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Artificial antigen presenting cell as well as preparation method and application thereof

A technology of artificial antigens and cells, applied in the fields of immunology and biochemistry, which can solve the problems of insufficient activity and short lifespan of NK cells

Active Publication Date: 2020-04-07
杭州中赢生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the above methods still have problems such as short lifespan and insufficient activity of the expanded NK cells.

Method used

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  • Artificial antigen presenting cell as well as preparation method and application thereof
  • Artificial antigen presenting cell as well as preparation method and application thereof
  • Artificial antigen presenting cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1 Preparation of Artificial Antigen Presenting Cells

[0105] (1) Artificially synthesized nucleic acid sequences of MICA, IL-12A, IL-12B, CD80 and CD4 transmembrane region SEQ ID NO: 6-10, cloned into a transposon with an antibiotic marker (puromycin resistance gene) In the vector, a transposon expression vector is constructed;

[0106] (2) Co-transfect K562 cells with the expression vector obtained in step (1) and the plasmid with transposase, add antibiotics for drug treatment, and obtain positive transfected cells;

[0107] (3) adding antibiotics to the positive transfected cells obtained in step (2) for drug treatment to obtain high-purity transfected K562 cells;

[0108] (4) Adding drugs and sorting twice by flow cytometry to obtain 100% positively transfected K562 cells. The positive cells have been stably passaged for more than 10 generations, and the genomes integrated with exogenous genes MICA, IL-12A, Stably transfected cell lines of IL-12B, CD80 an...

Embodiment 2

[0110] Example 2 Expression of MICA, IL-12A, IL-12B and CD80 by artificial antigen-presenting cells

[0111] The artificial antigen-presenting cells prepared in Example 1 were detected by flow cytometry, and the flow cytometry results are shown in Figure 2 (A), Figure 2 (B), Figure 2 (C) and Figure 2 (D), illustrating MICA, IL-12A, IL-12B and CD80 were successfully expressed on artificial antigen-presenting cells.

Embodiment 3

[0112] Example 3 Expansion Experiment of Artificial Antigen Presenting Cells to NK Cells

[0113] Peripheral blood mononuclear cells (PBMC) donated by healthy volunteers were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and the cells were counted after 24 hours of culture, and the cell concentration was adjusted to 2×10 6 cells / well, add an equal proportion of artificial antigen-presenting cells to the culture medium on day 0, day 7, and day 14, and add IL-2 at a concentration of 50 U / mL, at 37°C, 5% CO 2 Co-cultivate in an incubator under 500Gy irradiation conditions, and set up a control group (artificial antigen-presenting cells only express MICA).

[0114] Such as image 3 As shown, the number of NK cells increased significantly under the combined action of K562 artificial antigen-presenting cells expressing MICA, IL-12 and CD80 (MICA-IL12-CD80) and IL-2, and the number of cells increased by about 21 days 1100 times, significantly higher than the contr...

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Abstract

The invention provides an artificial antigen presenting cell as well as a preparation method and an application thereof. Wherein the artificial antigen presenting cell expresses membrane proteins MICA, IL-12 and CD80, the artificial antigen presenting cell is used for culturing NK cells or peripheral blood mononuclear cells containing the NK cells, the number of the NK cells can be remarkably increased, the service life of the NK cells is prolonged, the activity of the NK cells is improved, and compared with an existing artificial antigen presenting cell for expressing MICA, the artificial antigen presenting cell has a stronger effect of amplifying and activating the NK cells.

Description

technical field [0001] The invention belongs to the technical field of immunology and biochemistry, and relates to an artificial antigen-presenting cell and its preparation method and application, in particular to an artificial antigen-presenting cell, its preparation method and its application in culturing NK cells. Background technique [0002] NK cells (natural killer cells, natural killer cells), also known as large granular lymphocytes, play innate and acquired immune functions together with γδT cells and NKT cells. On the one hand, when the body is infected or traumatized, NK cells, as defenders, quickly, extensively and specifically recognize antigens in a non-peptide-MHC way, clear pathogenic microorganisms and mutated cells in time, and play an inherent immune role; on the other hand, NK cells participate in the adaptive immune response, affecting the effector functions of αβ T cells and B cells. [0003] During the occurrence and development of tumors, NK cells ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783C12N15/85C12N5/0786A61K35/17A61P35/00
CPCC07K14/5434C07K14/70532C07K14/47C12N5/0646C12N5/0645C12N15/85A61K35/17A61P35/00
Inventor 王冶陶吴忠福彭昉俞英豪
Owner 杭州中赢生物医疗科技有限公司
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