An Improved Method Of Human NK Cell Expansion In Vitro And Preliminary Study Of CAR-NK Cell Immunotherapy Technology

Natural killer(NK) cell adoptive transfer is a promising approach for cancer immunotherapy; however, its development has been hindered by the lack of efficient methods to produce large numbers of functional NK cells. Interleukin(IL)-21 is an important modulator of natural killer(NK) cell function. This study is to establish an improved approach for human NK cell ex vivo expansion. Accordingly, we engineered the cell line p APC-mb IL21 based on p APC which was constructed by Persongen previously with co-expressing IL15 and CD137 L, to establish membrane-bound interleukin(mb IL)-21 expressed on the cell surface, and used these engineered cells(named p APCNK) to expand NK cells from the peripheral blood mononuclear cells. The peripheral blood mononuclear cells(peripheral blood mononuclear cell, PBMC) from four healthy donors were isolated and stimulated with p APCNK and IL2 simultaneously. Proliferated cells were counted and the NK cell phenotype were determined by flow cytometry analysis. Then the cytotoxicity of expanded NK cells to K562 and RPMI-8226 was examined by CFSE/7-AAD assay. After culturing for three weeks, NK cells were significantly amplified and the average multiples were 409.4. The purity of the NK cells(CD3-CD56+) cells increased from less than 30% to above 90%, meanwhile the cytotoxic activity to K562 was reached to 85.2%.NK cell has the non-antigen-specific killing features, which limits its clinical treatment significantly. In recent years, adoptive cell immunotherapy especially the chimeric antigen receptor(CAR) modified T cell technologies have sprung up, which show good targeting, destruction and persistence both in vitro and in clinical trials, displaying great application potential and development prospects. Thus based on NK cell expansion system, additional study was pursed to develop the CAR-NK cell therapy technology to increase NK cell activity and endow NK cell with the tumor antigen specific cytotoxicity. In this study, we chose HER2 as a target to explore the possibility of CAR-NK cell therapy technology, and accumulated a preliminary experimental data which provides a basis for further improvement of the CAR-NK cell therapy.