Biological Engineering of Natural Killer Cells for Cellular Therapy Against Cancer

Abstract

Despite its success, CAR T cellular therapy is still a last-line cancer treatment due to deleterious side effects and financial costs. Cytokine release syndrome (CRS) and neurotoxicity frequently occur due to extensive cytokine production and immune stimulation by CAR T cells. Furthermore, this therapy requires autologous T cells, resulting in a time-gap between collection and infusion that some patients do not survive. Expanded, unmodified natural killer (NK) cells have been shown to exert anti-tumor activity in patients, even in an allogeneic setting, without inducing CRS, GvHD, or neurotoxicity. We believe NK cells are poised to overcome the limitations of CAR T cells, but genetic modification of NK cells has been an obstacle due to resistance to viral transduction and CRISPR/Cas9 editing. Here we developed a platform to efficiently expand and genetically engineer primary human NK cells. We transduced NK cells with 40% transduction efficiency and 73% more survival than previous protocols by using gibbon ape leukemia virus (GALV)-pseudotyped lentivirus and a TBK1 inhibitor without the need for repeated rounds of infection or transduction enhancers (like RetroNectin). We found that the gold standard method of expanding and activating NK cells with modified HLA class I-negative K562 cell lines is actually deleterious for NK cell expansion. By culturing NK cells in a cocktail of cytokines (IL-2, IL-18, IL-21, IL-12, and IL-15) with NKp46 beads we generated, we were able to consistently expand NK cells 1000-fold over 28 days and increase their cytotoxicity. Using this new platform, we propose to rationally design a superior allogeneic, over-the-counter NK cellular therapy product. We designed novel NK cell-tailored CAR constructs bearing transmembrane domains that recruit naturally expressed signaling adaptors to activate NK cells. Finally, we propose a novel technique that incorporates our methods and findings to unbiasedly probe pathways of tumor cell recognition, cytolytic activity, persistence, and survival of NK cells via the first-of-its-kind CRISPR/Cas9 knock-out screens directly on NK cells.