Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells

V gamma 9V delta 2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of V gamma 9V delta 2 T cells. Furthermore, V gamma 9V delta 2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of V gamma 9V delta 2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove alpha beta T lymphocytes. The method exhibited high expansion folds and a specific enrichment of V gamma 9V delta 2 T cells. Expanded V gamma 9V delta 2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)-engrafted V gamma 9V delta 2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional V gamma 9V delta 2 T cells in massive numbers suitable for clinical application in an allogeneic setting.