IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors

Abstract

Chimeric antigen receptor (CAR)-redirected Tcell therapy often fails to control tumors in the long term due to selecting cancer cells that downregulated or lost CAR targeted antigen. To reprogram the functional capacities specifically of engineered CAR Tcells, we inserted IL12 into the extracellular moiety of a CD28-ζ CAR; both the CAR endodomain and IL12 were functionally active, as indicated by antigen-redirected effector functions and STAT4 phosphorylation, respectively. The IL12-CAR reprogrammed CD8+ Tcells toward a so far not recognized natural killer (NK) cell-like signature and a CD94+CD56+CD62Lhigh phenotype closely similar, but not identical, to NK and cytokine induced killer (CIK) cells. In contrast to conventional CAR Tcells, IL12-CAR Tcells acquired antigen-independent, human leukocyte antigen E (HLA-E) restricted cytotoxic capacities eliminating antigen-negative cancer cells in addition to eliminating cancer cells with CAR cognate antigen. Simultaneous signaling through both the CAR endodomain and IL12 were required for inducing maximal NK-like cytotoxicity; adding IL12 to conventional CAR Tcells was not sufficient. Antigen-negative tumors were attacked by IL12-CAR Tcells, but not by conventional CAR Tcells. Overall, we present a prototype of a new family of CARs that augments tumor recognition and elimination through expanded functional capacities by an appropriate cytokine integrated into the CAR exodomain.