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Artificial antigen presenting cell and application thereof in NK (natural killer) cell amplification

An artificial antigen and NK cell technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of NK cell proliferation limitation, high cytotoxicity, etc., and achieve long-lasting expansion, high purity, Amplification method simple effect

Inactive Publication Date: 2012-07-11
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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AI Technical Summary

Problems solved by technology

In previous studies, researchers used genetically modified K562 cells as artificial antigen-presenting cells, such as K562 cells transformed with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15). NK cell expansion, this method can obtain NK cells with high cytotoxicity, but the proliferation of NK cells is limited, and the cells no longer expand after 5-6 weeks

Method used

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  • Artificial antigen presenting cell and application thereof in NK (natural killer) cell amplification
  • Artificial antigen presenting cell and application thereof in NK (natural killer) cell amplification
  • Artificial antigen presenting cell and application thereof in NK (natural killer) cell amplification

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Embodiment 1

[0031] Example 1. Preparation method of artificial antigen-presenting cell 4-1BBL-mIL-21-K562

[0032] 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.

[0033] 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).

[0034] 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-mIL-21-K562 artificial antigen-presenting cells.

Embodiment 2

[0035] Embodiment 2, NK cell expansion method

[0036] 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.

[0037] 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).

[0038] 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-mIL-21-K562 artificial antigen-presenting cells.

[0039] 4. Co-transfect 4-1BBL-K562 cells with IL-15-Fc(CoOp)-pSBSO and transposase SB11, and sort by flow cytometry to establish 4-1BBL-mIL-15-K562 artificial antigen-presenting cells.

[0040] 5. Separation of human peripheral blood lymphocytes with lymphocyte separation medium.

[0041] 6. Artificia...

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Abstract

The invention provides an in vitro amplification method for efficient and highly cytotoxic natural killer (NK) cells. Novel artificial antigen presenting cells 4-1BBL-mIL-21-aAPC, such as 4-1BBL-mIL-21-K562 cells and the like, are constructed through stably expressing 4-1BB ligands (4-1BBL) and membrane immobilized interleukin 21 (mIL-21) on the surfaces of cell membranes, and by using the novel artificial antigen presenting cells as feeder cells for amplification, the NK cells are directly amplified from peripheral blood lymphocytes. Flow cytometry, cytotoxicity test and the like suggest that the amplified cells NK have high purity and strong cytotoxicity and have obvious killing effect on tumor cells.

Description

technical field [0001] The invention relates to the field of disease immunotherapy, in particular to a method for expanding NK cells in vitro 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 initial (innate) immunity and subsequent (adaptive) immunity, and play an important role in the body's immune surveillance against infectious diseases and malignant tumors. important role. 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 hottest and most promising treatments for malignant tumors. [0003] Although secondary NK cell immunotherapy has good anti-tumor effects and great application prospects, NK cell immunothera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/09C12N5/0783
Inventor 朱诗国余健秀王艳丽
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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