Feeder cell-free production of CAR-NK cells via activation of PKC and calcium signaling pathways.

Abstract

Ex vivo manufacturing of CAR-NK cells is a complex, multistep process involving NK cell activation, genetic modification, and cellular expansion. Currently, tumor cell-derived feeder cells (e.g., K562) are commonly used to manufacture CAR-NK cells. However, the feeder cell-based (FCB) method presents several risks to the CAR-NK product, including contamination, residual impurities, immunogenicity, and lot-to-lot variability. Additionally, establishing and maintaining feeder cells increases costs, complicates scaling up production, and requires rigorous quality control to ensure safety. In contrast, the feeder cell-free (FCF) method can mitigate these risks. We used phorbol ester (PMA) and calcium ionophore (ionomycin) to develop a novel FCF method to manufacture CAR-NK cells and compared their critical quality attributes (CQAs) with those produced using a traditional FCB method. In small-scale production, most CQAs were comparable between the two methods, except for CAR expression, which was lower in the FCF method. However, in large-scale production, CAR expression was comparable between the two methods, and the FCF method significantly improved CAR-NK potency while reducing CAR-NK-mediated myeloid cell activation. Our data suggest that the FCF method provides a robust and scalable alternative for producing safer and high-quality CAR-NK cells.