A culture medium for improving the ability of CIK cells to kill tumor cells, a method and application thereof

Abstract

The present invention discloses a culture fluid, method and application for improving the ability of CIK cells to kill tumor cells, and specifically relates to the field of biomedical technology. The culture fluid includes a CIK basic culture fluid and an induction drug, wherein the induction drug includes vitamin C, basic fibroblast growth factor, hepatocyte growth factor, glutathione, luteolin, interleukin-9 and interleukin-13. A variety of biological factors are added to the culture fluid of the present invention, which can efficiently amplify and activate CIK cells, thereby obtaining a large number of highly active immune cells mainly composed of CIK cells, which have significant therapeutic effects in the treatment of anti-tumor and related diseases; the culture method of CIK cells described in the present invention is safe and stable, and the time required is short, which greatly improves the CIK cell culture cycle and is conducive to large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a culture fluid, method and application for improving the ability of CIK cells to kill tumor cells. Background Art

[0002] CIK (cytokine-induced killing) therapy is an adoptive immune cell therapy. Current studies have shown that it has certain therapeutic effects in anti-tumor. This therapy can be combined with other treatments, such as combining immune checkpoint inhibitors (ICIs) represented by PD-1 antibodies with CIK therapy, which has a synergistic effect because PD-1 monoclonal antibodies can restore the activity of T cells, and CIK cells are a subpopulation of T cells.

[0003] In addition, CIK therapy is also used as an adjuvant treatment in the treatment of thyroid cancer. Thyroid cancer patients undergo CIK therapy after surgery to remove residual thyroid tissue during surgery and reduce the recurrence rate. Therefore, CIK can be used as an adjuvant treatment for cancer, but the specific method of use and effect still need to be determined according to the patient's specific situation and cancer type.

[0004] Although existing CIK (cytokine-induced killer) cells are effective in treating cancer, they also have some defects: CIK cells do not have tumor antigen specificity, which means that they cannot accurately identify and attack tumor cells, and may cause damage to normal cells and induce side effects. This lack of specificity has greatly limited the application of CIK cells in treating cancer; CIK cell therapy also has the problem of individual differences. The immune system responses and tumor characteristics of different patients are different, and the effects obtained vary greatly. Therefore, the effect of CIK cell therapy may vary from person to person, and it is difficult to guarantee universal effectiveness.

[0005] In summary, although the existing CIK cell-assisted cancer therapy has certain effects, it still has defects such as lack of specificity, unclear targeting and individual differences. Further research and improvement are needed to improve its efficacy and safety. Summary of the invention

[0006] To this end, the present invention provides a culture solution, method and application for improving the ability of CIK cells to kill tumor cells, so as to solve the above-mentioned problems.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] According to the first aspect of the present invention, a culture medium for improving the ability of CIK cells to kill tumor cells is provided, the culture medium comprising CIK basic culture medium and induction drugs, wherein the induction drugs comprise vitamin C, basic fibroblast growth factor, hepatocyte growth factor, glutathione, luteolin, interleukin-9 and interleukin-13.

[0009] Furthermore, the induction drugs include vitamin C 0.01-0.1 μL / ml, basic fibroblast growth factor 0.3-1.0 μL / ml, hepatocyte growth factor 0.2-0.8 μL / ml, glutathione 0.01-0.1 μL / ml, luteolin 0.4-1.0 μL / ml, interleukin-9 0.2-0.8 μL / ml and interleukin-13 0.2-0.8 μL / ml.

[0010] Furthermore, the induction drugs include vitamin C 0.01 μL / ml, basic fibroblast growth factor 0.3 μL / ml, hepatocyte growth factor 0.2 μL / ml, glutathione 0.01 μL / ml, luteolin 0.4 μL / ml, interleukin-9 0.2 μL / ml and interleukin-13 0.2 μL / ml.

[0011] Function of each component:

[0012] Vitamin C: It is a powerful antioxidant that can neutralize free radicals and promote the synthesis of collagen. At the same time, vitamin C can participate in biological processes such as inflammation, tumor formation, and cell apoptosis by inhibiting the activation of NFkβ signals.

[0013] BFGF (basic fibroblast growth factor): basic fibroblast growth factor can promote cell growth, repair and replenish nutrition.

[0014] HGF (Hepatocyte Growth Factor): It can promote the occurrence, survival and regeneration of tissue cells and inhibit cell apoptosis. HGF can inhibit the programmed death (apoptosis) of endothelial cells in a high-sugar environment and promote the repair of vascular endothelial cells. It can also regulate the synthesis of collagen fibers and inflammatory reactions, and plays an important role in promoting wound healing and preventing and treating tissue fibrosis.

[0015] Glutathione: is a natural antioxidant found inside cells that neutralizes free radicals and aids in detoxification.

[0016] Baicalin: Baicalin is a flavonoid compound extracted and separated from the root of Scutellaria baicalensis. It has significant biological activity, antibacterial, diuretic, anti-inflammatory, anti-allergic and antispasmodic effects, and has strong anti-cancer effects and other physiological effects. It has an important position in clinical medicine. Anti-pathogen effect: Baicalin has a wide range of antibacterial effects and has a strong effect on Staphylococcus aureus and Pseudomonas aeruginosa.

[0017] Interleukin-9 (IL-9): The main functions of IL-9 include regulating the growth and differentiation of B cells, enhancing the killing effect of CTL and CIK cells, stimulating the proliferation and differentiation of hematopoietic stem cells, and promoting the synthesis of acute phase proteins by hepatocytes.

[0018] Interleukin-13 (IL-13): IL-13 can induce monocyte differentiation and enhance the expression of MHC class II molecules; inhibit LPS-induced monokine secretion and control inflammatory response; induce B cell proliferation and synthesis of IgE antibodies, enhance the expression of MHC class II molecules, CD23 and CD72 on the surface of B cells; cooperate with IL-2 to stimulate CIK cells to produce IFN, thereby promoting monocyte-macrophage activation and TH1 cell immune response. Recently, it has been found that IL-13 also has multiple functions such as inhibiting HIV-1 replication in macrophages and inducing IL-1RA gene expression and protein synthesis in neutrophils.

[0019] Recombinant human interleukin-2 (IL-2): IL-2 is a multifunctional cytokine. Lymphokine-activated LAK cells receive signal induction, which accelerates the proliferation of B lymphocytes, thereby promoting antibody secretion and increasing the activity of autophagic cells. It has clinical pharmacological effects such as antiviral, immunity enhancement, and anti-tumor.

[0020] Furthermore, the CIK basal culture medium is CIK culture medium RPMI-1640.

[0021] According to a second aspect of the present invention, a method for improving the ability of CIK cells to kill tumor cells is provided, characterized in that the CIK cells are induced to culture using the culture fluid described in any one of claims 1-4.

[0022] Furthermore, the method includes: resuspending peripheral blood mononuclear cells with CIK basic culture medium, adding inactivated autologous plasma and recombinant human IL-2 and induction drugs into a culture bottle, placing it in an incubator for culture, and then regularly replenishing CIK basic culture medium and recombinant human IL-2. After the culture is completed, the CIK cells obtained can improve their ability to kill tumor cells.

[0023] Furthermore, the CIK basal culture medium is CIK culture medium RPMI-1640.

[0024] According to the third aspect of the present invention, there is provided use of the culture solution as described above in the preparation of anti-tumor drugs.

[0025] The fourth aspect of the present invention provides use of the above method in preparing anti-tumor drugs.

[0026] The present invention has the following advantages:

[0027] 1. The culture medium of the present invention contains a variety of biological factors, which can efficiently amplify and activate CIK cells, thereby obtaining a large number of highly active immune cells mainly composed of CIK cells, which have significant therapeutic effects in the treatment of tumors and related diseases.

[0028] 2. The CIK cell culture method of the present invention is safe and stable, requires a short time, greatly improves the CIK cell culture cycle, and is conducive to large-scale production.

[0029] 3. The CIK cells obtained using the method of the present invention have strong in vitro killing ability, and their in vivo anti-cancer effect is significantly higher than the survival rate and average survival period in the animal model after injection of IL-2 or recombinant human CD73 protein alone. Therefore, this technology is expected to be applied to the treatment of clinical cancer.

[0030] 4. The CIK cells amplified by the method of the present invention have a significant anti-tumor effect. Experiments show that when the effector-target ratio of the CIK cells obtained by the present invention to A549 is 40:1, the killing rate can reach more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0032] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0033] Figure 1 A graph of CIK cell counts in a control group and an induced group provided in Experimental Example 1 of the present invention; wherein A-control group; B-induced group;

[0034] Figure 2 The CIK cell flow cytometry test of the control group and the induced group provided in Experimental Example 1 of the present invention; wherein A-control group; B-induced group;

[0035] Figure 3 The CIK cell killing test of the control group and the induced group provided in Experimental Example 1 of the present invention; wherein A-control group; B-induced group. DETAILED DESCRIPTION

[0036] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] This embodiment provides a method for cultivating CIK cells to improve their ability to kill tumor cells:

[0039] 1. Draw 50 ml of peripheral blood (donated) and dilute it with sterile PBS (volume dilution ratio is 1:1), add 20 ml of human lymphocyte separation solution (commercially available) to each 50 ml centrifuge tube, slowly add 50 ml of diluted peripheral blood along the tube wall to avoid destroying the aqueous phase of the separation solution, and centrifuge at 3000 rpm for 30 minutes; after centrifugation, carefully aspirate the upper plasma into another 50 ml centrifuge tube and inactivate it (bath in a 56°C water bath for 20 minutes. The purpose of extinguishing the fire is to remove heat-sensitive substances such as complement in the serum and to inactivate mycoplasma that may exist in the serum) for standby use, then aspirate the light yellow mononuclear cell layer obtained after centrifugation into a 15 ml centrifuge tube, centrifuge at 2000 rpm for 5 minutes, wash with sterile PBS (commercially available) after the end, and centrifuge 3 times in total. The precipitate is the peripheral blood mononuclear cells.

[0040] 2. Resuspend the mononuclear cells in 40 ml of common CIK culture medium RPMI-1640 (commercially available), add inactivated autologous plasma and recombinant human IL-2 (commercially available) and induction drugs (induction drugs are: vitamin C 0.01 μL / ml, basic fibroblast growth factor (BFGF) 0.3 μL / ml, hepatocyte growth factor (HGF) 0.2 μL / ml, glutathione 0.01 μL / ml, luteolin 0.4 μL / ml, interleukin-9 (IL-9) 0.2 μL / ml, interleukin-13 (IL-13) 0.2 μL / ml), mix well and add to a 75 cm^2 culture flask, place at 37°C, 5% (v / v) CO2 Culture the cells statically in a cell culture incubator.

[0041] 3. On the third day, CIK culture medium (to supplement the nutrients required for CIK cell culture) and recombinant human IL-2 (mainly to activate CIK cells and enhance the therapeutic effect of CIK cells) were added, and the culture medium in the culture bottle was evenly distributed to two large culture bags for continued culture. 100 ml of CIK culture medium and recombinant human IL-2 were added to each large culture bag every 3-4 days. The total culture period was about 14 days. All operations were carried out in a standard GMP laboratory, and quality monitoring was carried out, including bacterial and fungal contamination detection, endotoxin detection, cell viability, cell flow cytometry, cell killing detection, etc.

[0042] Comparative Example

[0043] Control group: CIK cells were cultured using the same culture method but without inducing drugs. Cell viability, cell flow cytometry, and cell killing were also tested.

[0044] Example 2

[0045] This example provides a method for cultivating CIK cells to improve their ability to kill tumor cells: except for the different amounts of inducing drugs used, everything else is completely the same as that of Example 1.

[0046] Induction drugs: The induction drugs include vitamin C 0.05 μL / ml, basic fibroblast growth factor 0.5 μL / ml, hepatocyte growth factor 0.4 μL / ml, glutathione 0.05 μL / ml, luteolin 0.8 μL / ml, interleukin-9 0.4 μL / ml and interleukin-13 0.6 μL / ml.

[0047] Example 3

[0048] This example provides a method for cultivating CIK cells to improve their ability to kill tumor cells: except for the different amounts of inducing drugs used, everything else is completely the same as that of Example 1.

[0049] Induction drugs: The induction drugs include vitamin C 0.1 μL / ml, basic fibroblast growth factor 1.0 μL / ml, hepatocyte growth factor 0.8 μL / ml, glutathione 0.1 μL / ml, luteolin 1.0 μL / ml, interleukin-9 0.8 μL / ml and interleukin-13 0.8 μL / ml.

[0050] Experimental example

[0051] The CIK cells of Example 1 and the comparative example were tested after being cultured for 14 days:

[0052] 1. CIK cell bacteria and fungi detection: negative.

[0053] 2. CIK cell endotoxin detection: <0.05EU.

[0054] 3. CIK cell control group and induced group viability test (such as Figure 1 shown):

[0055] Control group: total cell concentration: 0.91×10^6 / ml, live cell concentration: 0.82×10^6 / ml, cell viability: 89.69%.

[0056] Induction group: total cell concentration: 1.47×10^6 / ml, live cell concentration: 1.44×10^6 / ml, cell viability: 98.61%.

[0057] The results showed that the induced group was higher than the control group.

[0058] 4. Flow cytometry of CIK control group and induced group, the main detection items are CD16 FITC / CD3 PercP / CD56APC / CD8 PE / CD45 V500 (such as Figure 2 shown);

[0059] The test results of the control group:

[0060] CD3+ / CD56-T cells occupy 53.75% of nucleated cells.

[0061] CD3- / CD56+CIK cells occupy 13.01% of nucleated cells.

[0062] CD3- / CD16+CD56+CIK cells occupy 8.31% of nucleated cells.

[0063] CD3- / CD16-CD56+CIK cells occupy 4.63% of nucleated cells.

[0064] CD3+ / CD56+CIKT cells occupy 28.41% of nucleated cells.

[0065] CD3+ / CD8+T cells occupy 16.60% of nucleated cells.

[0066] CD3+ / CD8- T cells occupy 38.15% of nuclear cells.

[0067] Results of the induction group test:

[0068] CD3+CD56-T occupies nuclear cells: 13.55%,

[0069] CD3- / CD56+CIK occupies nuclear cells: 85.12%,

[0070] CD3- / CD16+CD56+CIK occupies nuclear cells: 78.62%,

[0071] CD3- / CD16-CD56+CIK occupies nuclear cells: 3.13%,

[0072] CD3+ / CD56+CIKT occupies nuclear cells: 2.52%,

[0073] CD3+ / CD8+T occupies nuclear cells: 6.40%,

[0074] CD3+ / CD8-T occupancy of nuclear cells: 5.98%.

[0075] From the flow cytometry results, the positive rate after induction was significantly higher than that in the control group.

[0076] 5. Cytotoxicity test of CIK cell control group and induced group

[0077] CIK cells (density of 1×10^6 cells / ml) and target cells K562 cells (density of 1×10^5 cells / ml) were mixed at a ratio of 40:1 and cultured in a 96-well plate with a total volume of 2 ml per well. Effector cell wells, target cell wells, and blank control wells were set up at the same time. The experimental wells were CIK cell wells and placed at 37°C and 5% (v / v) CO 2 After culturing in the cell culture incubator for 24 hours, add 20 μl of CCK-8 reagent, incubate in the cell culture incubator for 4 hours, and then measure the absorbance (A) value at 450 nm.

[0078] The killing activity of CIK cells was calculated according to the formula: killing rate = [1-9 (A value of experimental well - A value of effector cell well) / A value of target cell well] × 100%.

[0079] Specific release Figure 3 As shown, the killing rate of the control group was 62.43%, and that of the induced group was 98.54%; the experiment showed that the killing rate of the obtained CIK cells against A549 reached more than 90% when the effector-target ratio was 40:1, which was higher than that of the control group.

[0080] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A culture medium for improving the ability of CIK cells to kill tumor cells, characterized in that: The culture fluid comprises a CIK basic culture fluid and an induction drug, wherein the induction drug comprises vitamin C, basic fibroblast growth factor, hepatocyte growth factor, glutathione, luteolin, interleukin-9 and interleukin-13; wherein the CIK basic culture fluid is CIK culture fluid RPMI-1640; and the induction drug consists of 0.01-0.1 μL / ml of vitamin C, 0.3-1.0 μL / ml of basic fibroblast growth factor, 0.2-0.8 μL / ml of hepatocyte growth factor, 0.01-0.1 μL / ml of glutathione, 0.4-1.0 μL / ml of luteolin, 0.2-0.8 μL / ml of interleukin-9 and 0.2-0.8 μL / ml of interleukin-13.

2. The culture solution for improving the ability of CIK cells to kill tumor cells according to claim 1, characterized in that: The induction drugs include vitamin C 0.01 μL / ml, basic fibroblast growth factor 0.3 μL / ml, hepatocyte growth factor 0.2 μL / ml, glutathione 0.01 μL / ml, luteolin 0.4 μL / ml, interleukin-9 0.2 μL / ml and interleukin-13 0.2 μL / ml.

3. A method for improving the ability of CIK cells to kill tumor cells, characterized in that: CIK cells are induced and cultured using the CIK basal culture fluid of claim 1 or 2; the CIK basal culture fluid is RPMI-1640; the induction drugs for the induction culture include vitamin C 0.01-0.1 μL / ml, basic fibroblast growth factor 0.3-1.0 μL / ml, hepatocyte growth factor 0.2-0.8 μL / ml, glutathione 0.01-0.1 μL / ml, luteolin 0.4-1.0 μL / ml, interleukin-9 0.2-0.8 μL / ml and interleukin-13 0.2-0.8 μL / ml.

4. A method for improving the ability of CIK cells to kill tumor cells according to claim 3, characterized in that: The method comprises: resuspending peripheral blood mononuclear cells with CIK basic culture fluid, adding inactivated autologous plasma and recombinant human IL-2 and induction drugs into a culture bottle, placing the bottle in an incubator for culture, and subsequently regularly supplementing CIK basic culture fluid and recombinant human IL-2. After the culture is completed, the CIK cells obtained can improve their ability to kill tumor cells.

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