3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR Tcells.

Abstract

Unlimited generation of chimeric antigen receptor (CAR) Tcells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR Tcell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ Tcell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory Tcells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR Tcell characteristics. Expanded iPSC CD19-CAR Tcells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR Tcells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR Tcells also mediated potent antitumor activity invivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR Tcells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.