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Method for amplifying and activating NK (Natural Killer) cells by K562 cells

A technology of NK cells and cells, applied in the field of immunology, can solve problems such as difficulty in obtaining a large number of NK cells, limited activation and growth multiples, and low killing function of NK cells

Inactive Publication Date: 2012-07-18
杭州中赢方舟生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The number and killing function of NK cells are directly related to the curative effect. The problem in the treatment is that it is very difficult to obtain a large number of NK cells
Traditionally used high-dose interleukin-2 activates limited growth folds, reduces telomerase activity, and lowers the killing function of expanded NK cells

Method used

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  • Method for amplifying and activating NK (Natural Killer) cells by K562 cells
  • Method for amplifying and activating NK (Natural Killer) cells by K562 cells
  • Method for amplifying and activating NK (Natural Killer) cells by K562 cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Expansion of NK cells.

[0039] Take NK cells (2x10 7 ) were cultured in RPMI1640 supplemented with 10% human serum. K562 trophoblast cells transfected with transmembrane interleukin 21, CD14, CD19, CD86 and CD137 (irradiated, 100Gy) and interleukin 2 (50 units / ml) were added to the culture medium and cultured for 7 days. Centrifuge after 7 days, resuspend with an equal amount of culture medium, add irradiated K562 trophoblast cells transfected with transmembrane interleukin 21, CD14, CD19, CD86 and CD137, and culture for another 7 days, as figure 1 shown. High-dose interleukin-2 (200 units / ml) served as the control group. Such as figure 1 As shown, the number of NK cells increased significantly under the combined action of K562 cells transfected with transmembrane interleukin-21, CD14, CD19, CD86 and CD137 and low dose of interleukin-2. An increase in cell number can be measured by the insertion of thymidine. Cells were cultured for 12 hours after thymi...

Embodiment 2

[0040] Example 2 Transmembrane interleukin 21-CD137 complex expansion of unpurified peripheral blood lymphocytes

[0041]K562 trophoblast cells transfected with transmembrane interleukin 21, CD14, CD19, CD86, and CD137 could expand not only purified NK cells, but also unpurified lymphocytes. PBMC from healthy donors were cultured in RPMI1640 supplemented with 10% human serum. K562 trophoblast cells transfected with transmembrane interleukin-21, CD14, CD19, CD86 and CD137 (irradiated, 100Gy) and low-dose interleukin-2 were added to the culture medium and co-cultured for 7 days. Centrifuge after 7 days, resuspend with an equal amount of culture medium, add trophoblast cells irradiated by 100Gy, and culture for another 7 days, as figure 2 shown. K562 trophoblast cells transfected with transmembrane interleukin 21 and CD137 were used as controls. The number of NK cells was significantly increased under the combined action of K562 trophoblasts transfected with transmembrane int...

Embodiment 3

[0043] Lysis of tumor cells and TAMs (tumor-associated macrophages) by expanded NK cells in Example 3

[0044] Lysis of tumor cells by NK cells expanded under the combined action of K562 trophoblast cells transfected with transmembrane interleukin-21, CD14, CD19, CD86 and CD137 and low-dose interleukin-2

[0045] Previous studies have shown that injecting interleukin-2 into mice with tumors can enhance the anti-tumor ability of mice and delay the lifespan of mice. In this example, K562 trophoblast cells transfected with transmembrane interleukin 21, CD14, CD19, CD86 and CD137 and NK cells expanded under the combined action of low-dose interleukin 2 were co-cultured with tumor cells and tumor-associated macrophages. To study the feasibility of using expanded NK cells for anti-tumor. In this example, K562 trophoblast cells transfected with transmembrane interleukin 21, CD14, CD19, CD86 and CD137 and NK cells amplified under the combined action of low-dose interleukin 2 were inj...

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Abstract

The invention belongs to the field of immunology and particularly provides a method for amplifying and activating a natural killer cell (NK) in an oriented way under the mutual effect of a K562 cell, which is transfected by transmembranes IL-21, CD14, CD19, CD86 and CD137, and a low-concentration interleukin 2. According to the invention, the method comprises the following steps: transcribing expression carriers, which are used for stably expressing CD8 alpha-interleukin 21, CD14, CD19, CD86 and CD137, by using K562 cell lines, wherein the CD8 alpha is a membrane protein expressed on a cell membrane, and the interleukin 21 is expressed on the cell membrane to become a transmembrane protein after the CD8 alpha gene is connected with the interleukin 21 gene; and then culturing the K562 cell for a section of time; and finally, obtaining a purified K562 cell by using a physical and chemical method and then culturing the NK cell. According to the invention, by using the amplified and activated NK cell, the immunity of the patient can be enhanced, the viruses and bacteria are resisted, and high efficiency is obtained. The method for amplifying and activating the natural killer cell, provided by the invention, medically has wide purposes.

Description

technical field [0001] The invention belongs to the field of immunology, and specifically refers to a method for using transmembrane IL-21, CD14, CD19, CD86 and CD137 transfected K562 cells and low-concentration interleukin-2 to act together to amplify and activate natural killer cells (NK). technical background [0002] NK cell therapy has a significant effect on the treatment of renal cell carcinoma, melanoma, lung cancer, colon cancer, breast cancer, bladder cancer, liver cancer and acute leukemia. At present, NK cell therapy is mainly used in combination with surgery, chemotherapy and radiotherapy in the United States. The number and killing function of NK cells are directly related to the curative effect. The problem in the treatment is that it is very difficult to obtain a large number of NK cells. Traditionally used high-dose interleukin-2 has limited growth folds, reduced telomerase activity, and reduced killing function of expanded NK cells. The company's previous...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17
CPCY02A50/30
Inventor 吴忠福徐以兵董生聚
Owner 杭州中赢方舟生物工程有限公司
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