A Nonviral piggyBac Transposon-Mediated Method to Generate Large-Scale CAR-NK Cells from Human Peripheral Blood Primary NK Cells

With the inherent antitumor function and unique “off-the-shelf” potential, genetically engineered human natural killer (NK) cells with chimeric antigen receptors (CARs) bear great promise for the treatment of multiple hematological malignancies and solid tumors. Current methods of producing large-scale CAR-NK cells mainly rely on mRNA transfection and viral vector transduction. However, mRNA CAR-NK cells were not stable in CAR expression while viral vector transduction mostly ended up with low efficiency. In this chapter, we described an optimized protocol to generate CAR-NK cells by using the piggyBac transposon system via electroporation and to further expand these engineered CAR-NK cells in a large scale together with artificial antigen-presenting feeder cells. This method can stably engineer human primary NK cells with high efficiency and supply sufficient scale of engineered CAR-NK cells for the future possible clinical applications.