at 5-20 ng / ml, and IL-18 at 10-50 ng / ml; The basal culture medium is selected from any one of DMEM, RPMI 1640, Corning 581 and X-VIVO.

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