Natural killer cell in-vitro amplification composition, amplification method and application

Abstract

The invention relates to an amplification method of natural killer cells, and belongs to the technical field of cell amplification. The amplification method comprises the following steps: S1, pre-coating a culture container with a monoclonal antibody combination, wherein the monoclonal antibody combination comprises CD56 or CD16, NKG2D and CD52; s2, inoculating umbilical cord blood or peripheral blood mononuclear cells into the pre-coated culture container, and culturing by adopting an activation culture medium containing cytokines to obtain activated cells; s3, transferring the activated cells into a serum-free amplification culture medium containing IL-2 and albumin for amplification culture; and S4, collecting the cells, regulating the cells to the density of 5 * 10 <-15 > * 10 / mL by using a solution containing albumin, standing for 0.5-1.5 hours, and filtering to obtain the natural killer cells. According to the amplification method, the in-vitro amplification multiple of the NK cells and the tumor killing efficiency can be improved.

Description

Technical Field

[0001] The present application relates to the field of cell expansion technology, and in particular to a natural killer cell in vitro expansion composition, expansion method and application. Background Art

[0002] Natural killer (NK) cells, as core effector cells of the innate immune system, have demonstrated significant value in the field of tumor immunotherapy. Their unique non-MHC-restricted killing mechanism and immunomodulatory function have demonstrated significant efficacy in clinical trials of solid tumors such as gastric cancer and liver cancer, with many patients with advanced metastatic cancer achieving long-term survival through NK cell therapy. However, NK cells only account for 5-15% of peripheral blood lymphocytes, and bottlenecks in their large-scale in vitro expansion technology have severely restricted their clinical application.

[0003] Most of the current mainstream expansion technologies are based on the expansion system of K562 trophoblast cells. Although high-fold expansion can be achieved, this tumor cell line still has safety risks such as secretion of exosomes to promote cancer and residual live cell contamination even after irradiation. The cell therapy guidelines issued by the National Institutes of Health (NIH) in 2019 clearly pointed out that the use of tumor-derived trophoblast cells may induce the risk of transplant tumorigenesis. The trophoblast-free expansion scheme based on the combination of antibodies and cytokines, although it avoids the risk of carcinogenesis, has process defects such as low amplification efficiency (200-500 times) and large batch-to-batch differences, which makes it difficult to meet the requirements of clinical-grade cell preparations for cell number (≥1×10 9 ) and cytotoxic activity (CD107a expression rate>50%). On the other hand, chemotherapy, as the cornerstone of solid tumor treatment, is often limited in its efficacy by acquired drug resistance and tumor recurrence. The NK cells obtained by existing expansion schemes lack the temporal synergistic design with chemotherapy drugs and cannot overcome the immunosuppressive barrier in the tumor microenvironment. These technical defects together make it difficult for existing NK cell therapy to be effectively integrated with standard chemotherapy regimens, severely limiting its application value in the comprehensive treatment of solid tumors. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the purpose of the embodiments of the present application includes providing a natural killer cell in vitro expansion composition, expansion method and application, so as to achieve large-scale in vitro expansion and culture of NK cells with high purity and high anti-tumor activity in a short period of time, and the NK cells expanded by the composition combined with chemotherapy drugs have a strong synergistic killing effect on tumor cells such as gastric and intestinal cells.

[0005] In a first aspect, an embodiment of the present application provides a method for expanding natural killer cells, comprising: S1. pre-coating a culture container with a monoclonal antibody combination, the monoclonal antibody combination comprising CD56 or CD16, NKG2D and CD52; S2. inoculating umbilical cord blood or peripheral blood mononuclear cells into the pre-coated culture container, and culturing them with an activation medium containing cytokines to obtain activated cells; the activation medium comprises a serum-free basal medium, albumin, phytohemagglutinin and nutrients; wherein the cytokines comprise one or more of IL-2, IL-12, IL-15 and IL-18; S3. transferring the activated cells into a serum-free expansion medium containing IL-2 and albumin for expansion culture; S4. collecting the cells and adjusting the density to 5×10-15×10 cells / mL with an albumin-containing solution, letting them stand for 0.5-1.5 hours, and then filtering to obtain natural killer cells; wherein the albumin comprises human serum albumin or recombinant human albumin.

[0006] The present application provides a method for amplifying natural killer cells, which significantly improves the amplification efficiency and anti-tumor activity of NK cells through monoclonal antibody combination activation, cytokine cascade reaction and optimized culture environment. Among them, in terms of cell activation, the composition adopts the synergistic mechanism of CD56 / CD16 monoclonal antibody, NKG2D monoclonal antibody and CD52 monoclonal antibody. CD56 / CD16 monoclonal antibody directly activates the killing function of NK cells through cross-linking, NKG2D monoclonal antibody simulates the tumor microenvironment to enhance the recognition ability of NK cells, and CD52 monoclonal antibody effectively removes mixed T / B lymphocytes and improves the purity of NK cells. This multi-target activation strategy not only improves the initial activation efficiency of NK cells, but also does not use tumor-derived trophoblast cells in this application, thus avoiding the carcinogenic risk that may be caused by traditional trophoblast cell culture. The sequential addition of cytokine combinations (such as IL-2, IL-12, IL-15 and IL-18) forms a dynamic regulatory network, which can synergistically promote NK cell proliferation and inhibit apoptosis on the one hand, and significantly enhance the killing function of NK cells on the other hand. This combination not only achieves an expansion multiple of over 3,000 times, far exceeding the 200-500 times of traditional methods, but also, in terms of the culture system, the serum-free culture medium, combined with albumin, phytohemagglutinin, and nutrients, provides stable metabolic support for NK cell expansion, significantly improving expansion efficiency. Furthermore, NK cells expanded by this expansion method exhibit significant synergistic effects with chemotherapy drugs.

[0007] In some embodiments of the present application, the concentrations of CD56 or CD16, NKG2D and CD52 are all 0.5-5 μg / mL.

[0008] The concentration ranges for each of the above monoclonal antibodies have been rigorously optimized to achieve optimal antibody-mediated activation: 0.5 μg / mL is the minimum effective concentration threshold, ensuring sufficient antibody binding to NK cell surface receptors (CD56 / CD16, NKG2D) and triggering sufficient activation signals; the upper limit of 5 μg / mL avoids the risk of activation-induced cell death (AICD) caused by excessive antibody cross-linking. Within this range, NK cell CD25 (IL-2Rα) expression is upregulated 3-5-fold, while apoptosis remains at an ideal level of <10%. Within the 0.5-5 μg / mL range, the three antibodies form a dynamic equilibrium: the CD56 / CD16 antibodies primarily activate cytotoxicity, the NKG2D antibodies enhance tumor recognition, and the CD52 antibodies maintain environmental purity. At concentrations below 0.5 μg / mL, CD52 lymphocyte clearance efficiency decreases (purity <90%); at concentrations above 5 μg / mL, steric hindrance between the antibodies may inhibit NKG2D activation.

[0009] In some embodiments of the present application, the concentration of IL-2 is 200-1500 IU / mL; and / or the concentration of IL-12 is 10-50 ng / mL; and / or the concentration of IL-15 is 20-150 ng / mL; and / or the concentration of IL-18 is 50-200 ng / mL.

[0010] In the above technical solution, the concentration range of IL-2 (200-1500 IU / mL), IL-12 (10-50 ng / mL), IL-15 (10-50 ng / mL) and IL-18 (50-200 ng / mL) can achieve the optimal balance between NK cell expansion efficiency and functional activity, so that each cytokine can play a good synergistic role, thereby significantly increasing the NK cell proliferation multiples.

[0011] In some embodiments of the present application, in the activation culture medium, the concentration of albumin is 0.02-0.1 g / mL; and / or the concentration of phytohemagglutinin is 0.05-0.2 μg / mL; and / or the concentration of nutrients is 0.5%-10%.

[0012] In some embodiments of the present application, the nutrients include platelet lysate or autologous plasma.

[0013] In some embodiments of the present application, the activation culture medium further comprises 1-10 μg / mL transferrin and 5-25 μg / mL human recombinant insulin.

[0014] In some embodiments of the present application, in step S3, the concentration of IL-2 in the serum-free expansion medium is 200-1500 IU / mL, and the concentration of albumin is 1-10 μg / μL.

[0015] In some embodiments of the present application, the expansion culture includes a first-stage expansion culture and a second-stage expansion culture; the first-stage expansion culture includes transferring the cells that have undergone activation culture in step S2 to an expansion culture medium containing IL-2 and nutrients for expansion culture; the second-stage expansion culture includes resuspending the cells that have undergone the first-stage expansion culture, supplementing the serum-free basal culture medium, IL-2 and nutrients into the expansion culture medium to continue the expansion culture.

[0016] The present application adopts appropriate expansion culture medium to respectively carry out first-stage expansion culture and second-stage expansion culture, so as to efficiently expand and culture umbilical cord blood or peripheral blood mononuclear cells.

[0017] In some embodiments of the present application, in step S4, the concentration of albumin in the solution is 1-15 μg / μL.

[0018] In some embodiments of the present application, the solution further comprises 500-1500 IU / mL of IL-2, 10-50 ng / mL of IL-12, 10-50 ng / mL of IL-15, or 50-200 ng / mL of IL-18.

[0019] The present application uses a solution containing albumin at an appropriate concentration as a washing reagent to resuspend the cells and adjust the cells to an appropriate concentration, thereby facilitating the acquisition of natural killer cells with high viability and high killing activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 is the ratio of NK cells prepared in Examples 1-3 and Comparative Examples 1-7;

[0022] Figure 2 is the viability of NK cells obtained in Examples 1-3 and Comparative Examples 1-7;

[0023] Figure 3 is the expansion multiple of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7;

[0024] Figure 4 The cytotoxic activity of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 against HCT-8 cells alone and in combination with the chemical drug oxaliplatin;

[0025] Figure 5 is the synergy index of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 combined with the chemical drug oxaliplatin;

[0026] Figure 6 The cytotoxic activity of NK cells prepared in Examples 1-3 and Comparative Examples 1-7 against HCT-8 cells alone and in combination with the chemical drug 5-Fu;

[0027] Figure 7 is the synergistic index of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 combined with the chemical drug 5-Fu;

[0028] Figure 8 The cytotoxic activity of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 against HCT-8 cells alone and in combination with the chemical drug capecitabine;

[0029] Figure 9 is the synergistic index of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 combined with the chemical drug capecitabine;

[0030] Figure 10 The anti-tumor activity of NK cells in Example 1 against the HCT-8 cell tumor model;

[0031] Figure 11 This is the synergistic anti-tumor activity of NK cells combined with the chemical drug 5-Fu in Example 1. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0033] A method for expanding natural killer cells according to an embodiment of the present application is described in detail below.

[0034] The present invention provides a method for expanding natural killer cells, comprising the following steps:

[0035] (1) Pre-coat the culture vessel with a monoclonal antibody combination that includes CD56 or CD16, NKG2D, and CD52.

[0036] Wherein, the concentration of CD56 or CD16, NKG2D and CD52 is 0.5-5 μg / mL. As an example, the concentration of each monoclonal antibody of CD56 or CD16, NKG2D and CD52 can be, but is not limited to, 0.5 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.5 μg / mL, 1.8 μg / mL, 2 μg / mL, 2.2 μg / mL, 2.5 μg / mL, 2.8 μg / mL, 3 μg / mL, 3.2 μg / mL, 3.5 μg / mL, 3.8 μg / mL, 4 μg / mL, 4.2 μg / mL, 4.5 μg / mL, 4.8 μg / mL, and 5 μg / mL. Pre-coating the culture container with a combination of multiple monoclonal antibodies at appropriate concentrations facilitates the subsequent activation and culture of umbilical cord blood mononuclear cells.

[0037] As an example, 0.5-5 μg / mL of CD56 or CD16 monoclonal antibodies, NKG2D monoclonal antibodies, and CD52 monoclonal antibodies were added to a 24-well plate and cultured at 4°C for activation.

[0038] Pre-coated culture vessels facilitate subsequent activation and culture of peripheral blood or umbilical cord blood mononuclear cells.

[0039] (2) Inoculating umbilical cord blood or peripheral blood mononuclear cells in a pre-coated culture container and culturing them in an activation medium containing cytokines to obtain activated cells; the activation medium comprises a serum-free basal medium, albumin, phytohemagglutinin, and nutrients; wherein the cytokines include one or more of IL-2, IL-12, IL-15, and IL-18.

[0040] In the present application, the concentration of IL-2 is 200-1500 IU / mL; and / or, the concentration of IL-12 is 10-50 ng / mL; and / or, the concentration of IL-15 is 10-50 ng / mL; and / or, the concentration of IL-18 is 50-200 ng / mL. By way of example, the concentration of IL-2 can be, but is not limited to, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, 1050 IU / mL, 1100 IU / mL, 1150 IU / mL, 1200 IU / mL, 1250 IU / mL, 1300 IU / mL, 1350 IU / mL, 1400 IU / mL, 1450 IU / mL, or 1500 IU / mL. The concentration of IL-12 may be, but is not limited to, 10 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 25 ng / mL, 28 ng / mL, 30 ng / mL, 35 ng / mL, 38 ng / mL, 40 ng / mL, 45 ng / mL, 48 ng / mL, and 50 ng / mL. The concentration of IL-15 may be, but is not limited to, 10 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 25 ng / mL, 28 ng / mL, 30 ng / mL, 35 ng / mL, 38 ng / mL, 40 ng / mL, 45 ng / mL, 48 ng / mL, and 50 ng / mL. The concentration of IL-18 can be, but is not limited to, 50 ng / mL, 55 ng / mL, 58 ng / mL, 60 ng / mL, 65 ng / mL, 68 ng / mL, 70 ng / mL, 75 ng / mL, 78 ng / mL, 80 ng / mL, 85 ng / mL, 88 ng / mL, 90 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, and 200 ng / mL.

[0041] In the present application, the nutrient comprises platelet lysate or autologous plasma, and the concentration of the nutrient is 0.5%-10%. As an example, the concentration of the nutrient can be, but is not limited to, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0042] As an embodiment, the activation medium may further include transferrin and human recombinant insulin. As an example, the activation medium may further include 1-10 μg / mL of transferrin and 5-25 μg / mL of human recombinant insulin, wherein the concentration of transferrin may be, but is not limited to, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL. The concentration of human recombinant insulin may be, but is not limited to, 5 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 15 μg / mL, 18 μg / mL, 20 μg / mL, 22 μg / mL, 25 μg / mL.

[0043] As an example, peripheral blood or umbilical cord blood is collected, PBMC or CBMC is separated, and the plate is inoculated on a 24-well plate coated with the antibody of step (1), and autologous plasma (10%), IL-2 (200-1500 IU / ml), IL-12 (10-50 ng / ml), IL-15 (10-50 ng / ml), IL-18 (50-200 ng / ml), transferrin (1-10 μg / ml), recombinant insulin (5-25 μg / ml), and phytohemagglutinin (0.05-0.2 μg / ml) are added. Subsequently, the 24-well plate is placed in a 37°C, 5% CO incubator for culture.

[0044] A multi-factor combination (IL-2 / 12 / 15 / 18) simulates the immune microenvironment in vivo and synergistically activates NK cell function. Autologous plasma provides natural growth factors (such as FGF and EGF) and anti-apoptotic factors (such as ApoA1), reducing the risk of immune rejection caused by foreign proteins.

[0045] In this application, the serum-free basal medium is ImmunoCult TM -XF T Cell Expansion Medium.

[0046] (3) The activated cells were transferred into serum-free expansion medium containing IL-2 and albumin for expansion culture.

[0047] As an example, ImmunoCult® was added to the 24-well plate in step (2) above. TM-XF T complete medium (containing 200-1500 IU / ml IL-2, 10% autologous plasma or 5-10% human recombinant albumin), and then the 24-well plate was placed in a 37°C, 5% CO incubator for culture.

[0048] As an example, the concentration of IL-2 in the expansion medium can be, but is not limited to, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, 1050 IU / mL, 1100 IU / mL, 1150 IU / mL, 1200 IU / mL, 1250 IU / mL, 1300 IU / mL, 1350 IU / mL, 1400 IU / mL, 1450 IU / mL, or 1500 IU / mL. The concentration of albumin can be, but is not limited to, 1 μg / μL, 2 μg / μL, 3 μg / μL, 4 μg / μL, 5 μg / μL, 6 μg / μL, 7 μg / μL, 8 μg / μL, 9 μg / μL, and 10 μg / μL. Using appropriate concentrations of IL-2 and albumin can efficiently expand and culture umbilical cord blood mononuclear cells.

[0049] The serum-free system reduces contamination risk and complies with clinical-grade cell preparation production regulations (e.g., FDA 21CFR Part 1271). Dynamic IL-2 supplementation maintains expansion kinetics and avoids cell exhaustion caused by overactivation (e.g., PD-1 upregulation).

[0050] (4) The cells were collected and adjusted to a density of 5 × 10-15 × 10 cells / mL using an albumin-containing solution. After standing for 0.5-1.5 h, the cells were filtered to obtain natural killer cells.

[0051] In this solution, the concentration of albumin is 1-15 μg / μL. As an example, the concentration of albumin in the solution can be, but is not limited to, 1 μg / μL, 2 μg / μL, 3 μg / μL, 4 μg / μL, 5 μg / μL, 6 μg / μL, 7 μg / μL, 8 μg / μL, 9 μg / μL, 10 μg / μL, 11 μg / μL, 12 μg / μL, 13 μg / μL, 14 μg / μL, and 15 μg / μL. Resuspending the cells in a washing reagent containing an appropriate concentration of albumin and adjusting the cells to an appropriate concentration facilitates obtaining natural killer cells that maintain high viability and high cytotoxic activity and are not prone to clumping after low-temperature cold chain air transportation.

[0052] In the present application, the above solution further comprises 500-1500 IU / mL of IL-2, 10-50 ng / mL of IL-12 or 10-200 ng / mL of IL-18. By way of example, the concentration of IL-2 in the wash reagent can be, but is not limited to, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, 1050 IU / mL, 1100 IU / mL, 1150 IU / mL, 1200 IU / mL, 1250 IU / mL, 1300 IU / mL, 1350 IU / mL, 1400 IU / mL, 1450 IU / mL, or 1500 IU / mL. The concentration of IL-12 in the solution can be, but is not limited to, 10 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 25 ng / mL, 28 ng / mL, 30 ng / mL, 35 ng / mL, 38 ng / mL, 40 ng / mL, 45 ng / mL, 48 ng / mL, and 50 ng / mL. The concentration of IL-18 in the solution can be, but is not limited to, 50 ng / mL, 55 ng / mL, 58 ng / mL, 60 ng / mL, 65 ng / mL, 68 ng / mL, 70 ng / mL, 75 ng / mL, 78 ng / mL, 80 ng / mL, 85 ng / mL, 88 ng / mL, 90 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, or 200 ng / mL. Resuspending the cells in a solution further comprising IL-2, IL-12, IL-15 or IL-18 at an appropriate concentration is beneficial for obtaining natural killer cells with high viability and high killing activity that are not prone to agglomeration.

[0053] In some embodiments of the present application, resuspending cells using the above solution and adjusting the cell density includes:

[0054] (1) Resuspend the cells in saline containing 1-5 μg / μL albumin and 500-1500 IU / mL IL-2, 10-50 ng / mL IL-12, or 50-200 ng / mL IL-18. Let stand for 1-15 min, centrifuge, discard the supernatant, and repeat this operation 1-2 times.

[0055] (2) Resuspend the cells in saline containing 1-3 μg / μL albumin, let it stand for 1-15 minutes, centrifuge, discard the supernatant, and repeat the operation 2-3 times; then resuspend the cells in saline containing 5-15 μg / μL albumin to adjust the cell density.

[0056] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0057] 1. The reagents and materials used in this application are as follows:

[0058] Reagents: Ficoll (Tianjin Haoyang), CD16 monoclonal antibody (Tongli Haiyuan), NKG2D monoclonal antibody (Jianwan protein), CD52 monoclonal antibody (Jianwan protein), ImmunoCult TM -XF T cell expansion medium (Stemcell), 1640 basal medium (Shanghai Darthill Biotechnology), fetal bovine serum (Ecosai), IL-2 (Jiangsu Jinsili Pharmaceutical), IL-12 (Jiangnan Protein), IL-15 (Beijing Kexin Biotechnology), IL-18 (Beijing Kexin Biotechnology), human serum albumin (Rongsheng), human recombinant albumin (Wuhan Heyuan Biotechnology), human recombinant insulin (Beijing Kexin Biotechnology), transferrin (Wuhan Heyuan Biotechnology), platelet lysate (Mill Creek), 5-Fu (MCE), oxaliplatin (MCE), capecitabine (MCE), CCK-8 (Shanghai Beyotime).

[0059] Cell line: HCT-8 (purchased from ATCC).

[0060] 2. The detection methods in the test examples of this application include:

[0061] (1) Cell counting: Gently resuspend the expanded cultured cells in physiological saline and transfer them to a 50 mL tube. Take 10.5 μL of the cell suspension and add 10.5 μL of AO / PI dye. Detect the cell viability and cell concentration on a Countstar instrument.

[0062] (2) Detection of cell surface markers:

[0063] 1. Take a sample of cells and count them. Centrifuge the cell suspension at 1000 rpm for 5 minutes and discard the supernatant.

[0064] 2. Resuspend with appropriate PBS (containing 1% BSA), wash the cells once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant;

[0065] 3. Resuspend the cells with appropriate PBS (containing 1% BSA) and adjust the cell concentration to about 2.0×10 7 / mL;

[0066] 4. Dispense the cell suspension from the previous step into 1.5mL EP tubes, 50μL each (10 cells per tube). 6 );

[0067] 5. Add 2 μL each of PerCP-CD45, FITC-CD3, and PE-CD56 antibodies at the same time, place the mixed tube in a 4°C refrigerator, and incubate in the dark for 30 minutes;

[0068] 6. Add 1 mL of PBS to each tube to resuspend and wash the cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant;

[0069] 7. Add 200 μL of PBS to each tube to resuspend the cells and detect them on the instrument.

[0070] (3) NK cells’ ability to kill tumor cells:

[0071] 1. Tumor cell treatment: Remove HCT-8 tumor cells, discard 1640 complete medium (1640 basal medium + fetal bovine serum + antibiotics), add 6mL DPBS to wash once, and discard the liquid; then add 1mL 0.25% trypsin (T25 culture flask) to digest, observe under the microscope that the digestion is single cells, add 6mL 1640 complete medium to neutralize and terminate the digestion; use a pipette to mix the cells and collect them into a 15mL centrifuge tube, centrifuge at 150g for 5 minutes, and discard the liquid; then add a small amount of 1640 complete medium to resuspend. Take the resuspended tumor cells and count them. The counting method refers to the "Countstar Cell Counter SOP" and dilute with complete medium to adjust the density to 5×10 4 / mL to a 15mL centrifuge tube, take a 96-well flat-bottom plate, and use a pipette to draw the diluted tumor cell suspension (5×10 4 / mL), 100 μL was plated in each well, and the cell plate was transferred to a CO2 incubator and incubated overnight;

[0072] 2. Prepare the corresponding chemotherapy drugs in 1640 complete medium. Add 100 μL of the chemotherapy drugs 5-Fu (1.67 μM), oxaliplatin (0.33 μM), and capecitabine (1.33 mM) to each well. Add 1640 complete medium to the remaining wells. Transfer the cell plate to a CO2 incubator and incubate for 48 hours.

[0073] 3. Effector (NK) cell treatment: Replace all 1640 complete medium containing drugs with new 1640 complete medium, gently aspirate the medium in all wells, and add 100 μL of 1640 complete medium to each well. Take NK cells and use ImmunoCult TM-XF medium + 1000 IU / mL IL-2, collect NK cells in a 15 mL centrifuge tube, centrifuge at 350 g for 5 min, resuspend in culture medium and count. The counting method refers to the "Countstar Cell Counter SOP" and the density is adjusted to 2.5 × 10 4 / mL was transferred to a 15mL centrifuge tube, 100μL was plated per well for NK cell plating, and NK culture medium was added to the remaining wells. The cell plate was transferred to a CO2 incubator and incubated for 24h;

[0074] 4. Detection: Remove the cells from the incubator, aspirate the culture medium from all wells, gently add 200 μL of DPBS to each well to wash once, remove the remaining NK cells, and prepare an appropriate amount of detection solution at a ratio of 50 μL complete tumor cell culture medium + 50 μL NK cell culture medium + 10 μL CCK-8 per well. Incubate in the dark in an incubator for 2 hours, set the detection wavelength to 450 nm, and detect using a microplate reader.

[0075] 5. Result evaluation: Cytotoxicity (%) = (1-(NK or chemical drug or NK combined with chemical drug well) / tumor cell well); Synergy index = cytotoxicity of NK combined with chemical drug / (cytotoxicity of NK + cytotoxicity of chemical drug - cytotoxicity of NK × cytotoxicity of chemical drug).

[0076] Example 1

[0077] This embodiment provides a method for expanding NK cells in vitro, comprising the following steps:

[0078] 1. Obtaining CBMC (mononuclear cells) from umbilical cord blood

[0079] (1) Umbilical cord blood (collected from Wenjiang District People's Hospital, Chengdu) was equally divided and transferred into 50 mL centrifuge tubes. After equalization, the tubes were centrifuged at 3000 rpm for 15 min.

[0080] (2) After centrifugation, discard the red blood cells at the bottom and transfer the white blood cell layer and upper plasma layer as completely as possible to a 50 mL centrifuge tube. Add physiological saline to 35 mL and resuspend thoroughly. Prepare four 15 mL centrifuge tubes and add 4 mL of Ficoll separation solution to each tube. Slowly add to the Ficoll separation solution surface, taking care not to disrupt the surface layer. Centrifuge at 2000 rpm for 20 min.

[0081] (3) After centrifugation, discard the supernatant, aspirate the buffy coat layer into a new 50 mL centrifuge tube, add physiological saline to 50 mL, resuspend, and centrifuge at 1700 rpm for 5 min; repeat the above steps, take a small amount for counting, centrifuge at 1500 rpm for 5 min, and discard the remaining supernatant;

[0082] (4) The CBMCs washed for the last time were treated with ImmunoCult TMResuspend in -XF T cell expansion medium and adjust the cell density to 1 × 10 6 -2×10 6 / mL to obtain umbilical cord blood mononuclear cells.

[0083] 2. Activation and Expansion of NK Cells

[0084] (1) 2 μg / mL of CD56 monoclonal antibody, NKG2D monoclonal antibody, and CD52 monoclonal antibody were added to 24-well plates for antibody pre-coating at 4°C overnight;

[0085] (2) The extracted CBMCs were inoculated into 0.6 mL of cells in a 24-well plate pre-coated with antibodies in step (1), and 500 IU / mL of IL-2, 20 ng / mL of IL-12, 20 ng / mL of IL-15, 100 ng / mL of IL-18, 2 μg / mL of transferrin, 10 μg / mL of human recombinant insulin, 2% platelet lysate, 5 μg / μL of human serum albumin, and 0.1 μg / mL of phytohemagglutinin were added, and the cells were activated and cultured in an incubator at 37°C and 5% CO2;

[0086] (3) On the fourth day after the activation culture, ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human albumin, and 2% platelet lysate was added to the 24-well plate. TM -XF T cell expansion complete medium 1.2mL, in a 37℃, 5% CO2 incubator for the first stage of expansion culture;

[0087] (4) On day 6 (calculated from the start of activation culture), gently resuspend the cells and transfer them to a T25 flask. Add ImmunoCult™ containing 500 IU / mL IL-2, 5 μg / μL human albumin, and 2% platelet lysate to the T25 flask. TM -3.6 mL of XFT cell expansion complete medium was used for the first stage of expansion culture in a 37°C, 5% CO2 incubator;

[0088] (5) On day 8 (calculated from the start of activation culture), add ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human albumin, and 2% platelet lysate to the T25 flask. TM -10.8 mL of XF T cell expansion complete medium was added and continued in a 37°C, 5% CO2 incubator for the first stage of expansion culture.

[0089] (6) On day 10 (calculated from the start of activation culture), gently resuspend the cells and transfer them to a T75 flask. Add ImmunoCult® containing 500 IU / mL IL-2 and 5 μg / μL human albumin to the T75 flask. TM -16.2 mL of XF T cell expansion complete medium was used for the second stage of expansion culture in a 37°C, 5% CO2 incubator;

[0090] (7) On day 12 (calculated from the start of activation culture), add ImmunoCult® containing 500 IU / mL IL-2 and 5 μg / μL human albumin to the T75 flask. TM -16.2 mL of XF T cell expansion complete medium was used for the second stage of expansion culture in a 37°C, 5% CO2 incubator;

[0091] (8) On day 14 (calculated from the start of activation culture), gently resuspend the cells and transfer them to a T182 flask. Add ImmunoCult® containing 500 IU / mL IL-2 and 5 μg / μL human albumin to the T182 flask. TM -24.3 mL of XF T cell expansion complete medium was used for the second stage of expansion culture in a 37°C, 5% CO2 incubator;

[0092] (9) On day 16 (calculated from the start of activation culture), add ImmunoCult® containing 500 IU / mL IL-2 and 5 μg / μL human albumin to the T182 flask. TM -24.3 mL of XF T cell expansion complete medium was used for the second stage of expansion culture in a 37°C, 5% CO2 incubator;

[0093] (10) On the 18th day (calculated from the start of activation culture), the cells were counted, the cell status was observed under a microscope, and the cell proliferation was calculated. The cell density reached 3×10 6 -6×10 6 Cells were collected and resuspended in saline containing 2 μg / μL human albumin and 800 IU / mL IL-2. After standing for 10 minutes, the cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. This was repeated twice. The cells were resuspended in saline containing 1 μg / μL human albumin. After standing for 10 minutes, the cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. This was repeated three times. Finally, the cells were resuspended in saline containing 10 μg / μL human albumin to adjust the cell density to 5×10 7 The cells were placed at room temperature for 0.5 h, filtered using a 70 μm filter, and placed at 4°C.

[0094] Example 2

[0095] This embodiment is basically the same as the first embodiment, except that:

[0096] (1) 2 μg / mL of CD16 monoclonal antibody, NKG2D monoclonal antibody, and CD52 monoclonal antibody were respectively prepared and added to a 24-well plate for antibody pre-coating at 4°C overnight.

[0097] Example 3

[0098] This embodiment is basically the same as embodiment 1, except that peripheral blood mononuclear cells are used in step (2).

[0099] The steps for extracting peripheral blood mononuclear cells (PBMC) are as follows:

[0100] (1) Blood separation

[0101] The peripheral blood (collected from volunteers of Chengdu Kangjing Biotechnology Co., Ltd.) was equally divided into centrifuge tubes and centrifuged at 800×g for 10 min at room temperature. The upper plasma layer was collected and inactivated for later use. The remaining blood components were made up to the original volume with normal saline, and then diluted and mixed with an equal volume of normal saline.

[0102] (2) Ficoll density gradient centrifugation

[0103] Slowly add the diluted blood cells into Ficoll separation solution, with the ratio of Ficoll separation solution to diluted blood cells being 1:1.5, and centrifuge at 500xg for 20 minutes at room temperature with slow ramping.

[0104] (3) PBMC washing and purification

[0105] After centrifugation, use a pipette to collect the mononuclear cells in the buffy coat into a new centrifuge tube; add 40 ml of normal saline and centrifuge at 400 x g for 5 min; remove the supernatant, add 40 ml of normal saline, mix, and centrifuge at 400 x g for 5 min; resuspend the cells in 10 ml of normal saline, take 20 μl of the suspension for cell counting, centrifuge the remaining suspension at 400 x g for 5 min, and remove the supernatant.

[0106] (4) Cell resuspension and culture medium adjustment

[0107] The PBMCs were washed for the last time and then treated with ImmunoCult TM Resuspend in -XF T cell expansion medium; adjust the cell density to 1 × 10 6 -2×10 6 Peripheral blood mononuclear cells (PBMC) were obtained.

[0108] Comparative Example 1

[0109] This comparative example is basically the same as Example 1, except that:

[0110] (1) Prepare 2 μg / mL CD52 monoclonal antibody and add it to 24-well plates for antibody pre-coating at 4°C overnight.

[0111] Comparative Example 2

[0112] This comparative example is basically the same as Example 1, except that:

[0113] (1) Prepare 2 μg / mL CD56 monoclonal antibody and add it to 24-well plates for antibody pre-coating at 4°C overnight.

[0114] Comparative Example 3

[0115] This comparative example is basically the same as Example 1, except that:

[0116] (1) Prepare 2 μg / mL of NKG2D monoclonal antibody and add it to 24-well plates for antibody pre-coating at 4°C overnight.

[0117] Comparative Example 4

[0118] This comparative example is basically the same as Example 1, except that:

[0119] (1) 2 μg / mL of CD52 monoclonal antibody and CD56 monoclonal antibody were added to 24-well plates for antibody pre-coating at 4°C overnight.

[0120] Comparative Example 5

[0121] This comparative example is basically the same as Example 1, except that:

[0122] (1) 2 μg / mL of CD56 monoclonal antibody and NKG2D monoclonal antibody were respectively prepared and added to 24-well plates for antibody pre-coating at 4°C overnight.

[0123] Comparative Example 6

[0124] This comparative example is basically the same as Example 1, except that:

[0125] (1) 2 μg / mL of CD52 monoclonal antibody and NKG2D monoclonal antibody were respectively prepared and added to 24-well plates for antibody pre-coating at 4°C overnight.

[0126] Comparative Example 7

[0127] This comparative example is basically the same as Example 1, except that:

[0128] (1) 2 μg / mL of CD16 monoclonal antibody, CD52 monoclonal antibody, and CD137 monoclonal antibody were respectively prepared and added to a 24-well plate for antibody pre-coating at 4°C overnight.

[0129] Some parameters of the above examples and comparative examples are detailed in Table 1.

[0130] Table 1

[0131] Group Nuclear cell source Antibody Example 1 Umbilical cord blood CD56, CD52, NKG2D Example 2 Umbilical cord blood CD16, CD52, NKG2D Example 3 peripheral blood CD56, CD52, NKG2D Comparative Example 1 Umbilical cord blood CD52 Comparative Example 2 Umbilical cord blood CD56 Comparative Example 3 Umbilical cord blood NKG2D Comparative Example 4 Umbilical cord blood CD52, CD56 Comparative Example 5 Umbilical cord blood CD56, NKG2D Comparative Example 6 Umbilical cord blood CD52, NKG2D Comparative Example 7 Umbilical cord blood CD16, CD52, CD137

[0132] Test Example 1

[0133] In this test example, the NK cells expanded and cultured to day 18 in Examples 1-3 and Comparative Examples 1-7 were tested using a Countstar instrument to detect cell viability and viable cell concentration, and the viable cell count was calculated, viable cell count = viable cell concentration × culture medium volume; cell surface markers were detected to determine the NK cell ratio; and the NK cell expansion fold was calculated, expansion fold = viable NK cell count after expansion / viable NK cell count before expansion; the above items were measured in three parallel groups. The measurement results of the above items are detailed in Table 2. Figure 1-Figure 3 ,in Figure 1 is the ratio of NK cells obtained in Examples 1-3 and Comparative Examples 1-7, Figure 2 is the NK cell viability obtained in Examples 1-3 and Comparative Examples 1-7, Figure 3 It is the expansion multiple of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7.

[0134] Table 2

[0135]

[0136]

[0137] From Table 2, and Figure 1-Figure 3 It can be seen that compared with comparative examples 1-7, the NK cells prepared in Examples 1-3 of the present application have high purity (above 90%), cell viability greater than 90%, and amplification multiple greater than 3000 times.

[0138] Test Example 2

[0139] This test example respectively detects the killing activity of NK cells expanded and cultured to day 18 against HCT-8 cells alone and in combination with the chemical drug 5-Fu and capecitabine, as well as the synergistic index. The test results of the above items are detailed in Figure 4-11 ,in, Figure 4 The cytotoxic activity of NK cells prepared in Examples 1-3 and Comparative Examples 1-7 against HCT-8 cells alone and in combination with the chemical drug 5-Fu; Figure 5 is the synergistic index of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 combined with the chemical drug 5-Fu; Figure 6The cytotoxic activity of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 against HCT-8 cells alone and in combination with the chemical drug oxaliplatin; Figure 7 is the synergy index of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 combined with the chemical drug oxaliplatin; Figure 8 The cytotoxic activity of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 against HCT-8 cells alone and in combination with the chemical drug capecitabine; Figure 9 is the synergistic index of the NK cells prepared in Examples 1-3 and Comparative Examples 1-7 combined with the chemical drug capecitabine; Figure 10 The anti-tumor activity of NK cells in Example 1 against the HCT-8 cell tumor model; Figure 11 This is the synergistic anti-tumor activity of NK cells combined with the chemical drug 5-Fu in Example 1.

[0140] from Figure 4-Figure 9 It can be seen that compared with Comparative Examples 1-6, the NK cells of Examples 1-3 have increased killing activity against HCT-8 cells. Compared with Comparative Examples 1-7, the NK cells of Examples 1-3 combined with chemotherapy drugs 5-Fu, oxaliplatin and capecitabine respectively have very significant synergistic anti-tumor activity against HCT-8 colorectal cancer cells, and the synergistic index is all >1.5.

[0141] from Figure 10 and Figure 11 It can be seen that the NK cells prepared in Example 1 of the present application and the chemical drug 5-Fu have a certain therapeutic effect on the tumor progression of HCT-8 when used alone. When the two are used simultaneously, the effect is more significant, indicating that the NK cells prepared in the present application have high synergy with existing chemotherapy drugs and have a significant therapeutic effect.

[0142] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

Claims

1. A method for expanding natural killer cells, characterized in that: include: S1. Pre-coating the culture vessel with a monoclonal antibody combination comprising CD56 or CD16, NKG2D, and CD52; S2. Inoculating umbilical cord blood or peripheral blood mononuclear cells in the pre-coated culture vessel and culturing them in an activation medium containing cytokines to obtain activated cells; the activation medium comprises a serum-free basal medium, albumin, phytohemagglutinin and nutrients; Wherein, the cytokines include one or more of IL-2, IL-12, IL-15 and IL-18; S3. The activated cells are transferred into a serum-free expansion medium containing the IL-2 and the albumin for expansion culture; S4. Collecting the cells and adjusting the density to 5×10-15×10 cells / mL with the albumin-containing solution, letting it stand for 0.5-1.5 hours, and then filtering to obtain the natural killer cells; Wherein, the albumin includes human serum albumin or human recombinant albumin.

2. The amplification method according to claim 1, characterized in that The concentrations of the CD56 or CD16, the NKG2D and the CD52 are all 0.5-5 μg / mL.

3. The amplification method according to claim 1, characterized in that The concentration of the IL-2 is 200-1500 IU / mL; and / or, the concentration of the IL-12 is 10-50 ng / mL; and / or, the concentration of the IL-15 is 10-50 ng / mL; and / or, the concentration of the IL-18 is 50-200 ng / mL.

4. The amplification method according to any one of claims 1 to 3, characterized in that In the activation culture medium, the concentration of the albumin is 2-10 μg / μL; and / or the concentration of the phytohemagglutinin is 0.05-0.2 μg / mL; and / or the concentration of the nutrients is 0.5%-10%.

5. The amplification method according to claim 4, characterized in that The nutrients include platelet lysate or autologous plasma.

6. The amplification method according to any one of claims 1 to 3, characterized in that The activation culture medium further comprises 1-10 μg / mL transferrin and 5-25 μg / mL human recombinant insulin.

7. The amplification method according to any one of claims 1 to 3, characterized in that In step S3, the concentration of IL-2 in the serum-free expansion medium is 200-1500 IU / mL, and the concentration of albumin is 1-10 μg / μL.

8. The amplification method according to claim 7, characterized in that The amplification culture includes a first-stage amplification culture and a second-stage amplification culture; The first stage of expansion culture includes transferring the cells that have undergone the activation culture in step S2 to an expansion culture medium containing the IL-2 and the nutrients for expansion culture; The second stage expansion culture includes resuspending the cells that have undergone the first stage expansion culture, supplementing the serum-free basal culture medium, the IL-2 and the nutrients into the expansion culture medium to continue the expansion culture.

9. The amplification method according to claim 8, characterized in that In step S4, the concentration of the albumin in the solution is 1-15 μg / μL.

10. The amplification method according to claim 9, characterized in that The solution also includes 500-1500 IU / mL of IL-2, 10-50 ng / mL of IL-12, 10-50 ng / mL of IL-15, or 50-200 ng / mL of IL-18.

Images