Method for improving proliferation and activity of cytokine-induced killer cells

Abstract

The invention discloses a method for improving proliferation and activity of cytokine-induced killer cells, and belongs to the technical field of cellular immunity. According to the method, the human peripheral blood mononuclear cells are firstly cultured in the culture medium containing the cell factors, then the obtained CIK cells are cultured in the culture medium containing the cell factors and the PP2A inhibitor, the amplification efficiency of the CIK cells is improved through the PP2A inhibitor, and the proportion of CD3 + CD56 + cells in the CIK cells is increased; according to the invention, a cell signal transduction pathway regulator PP2A inhibitor is ingeniously introduced, and culture conditions are optimized, so that the amplification efficiency of CIK cells is remarkably improved, and the proportion of functional subgroups is improved. Meanwhile, on the basis of improving the cell performance, the preparation cost of the CIK cell is relatively reduced, and the CIK cell is particularly suitable for preparing a CIK cell preparation required by adoptive immunotherapy and can be widely applied to treatment of solid tumors and blood system tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cellular immunology, and particularly relates to a method for improving the proliferation and activity of cytokine-induced killer cells. BACKGROUND

[0002] Cytokine-induced killer cells (CIK) are a heterogeneous population of immune cells induced by multiple cytokines, which have the characteristics of non-MHC restricted killing of tumor cells. Clinical studies have shown that cell therapy based on CIK cells greatly improves the quality of life, overall survival time and total survival of patients, making it a subject of great interest.

[0003] However, the existing CIK cell culture technology has many limitations, mainly manifested in low in vitro expansion efficiency, imbalance of cell subpopulations, and insufficient cell activity. First, in the culture process of CIK cells, the dependence on cytokines is extremely high. Although high concentrations of IL-2 can significantly improve the culture efficiency, the high cost and the problem of promoting the proliferation of regulatory T cells (Treg) to inhibit the anti-tumor effect cannot be ignored. Second, the expansion of CIK cells is limited, and under the traditional method, it can only reach an expansion of 10-20 times, which is difficult to meet the demand for large-dose cell infusion in clinical treatment. In addition, the imbalance of cell subpopulations is also a problem, especially the proportion of CD3+CD56+ cells is low (less than 30%), which affects the overall killing activity of CIK cells. Furthermore, metabolic stress is an important factor restricting the culture of CIK cells. In the long-term culture process, the accumulation of lactic acid and the depletion of nutrients will lead to cell apoptosis. 3+ CD 56+ The proportion of CD3+CD56+ cells is low (less than 30%), which affects the overall killing activity of CIK cells. Furthermore, metabolic stress is an important factor restricting the culture of CIK cells. In the long-term culture process, the accumulation of lactic acid and the depletion of nutrients will lead to cell apoptosis.

[0004] Protein phosphatase 2A (PP2A) is a serine / threonine phosphatase widely distributed in organisms, which can regulate the phosphorylation state of various proteins through ATM / ATR-dependent and independent pathways, and plays a crucial role in cell signaling, cell cycle progression, and cell growth regulation. In the immune system, PP2A deeply affects the activation, differentiation and function of immune cells through dephosphorylation of various signaling molecules. Studies have shown that in T cells, PP2A regulates the proliferation and survival of T cells by dephosphorylating key signaling molecules such as AKT and mTOR. In addition, PP2A is also involved in the negative regulation of B cell receptor signaling pathway, thereby affecting the activation of B cells and the production of antibodies. The expression level of PP2A has a significant impact on regulating the function of Treg cells. Inhibition of PP2A activity can lead to suppression of Treg cell function, thereby triggering the enhancement of autoimmune response. In dendritic cells, changes in PP2A activity can affect their antigen presentation function and secretion of cytokines, thereby regulating the immune response of T cells. SUMMARY

[0005] In view of this, the purpose of the present application is to provide a method for improving the proliferation and activity of cytokine-induced killer cells (CIK), which ingeniously introduces a cell signaling pathway regulator to optimize the culture conditions, so as to significantly improve the expansion efficiency of CIK cells and improve the proportion of functional subgroups. At the same time, on the basis of improving cell performance, the preparation cost of CIK cells is relatively reduced, which is especially suitable for the preparation of CIK cell preparations required for adoptive immunotherapy, and can be widely used in the treatment of solid tumors and hematological tumors.

[0006] In order to achieve the above purpose, the present application provides the following technical scheme: In a first aspect, the present application provides a culture system for improving the proliferation and activity of cytokine-induced killer cells, comprising a basic culture medium, cytokines and a cell signaling pathway regulator; the cytokines include IL-2 and IFN-γ, and the cell signaling pathway regulator is a PP2A inhibitor.

[0007] Further, the final concentration of IL-2 is 100-1000 U / mL, and the final concentration of IFN-γ is 500-1500 U / mL.

[0008] Further, the PP2A inhibitor is LB100, and the final concentration is 0.2-2 μM, preferably 0.5-1 μM.

[0009] Further, the basic culture medium includes RPMI 1640 medium and DMEM medium.

[0010] In a second aspect, the present application provides the use of the culture system as described above in the preparation of CIK cell preparation for adoptive immunotherapy.

[0011] In a third aspect, the present application provides a method for improving the proliferation and activity of cytokine-induced killer cells (CIK cells), by culturing the CIK cells in the presence of a PP2A inhibitor.

[0012] Further, the culture condition is 37℃, 5% CO2 for 1-5 days.

[0013] Further, the method comprises culturing human peripheral blood mononuclear cells in a culture medium containing cytokines, and then culturing the obtained CIK cells in a culture medium containing cytokines and a PP2A inhibitor, the PP2A inhibitor improving the expansion efficiency of the CIK cells and increasing the proportion of CD 3+ CD 56+ cells in the CIK cells.

[0014] Further, the cytokines include IL-2 and IFN-γ, the final concentration of IL-2 is 100-1000 U / mL, and the final concentration of IFN-γ is 500-1500 U / mL.

[0015] Further, the PP2A inhibitor is LB100, and the final concentration is 0.2-2 μM, preferably 0.5-1 μM.

[0016] Compared with the prior art, the present application has the following beneficial effects: 1. Significantly improve the expansion efficiency: after being cultured by the culture system of the present application, the expansion efficiency of the CIK cells is significantly improved compared with the traditional culture system, which can fully meet the demand for large cell infusion in clinical treatment.

[0017] 2. Improve cell activity: by precisely regulating the cell signaling pathway and metabolic state, the culture system of the present application significantly improves the proportion of CD 3+ CD 56+ cell subpopulation to more than 40-50%, greatly enhancing the overall killing activity of the CIK cells. At the same time, the cell survival cycle is significantly prolonged, and the cell apoptosis is significantly reduced, which effectively guarantees the quality and function of the cells.

[0018] 3. Simple operation and cost reduction: The present application uses a low-dose cytokine combination, which significantly reduces the use amount of expensive cytokines, thereby effectively reducing the culture cost. In addition, through the overall optimization of the culture system, the success rate and cell quality of cell culture are significantly improved, the resource waste caused by cell culture failure is reduced, and the preparation cost is further reduced from multiple aspects.

[0019] 4. Large-scale preparation of autologous or allogeneic CIK cells: The method of the present application has high expansion capacity and good cell activity maintenance capacity, which is very suitable for large-scale preparation of autologous or allogeneic CIK cells, and provides sufficient cell source for clinical cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced as follows.

[0021] Figure 1 CCK8 activity detection results of CIK cells under different concentrations of LB100 in Example 5.

[0022] Figure 2 CD 3+ CD 56+ Proportion detection results of cells in each group of CIK cells in Example 6.

[0023] Figure 3 Enzyme expression level detection results in the metabolic pathway of CIK cells under the action of LB100 in Example 7. DETAILED DESCRIPTION

[0024] The present application will be described in detail below in combination with the embodiments, but the embodiments of the present application are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present application, and for those skilled in the art, other similar embodiments can be obtained without creative labor, which fall within the protection scope of the present application.

[0025] The experimental apparatus and reagents used in the embodiments are as follows: 1. Experimental apparatus Low-speed centrifuge (Beckman, USA), CO2 cell incubator (Sanyo, Japan), pipette (Thermo, USA), graduated pipette (Kimble, USA), 75cm 2Culture flask (USA, Corning), 15ml centrifuge tube (USA, Corning), 50ml centrifuge tube (USA, Corning), cell scraper (USA, Corning), cell filter (USA, Corning), inverted microscope (Japan, Nikon), cryopreservation tube (USA, Corning), 96-well cell culture plate (USA, Corning), microplate reader (USA, BioTek), flow cytometer (USA, BD).

[0026] 2. Experimental reagents Ficoll-Hypaque cell separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd.), sodium chloride injection (Shijiazhuang Four Pharmaceutical Company), heparin sodium (Shanghai First Biochemical Pharmaceutical Co., Ltd.), IFN-γ (Beijing Sihuan Biological Pharmaceutical Co., Ltd.), IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.), purified grade anti-human CD3 monoclonal antibody (USA, eBioscience), serum-free medium (USA, Thermo), human serum albumin (Shanxi Kangbao Biological Products Co., Ltd.), FITC-labeled mouse anti-human CD3 monoclonal antibody (USA, BD), Hank's solution (preparation method: 1. A solution: NaCl 160g, KCl 8g, MgSO4·7H2O 2g, MgCl2·6H2O 2g added to 800ml double distilled water, dissolved. CaCl2 2.8g dissolved in 100ml double distilled water, after dissolving, added to the above 800ml solution, made up with double distilled water to 1000ml. 2. B solution: Na2HPO4·12H2O 3.04g, KH2PO4 1.2g, glucose 20g added to 800ml double distilled water, dissolved, added 0.4% phenol red solution 100ml, the above two solutions were mixed, double distilled water was added to 1000ml. When used, filter through 1 part of A solution, 1 part of B solution, and 18 parts of double distilled water, sterilize for 8 pounds 20min, store at 4℃), NaCl (Guojia Lu test), KCl (Guojia Lu test), MgSO4·7H2O (Tianjin Chuyuan Chemical Co., Ltd.), MgCl2·6H2O (Tianjin Chuyuan Chemical Co., Ltd.), CaCl2 (Tianjin Bodhi Chemical Co., Ltd.), Na2HPO4·12H2O (Tianjin Guangfu Science and Technology Development Co., Ltd.), KH2PO4 (Tianjin Chuyuan Chemical Co., Ltd.), glucose (Guojia Lu test), phenol red (Shanghai Baoman Biological Technology Co., Ltd.), LB100 (selleck).

[0027] Example 1: CD3 monoclonal antibody coating In this example, the purified grade anti-human CD3 monoclonal antibody was diluted to a concentration of 50ng / ml with 0.01M pH9.6 PBS solution, and the anti-human CD3 monoclonal antibody solution was added to 75cm 2In each culture flask, add 30-50 ml of culture medium and incubate at 4°C for 1 to 7 days. Before use, aspirate all PBS solution. Alternatively, remove PBS by freeze-drying to obtain cell culture flasks coated with anti-human CD3 monoclonal antibody, which can be stored for a longer period at 4°C. Before cell culture, add 20 ml of culture medium to wash the flask walls, lay the flask horizontally so that the culture medium covers the coated side of the flask wall, let stand for 10 minutes, then aspirate the culture medium and set aside.

[0028] Example 2: Preparation of LB100 solution, a PP2A protein phosphatase inhibitor The PP2A inhibitor LB100 was dissolved in ddH2O to prepare a stock solution with a concentration of 5 mg / ml.

[0029] Example 3: In vitro expansion of CIK cells Inject 50ml of heparin-anticoagulated blood into a 50ml centrifuge tube A, centrifuge at 3000rpm for 5min at 4℃, transfer the supernatant plasma into a 50ml centrifuge tube B, incubate at 56℃ for 30min, then centrifuge at 3000rpm for 15min at 4℃, collect the supernatant, and store at 4℃ for later use. Add Hank's solution or physiological saline to centrifuge tube A to a final volume of 48 ml and mix well. Slowly add the blood cell suspension to a 15 ml centrifuge tube containing 4 ml of Ficoll-Hypaque (density: 1.077±0.001), 8 ml / tube, and centrifuge at 4°C and 3000 rpm for 20 min. Collect the mononuclear cells (PBMCs) from the interfacial layer, combine them, and transfer them to a 50 ml centrifuge tube. Add Hank's solution or physiological saline to a final volume of 50 ml, centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and repeat 3 times. Add 10 ml of serum-free culture medium to suspend the precipitated cells and disperse them into a cell suspension. Add the cell suspension to a 75 cm⁻¹ centrifuge tube coated with the anti-human CD3 monoclonal antibody as described in Example 1. 2 Add 8 ml of plasma from centrifuge tube B to the culture flask, and add serum-free culture medium to a final volume of 80 ml. Incubate at 37°C and 5% CO2 for 24 hours. Add IFN-γ to the culture flask to a final concentration of 1000 U / ml, and continue incubating at 37°C and 5% CO2 for approximately 4-6 days. When the cells have reached full confluence in the culture flask under a microscope, use a graduated pipette and the culture medium in the flask to flush the cells off the flask wall. Transfer the cell suspension to a 1500-2000 ml cell culture bag, add 500 ml of serum-free culture medium, 10 ml of plasma from centrifuge tube B, and IL-2 (final concentration 500 U / ml). Incubate at 37°C and 5% CO2 for 4-5 days. Add 1000 ml of serum-free culture medium and IL-2 (final concentration 500 U / ml), and incubate at 37°C and 5% CO2 for 2 days.

[0030] Example 4: Grouping and In Vitro Induction Amplification After passage, PBMC cells from Example 3 were taken, the culture medium was discarded, and after trypsin digestion, they were resuspended in serum-free culture medium to prepare a concentration of 1.0 × 10⁻⁶ cells / mL. 6 Cell suspensions of / mL were further cultured according to the following groups: Conventional group: Add IL-2 to a final concentration of 500 U / ml and continue culturing for 48 h; LB100 group: IL-2 with a final concentration of 500 U / ml and LB100 with final concentrations of 0.25, 0.5, 0.75, 1 and 2 μM were added and cultured for 48 h.

[0031] Example 5: Determination of cell proliferation rate and cell viability Two groups of CIK cells from Example 4 were taken respectively, and cell viability was detected in each group using the CCK8 assay. The results are as follows: Figure 1 As shown, the addition of LB100 to the culture medium significantly increased the cell activity of CIK cells, indicating that the method of the present invention promotes the expansion of CIK cells.

[0032] Example 6: Determination of the proportion of CD3+CD56+ cells CD 3+ CD 56+ Cell proportions were determined by flow cytometry. Specifically, cells from the conventional group (veh) and the LB100 group (1 μM) in Example 4 were collected and cultured for 4 days. Cells were washed 2-3 times with PBS and resuspended, then the cell concentration was adjusted to 5 × 10⁻⁶ cells / day. 6 / mL, 100μL of cell suspension was taken into a flow cytometry tube, and cells were labeled with FITC-CD3 and PE-CD56 flow cytometry antibodies. After incubation at 4℃ for 30 min, cell phenotype analysis was performed using flow cytometry. The results are as follows: Figure 2 As shown, adding LB100 to the culture medium can effectively promote CD4+ in cells. 3+ CD 56+ An increase in the proportion of cells.

[0033] Example 7: Detection of enzyme expression levels in the metabolic pathway of CIK cells under the action of LB100. CIK cells from Example 4 were treated with different concentrations of LB100 (0, 500 nM, and 2000 nM) for 24 hours. Cell proteins were then extracted and analyzed by Western blotting. The results are as follows: Figure 3 As shown, the addition of LB100 to the culture medium can effectively promote the expression of hexokinase (glucokinase II) and PGK1 (pentose phosphate kinase 1) in the glycolysis signaling pathway in cells.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture system for enhancing the proliferation and activity of cytokine-induced cytotoxic cells, characterized in that, It contains basal culture medium, cytokines, and cell signaling pathway regulators; the cytokines include IL-2 and IFN-γ, and the cell signaling pathway regulators are PP2A inhibitors.

2. The culture system according to claim 1, characterized in that, The final concentration of IL-2 is 100~1000 U / mL, and the final concentration of IFN-γ is 500~1500 U / mL.

3. The culture system according to claim 1, characterized in that, The PP2A inhibitor is LB100, with a final concentration of 0.2~2μM, preferably 0.5~1μM.

4. The culture system according to claim 1, characterized in that, The basal culture media include RPMI 1640 medium and DMEM medium.

5. The use of the culture system according to any one of claims 1-4 in the preparation of CIK cell preparations required for adoptive immunotherapy.

6. A method for enhancing the proliferation and activity of cytokine-induced killer cells (CIK cells), characterized in that, The method involves culturing CIK cells in the presence of a PP2A inhibitor.

7. The method according to claim 6, characterized in that, The culture conditions are 37℃ and 5% CO2 for 1 to 5 days.

8. The method according to claim 6, characterized in that, The method involves culturing human peripheral blood mononuclear cells in a medium containing cytokines, and then culturing the resulting CIK cells in a medium containing cytokines and a PP2A inhibitor. The PP2A inhibitor enhances the expansion efficiency of CIK cells and increases CD4+ in CIK cells. 3+ CD 56+ The proportion of cells.

9. The method according to claim 8, characterized in that, The cytokines include IL-2 and IFN-γ, with a final concentration of 100-1000 U / mL for IL-2 and a final concentration of 500-1500 U / mL for IFN-γ.

10. The method according to claim 8, characterized in that, The PP2A inhibitor is LB100, with a final concentration of 0.2~2μM, preferably 0.5~1μM.

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