Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation

A technology of genetic engineering and NK cells, which is applied in the field of in vitro preparation of NK cells in NK cell immunotherapy, can solve the problems of NK cell proliferation limitation, etc., and achieve the effect of long duration of expansion, simple amplification method and high purity

Inactive Publication Date: 2013-02-06
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In previous studies, researchers used CD64 and CD86 to modify K562 cells to construct artificial antigen presenting cells (Antigen present cell, aAPC), and then 4-1BBL (CD137L) and membrane-fixed IL15 (membrane-bound IL-15, mIL -15) etc. into the cells, construct CD64-CD86-4-1BBL-mIL15-aAPC, and use the artificial antigen-presenting cells to expand NK cells in vitro. This method can obtain NK cells with strong cytotoxicity, However, NK cell proliferation is limited, and the cells no longer expand after 5-6 weeks

Method used

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  • Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation
  • Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation
  • Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation method of genetic engineering cell 4-1BBL-mbIL-21-K562

[0035] 1. Purchase the 4-1BBL / PCR4 TOPO plasmid from Open Biosysems (Thermo Fisher Scientific, CO, USA), amplify it by PCR, and insert it into the Nhe I-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct 4-1BBL / pSBSO transposon.

[0036] 2. Co-transfect K562 cells with 4-1BBL / pSBSO transposon and transposase SB11, and sort by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).

[0037] 3. IL-21-Fc(CoOp)-pSBSO and transposase SB11 were co-transfected into 4-1BBL-K562 cells and sorted by flow cytometry to establish 4-1BBL-mbIL-21-K562 genetically engineered cells. Flow cytometry analysis confirmed that 4-1BBL-mbIL-21-K562 cell surface had obvious expression of 4-1BBL and IL-21 ( figure 2 ).

Embodiment 2

[0038] Embodiment 2, NK cell expansion method

[0039] 1. Purchase the 4-1BBL / PCR4 TOPO plasmid from Open Biosysems (Thermo Fisher Scientific, CO, USA), amplify it by PCR, and insert it into the Nhe I-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct 4-1BBL / pSBSO transposon.

[0040] 2. Co-transfect K562 cells with 4-1BBL / pSBSO transposon and transposase SB11, and sort by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).

[0041] 3. IL-21-Fc(CoOp)-pSBSO and transposase SB11 were co-transfected into 4-1BBL-K562 cells and sorted by flow cytometry to establish 4-1BBL-mbIL-21-K562 genetically engineered cells.

[0042] 4. Separate human peripheral blood mononuclear cells with lymphocyte separation medium.

[0043]5. The genetically engineered cells were irradiated with 100Gy radiation, or treated with 15ug / mL mitomycin for 4 hours; after that, the cells were washed 2~3 times with PBS.

[0044] 6. Pretreated genetic...

Embodiment 3

[0047] Example 3, NK cell expansion mechanism research

[0048] 1. Purchase the 4-1BBL / PCR4 TOPO plasmid from Open Biosysems (Thermo Fisher Scientific, CO, USA), amplify it by PCR, and insert it into the Nhe I-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct 4-1BBL / pSBSO transposon.

[0049] 2. Co-transfect K562 cells with 4-1BBL / pSBSO transposon and transposase SB11, and sort by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).

[0050] 3. IL-21-Fc(CoOp)-pSBSO and transposase SB11 were co-transfected into 4-1BBL-K562 cells and sorted by flow cytometry to establish 4-1BBL-mbIL-21-K562 genetically engineered cells.

[0051] 4. Separate human peripheral blood mononuclear cells with lymphocyte separation medium.

[0052] 5. The genetically engineered cells were irradiated with 100Gy radiation, or treated with 15μg / mL mitomycin for 4 hours; after that, the cells were washed 2~3 times with PBS.

[0053] 6. Pretreat...

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PUM

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Abstract

The invention discloses a gene engineering cell and application thereof in NK (Nature Killer) cell proliferation, and provides an in vitro proliferation method of an NK cell with high efficiency and high cytotoxicity. A 4-1BB ligand (4-1BBL) and a membrane fixed interleukin 21 (mbIl-21) are stably expressed on the surface of a cell membrane by adopting a genetic engineering technology for constructing 4-1BBL-mbIl-21-gene engineering cells (4-1BBL-mbIl-21-Gene Engineering Cells, 4-1BBL-mbIl-21-GEC), such as 4-1BBL-mbIl-21-K562 cells, to be used as feeder cells for proliferation, and the NK cells are directly proliferated from peripheral blood monouclear cells, thus the operation is simpler and easier. Studies such as cell counting, flow cytometry and cytotoxicity tests indicate that high cell proliferation times, high NK cell purity, strong cytotoxicity and remarkable cancer cell killer effect are achieved.

Description

technical field [0001] The invention relates to disease immunotherapy, in particular to the in vitro preparation of NK cells in NK cell immunotherapy. Background technique [0002] NK cells (natural killer cells, natural killer cells) are a type of large granular lymphocytes, which are an important bridge between the body's innate immunity and adaptive immunity, and play an important role in the body's immune surveillance against infectious diseases and malignant tumors. Secondary NK cell transplantation can produce a good anti-tumor effect, but itself is rarely rejected by the recipient, and will not cause any GVHD (Graft Versus Host Disease). Therefore, secondary NK cell therapy (Adoptive NK cell therapy) has become one of the most popular and promising methods for the treatment of malignant tumors. [0003] Although secondary NK cell immunotherapy has good anti-tumor effects and great application prospects, NK cell immunotherapy is still difficult for routine clinical ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783
Inventor 朱诗国王艳丽
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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