A method for preparing NK cells with stable process and enhanced specific killing function and its application

Abstract

The present invention belongs to the field of cell culture technology, and specifically relates to a method for preparing NK cells with stable process and enhanced specific killing function and its application. The present invention uses transferrin, IL2, IL15, and Lymactin-NK antibodies to synergistically activate NK cells to achieve efficient in vitro amplification, wherein the Lymactin-NK antibodies do not need to be pre-coated, and the operation is simple. The positive rate of NK cells obtained by culturing different samples through the present invention is consistent, and the process stability is good. The NK cells obtained by culturing through the above-mentioned technical scheme have better specific killing effect on breast cancer. The present invention has simple operating steps, strong operability, and is easy to promote. At the same time, the effect is excellent and has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and specifically relates to a method for preparing NK cells with stable process and enhanced specific killing function and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Natural Killer Cell (NK) cells are a type of natural cytotoxic lymphocytes that can kill tumor cells through a variety of mechanisms without prior sensitization. NK cells do not have TCR, will not attack the host when used as allogeneic cells, and will hardly cause graft-versus-host disease. Multiple clinical results have shown that NK cell immunotherapy has good safety and tolerability in the treatment of hematological tumors and solid tumors. These advantages make NK cells have the potential to become a candidate product for universal cell therapy.

[0004] In the prior art, there are reports on the use of CD16 monoclonal antibodies, HER2 monoclonal antibodies, different cytokines and other methods to expand NK cells in vitro. However, due to differences between different samples, the positive rate of NK cells obtained by culture fluctuates widely, the process stability is poor, and the killing effect is reduced. Therefore, it is urgent to obtain a preparation method with stable process, improve the killing effect, and enhance the effectiveness of NK cell therapy. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for preparing NK cells with stable process and enhanced specific killing function and its application. Specifically, the present invention effectively realizes the efficient in vitro amplification of NK cells by optimizing the culture process, and the process is stable, and the cultured NK cells have better specific killing effect on breast cancer. Based on the above research results, the present invention is completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing NK cells with stable process and enhanced specific killing function, the preparation method comprising:

[0008] Peripheral blood mononuclear cells are added to an activation medium, and then IL15 and Lymactin-NK antibodies are added thereto, and autologous plasma is added thereto for expansion culture for 14 to 21 days; wherein transferrin and IL2 are added to the activation medium.

[0009] During the culture process, rehydration treatment was performed every 2 to 4 days. Specifically, the cell density was adjusted to 0.8 to 2.0 × 10 6 / mL, and supplemented with 5% to 10% autologous plasma.

[0010] In the present invention, NK cells can be harvested after culturing for 14 to 21 days.

[0011] The second aspect of the present invention provides the application of the above preparation method in any one or more of the following:

[0012] a1) Obtaining natural killer cells;

[0013] a2) Tumor immunotherapy or preparation of tumor immunotherapy drugs.

[0014] Beneficial technical effects of one or more of the above technical solutions:

[0015] The above technical solution uses transferrin, IL2, IL15, and Lymactin-NK antibody to synergistically activate NK cells to achieve efficient in vitro amplification, wherein the Lymactin-NK antibody does not need to be pre-coated and is easy to operate. The positive rate of NK cells obtained by culturing different samples through the present invention is consistent, and the process stability is good. The NK cells obtained by culturing through the above technical solution have better specific killing effect on breast cancer.

[0016] The above technical solution has simple operation steps, strong operability, is easy to promote, and has excellent effects and good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 These are the flow cytometry results of NK cells obtained by culturing in Examples 1-3 of the present invention and the comparative example.

[0019] Figure 2 It is a bar graph of the test results of NK cells obtained by culturing sample 1 of the present invention killing A549 (human non-small cell lung cancer cell line), Hela (human cervical cancer cell line), and SKBR3 (human breast cancer cell line) in vitro.

[0020] Figure 3 It is a bar graph of the test results of NK cells obtained by culturing sample 2 of the present invention killing A549 (human non-small cell lung cancer cell line), Hela (human cervical cancer cell line), and SKBR3 (human breast cancer cell line) in vitro.

[0021] Figure 4 It is a bar graph of the test results of NK cells obtained by culturing sample 3 of the present invention killing A549 (human non-small cell lung cancer cell line), Hela (human cervical cancer cell line), and SKBR3 (human breast cancer cell line) in vitro. DETAILED DESCRIPTION

[0022] The present invention provides a method for preparing NK cells with stable process and enhanced specific killing function. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0023] 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. If the experimental method of specific conditions is not specified in the following specific implementation methods, it is usually based on the conventional methods and conditions in the art, and such techniques and conditions are fully explained in the literature.

[0024] Term explanation: Natural Killer Cells (NK cells) are an important part of the immune system. They belong to the large granular lymphocyte subset and originate from the bone marrow. They are important cells in the body's immune defense response and immune surveillance. NK cells play a pivotal role in immune response, especially in identifying and clearing tumors and virus-infected cells.

[0025] NK cells have non-specific cytotoxic effects. They can spontaneously dissolve a variety of tumor cells and virus-infected cells without relying on antigen stimulation. This effect is mainly achieved by secreting perforin and tumor necrosis factor, which can destroy target cells. NK cells can also bind to the receptors of target cells, causing them to undergo apoptosis and promoting the production of new cells, thereby balancing the functions of organs and the immune system.

[0026] Peripheral Blood Mononuclear Cells (PBMCs) refer to a group of cells with a single nucleus separated from peripheral blood, mainly including lymphocytes (T cells, B cells and NK cells) and monocytes. These cells play a vital role in the human immune system and participate in a variety of biological processes such as immune response, immune regulation and inflammatory response.

[0027] PBMCs have a wide range of application value in medical research and clinical applications. By analyzing peripheral blood mononuclear cells, we can understand the state and function of the immune system, thereby diagnosing diseases, evaluating conditions and predicting disease progression. In addition, PBMCs can also be used as a source of cell therapy for the preparation of immune cell therapy, stem cell therapy, etc., providing new ideas and methods for the treatment of various diseases such as tumors and autoimmune diseases.

[0028] As mentioned above, there are reports in the prior art of using CD16 monoclonal antibodies, HER2 monoclonal antibodies, different cytokines and other methods to expand NK cells in vitro. However, due to differences between different samples, the positive rate of NK cells obtained by culture fluctuates widely, the process stability is poor, and the killing effect is reduced.

[0029] In view of this, in a typical embodiment of the present invention, a method for preparing NK cells with stable process and enhanced specific killing function is provided, and the preparation method comprises:

[0030] Peripheral blood mononuclear cells are added to an activation medium, and then IL15 and Lymactin-NK antibodies are added thereto, and autologous plasma is added thereto for expansion culture for 14 to 21 days; wherein transferrin and IL2 are added to the activation medium.

[0031] In another specific embodiment of the present invention, the activation medium is a serum-free medium, the final concentration of transferrin in the activation medium is 10-100 ug / mL, and the final concentration of IL2 is 100-1000 IU / mL.

[0032] Wherein, the peripheral blood mononuclear cells are of human origin, and after adding the activation medium, the cell density of the peripheral blood mononuclear cells is controlled to be 1 to 3×10 6 / mL; and the final concentration of IL15 is controlled at 10-100 ng / ml, and the final concentration of Lymactin-NK antibody is controlled at 0.1-2.0 mg / ml.

[0033] The present invention has found through research that transferrin is an important carrier of iron ions. When added to serum-free culture medium, it can effectively transfer iron and promote the growth of NK cells.

[0034] IL2 can react with IL-2R on the surface of NK cells. IL15 has a similar structure to IL2 and transmits signals through a complex composed of IL-2 / IL-15 receptor β chain (CD122) and universal γ chain (gamma-c, cd132), synergistically regulating the activation and proliferation of NK cells.

[0035] Lymactin-NK antibody is a humanized monoclonal antibody derived from recombinant DNA. NKG2D activating receptors are expressed on the surface of NK cells. Lymactin-NK antibody binds to NKG2D activating receptors, specifically activates NK cell function, and enhances cell killing activity. When the preparation method of the present invention is used, no pre-coating is required, and the operation is simple.

[0036] The volume fraction of the autologous plasma supplemented is controlled to be 5-10%.

[0037] Furthermore, during the culture process, the cell density was adjusted to 0.8-2.0×10 6 / mL, and supplemented with 5% to 10% autologous plasma.

[0038] Furthermore, the specific method of the fluid replenishment treatment includes:

[0039] During the first 7 days of the culture process, the cell density was adjusted to 0.8-2.0×10 cells / mL using an activated medium. 6 / mL, and add IL15 to control the final concentration of IL15 to 10-100 ng / mL; and add 5%-10% volume fraction of autologous plasma;

[0040] During the 8th to 21st day of the culture process, the cell density was adjusted to 0.8-2.0×10 using activation medium every 2-4 days. 6 / mL.

[0041] In the present invention, NK cells can be harvested after culturing for 14 to 21 days.

[0042] In another specific embodiment of the present invention, the application of the above preparation method in any one or more of the following is provided:

[0043] a1) Obtaining natural killer cells;

[0044] a2) Tumor immunotherapy or preparation of tumor immunotherapy drugs.

[0045] The tumor may be non-small cell lung cancer, cervical cancer and breast cancer, more preferably breast cancer.

[0046] It should be noted that the natural killer cells obtained by culturing using the first aspect have better specific killing activity, good stability between different samples, high and relatively consistent positive rate, which is more conducive to their application in clinical treatment.

[0047] The present invention is further explained by the following examples, but it does not limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Samples 1-3 in the examples are derived from different individuals.

[0048] Example 1: Sample 1 NK cell preparation

[0049] (1) Preparation of activation medium: Add transferrin and IL2 to GT-T551 H3 serum-free medium to make the final concentration of transferrin 12.5 ug / mL and the final concentration of IL2 200 IU / mL.

[0050] (2) Day 0 inoculation: Peripheral blood mononuclear cells from sample 1 were added to a certain volume of the above activation medium at a density of 2.0×10 6 / mL. Then add IL15 and Lymactin-NK antibody. Make the final concentration of IL15 50ng / ml and Lymactin-NK antibody 0.25mg / ml. Supplement with 10% volume fraction of autologous plasma. Mix well and culture at 37℃, 5% CO2.

[0051] (3) Fluid replenishment: On the 3rd, 5th, and 7th days of culture, the cell density was adjusted to 1.0 × 10 6 / mL, and supplemented with IL15, with a final concentration of 50ng / mL. And supplemented with 5% volume fraction of autologous plasma. On the 9th and 11th days of the culture process, the cell density was adjusted to 1.0×10 6 / mL.

[0052] (4) Cell harvesting: NK cells were harvested on day 14 of culture and analyzed by flow cytometry.

[0053] The experimental results showed that the NK cell positive rate was 95.14%.

[0054] Example 2: Sample 2 NK cell preparation

[0055] (1) Preparation of activation medium: Add transferrin and IL2 to GT-T551 H3 serum-free medium to make the final concentration of transferrin 12.5 ug / mL and the final concentration of IL2 200 IU / mL.

[0056] (2) Day 0 inoculation: Peripheral blood mononuclear cells from sample 2 were added to a certain volume of the above activation medium at a density of 2.0×10 6 / mL. Then add IL15 and Lymactin-NK antibody. Make the final concentration of IL15 50ng / ml and Lymactin-NK antibody 0.25mg / ml. Supplement with 10% volume fraction of autologous plasma. Mix well and culture at 37℃, 5% CO2.

[0057] (3) Fluid replenishment: On the 3rd, 5th, and 7th days of culture, the cell density was adjusted to 1.0 × 10 6 / mL, and supplemented with IL15, with a final concentration of 50ng / mL. And supplemented with 5% volume fraction of autologous plasma. On the 9th and 11th days of the culture process, the cell density was adjusted to 1.0×10 6 / mL.

[0058] (4) Cell harvesting: NK cells were harvested on day 14 of culture and analyzed by flow cytometry.

[0059] The experimental results showed that the NK cell positive rate was 94.15%.

[0060] Example 3: Sample 3 NK cell preparation

[0061] (1) Preparation of activation medium: Add transferrin and IL2 to GT-T551 H3 serum-free medium to make the final concentration of transferrin 12.5 ug / mL and the final concentration of IL2 200 IU / mL.

[0062] (2) Day 0 inoculation: Peripheral blood mononuclear cells from sample 3 were added to a certain volume of the above activation medium at a density of 2.0×10 6 / mL. Then add IL15 and Lymactin-NK antibody. Make the final concentration of IL15 50ng / ml and Lymactin-NK antibody 0.25mg / ml. Supplement with 10% volume fraction of autologous plasma. Mix well and culture at 37℃, 5% CO2.

[0063] (3) Fluid replenishment: On the 3rd, 5th, and 7th days of culture, the cell density was adjusted to 1.0 × 10 6 / mL, and supplemented with IL15, with a final concentration of 50ng / mL. And supplemented with 5% volume fraction of autologous plasma. On the 9th and 11th days of the culture process, the cell density was adjusted to 1.0×10 6 / mL.

[0064] (4) Cell harvesting: NK cells were harvested on day 14 of culture and analyzed by flow cytometry.

[0065] The experimental results showed that the NK cell positive rate was 97.02%.

[0066] Comparative Example

[0067] The commercially available NK cell expansion product was used to culture Sample 1, Sample 2, and Sample 3 according to the instructions, and the cells were harvested and subjected to flow cytometry testing.

[0068] The experimental results showed that the NK cell positivity rate of sample 1 was 81.74%, the NK cell positivity rate of sample 2 was 91.58%, and the NK cell positivity rate of sample 3 was 74.06%, with large fluctuations between samples.

[0069] Effect verification

[0070] NK cells kill A549 (human non-small cell lung cancer cell line), Hela (human cervical cancer cell line), and SKBR3 (human breast cancer cell line) in vitro, as follows:

[0071] The NK cells obtained by culture in Example 1, Example 2, Example 3 and the comparative example were used as effector cells, and A549, Hela and SKBR3 were used as target cells respectively. Under the condition of effector-target ratio of 10:1, the operation was performed according to the instructions of the lactate dehydrogenase cytotoxicity detection kit to calculate the killing capacity of NK cells.

[0072] The experimental results show that the NK cells obtained by culturing samples 1, 2 and 3 kill SKBR3 cells in vitro by more than 80%, which is significantly higher than A549 cells and Hela cells. The killing effect of the present invention on SKBR3 cells is significantly higher than that of commercially available products.

[0073] The NK cells obtained by the culture of the present invention have good stability among different samples and have a stronger specific killing effect on breast cancer.

[0074] Table 1 Specific data of killing test of sample 1

[0075] / The present invention Commercially available products A549 42.54％ 39.44％ Hela 50.78％ 44.31％ SKBR3 84.29％ 51.45％

[0076] Table 2 Specific data of killing test of sample 2

[0077] / The present invention Commercially available products A549 44.51％ 40.06％ Hela 52.78％ 42.31％ SKBR3 82.84％ 52.95％

[0078] Table 3 Specific data of killing test of sample 3

[0079]

[0080]

[0081] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing NK cells with stable process and enhanced specific killing function is used in the following applications: a1) Obtaining natural killer cells; a2) Preparation of breast cancer immunotherapy drugs; The NK cell preparation method comprises the following steps: (1) Preparation of activation medium: Add transferrin and IL2 to serum-free medium to make the final concentration of transferrin 12.5ug / mL and the final concentration of IL2 200 IU / mL; (2) Day 0 inoculation: peripheral blood mononuclear cells were added to the activation medium at a density of 1-3×10 6 / mL, then add IL15 and Lymactin-NK antibody to make the final concentration of IL15 50ng / ml and Lymactin-NK antibody 0.25mg / ml, and add 5-10% volume fraction of autologous plasma for expansion culture; (3) Fluid replenishment: On the 3rd, 5th, and 7th days of culture, the activation medium was used to adjust the cell density to 0.8-2.0×10 6 / mL, supplemented with IL15 with a final concentration of 50ng / mL, and supplemented with autologous plasma with a volume fraction of 5-10%. On the 9th and 11th days of the culture process, the activation medium was used to adjust the cell density to 0.8-2.0×10 6 Pieces / mL; (4) Cell harvesting: After culturing for 14 days, NK cells were harvested.

2. The use according to claim 1, characterized in that The peripheral blood mononuclear cells are human peripheral blood mononuclear cells.

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