A Feeder Cell-Free System for Chimeric Antigen Receptor Gene Transduction Into Natural Killer Cells

Anti-CD19 chimeric antigen receptor (CAR)-natural killer (NK) cells are expected to demonstrate anti-CD19 CAR-T-cell-like efficacy against relapsed and refractory B-cell malignancies and autoimmune diseases, with fewer adverse events and the added advantage of permitting the use of allogeneic cells. However, the methodology for generating CAR-NK cells remains under development. Although various cell sources and expansion methods are available, feeder cells derived from cancerous tissue have been most commonly employed to promote ex vivo expansion of NK cells. In the protocol described herein, NK cells are expanded from adult peripheral blood mononuclear cells using CD2- and NKp46-specific stimulating antibodies in combination with multiple cytokines. The activated NK cells can be genetically modified using a retroviral vector. Subsequent culture of these cells yields large numbers of anti-CD19 CAR-NK cells. The current method, which enables feeder-free, large-scale generation of anti-CD19 CAR-NK cells, eliminates the risk of tumor cell contamination and may facilitate safer clinical application. Key features • This method for expanding human primary NK cells ex vivo uses stimulatory antibodies and multiple cytokines, without requiring feeder cells, usually derived from cancerous tissue. • NK cells are selectively expanded from unsorted peripheral blood mononuclear cells. • Retroviral vector efficiently mediates gene transfer into NK cells stimulated with the current method. • Although the cells were not sorted, gene transfer into T cells is minimal.