Parallel processing of T cells and natural killer cells to enhance in vivoCAR T cell activity against blood and solid cancers.

OBJECTIVES: Despite their anti-cancer properties, natural killer (NK) cells are discarded during CAR T cell manufacturing. NK cells display promising responses as a cell therapy, exhibiting remarkable safety at high doses. Moreover, NK cells have complementary cytokine profiles and distinct antigen-recognition pathways to supplement CAR T cell therapies. We demonstrate parallel expansion of NK cells from the normally discarded PBMC of CAR T cell production for downstream combination therapies against solid and blood-borne malignancies. METHODS: CAR T cells were manufactured from buffy coats with initial isolation of T cells from PBMC. NK cells were rapidly expanded in parallel by stimulating the remaining PBMC with membrane-bound (mb)IL-21 + mbIL-15 feeder cells. Expanded NK cells were assessed through spectral flow cytometry and applied as a pretreatment prior to CAR Tcell infusion against xenograft tumor models in NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Applying feeder cells to residual PBMC stimulated 5000-fold expansions of pure NK cells. Expanded NK cells expressed various activation receptors and displayed strong in vitro cytotoxicity, which was further improved in CAR T-conditioned medium. A pretreatment of NK cells enhanced existing CAR T cell therapies, improving in vivo tumor clearance and survival against breast cancer, acute lymphoblastic leukaemia and non-Hodgkin's lymphoma. While NK cells eliminated CD19-/- variants in vitro, they were unable to suppress antigen-escape relapse in vivo. CONCLUSION: This study encourages the optimal use of leukapheresis product. By recycling discarded PBMC into NK cells, CAR T cell therapies can be effectively enhanced, improving tumor clearance and survival.