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

Chimeric antigen receptor (CAR)-redirected T cell therapy often fails to control tumors in the long-term due to selecting cancer cells that down-regulated or lost CAR targeted antigen. To reprogram the functional capacities of CAR T cells, we inserted IL12 into the extracellular moiety of a CD28- CAR; both the CAR and IL12 were functional indicated by antigen-redirected activation and STAT4 phosphorylation, respectively. CD8+ T cells engineered with a IL12-CAR gained a so far not recognized NK cell-like RNA expression signature and a CD94+CD56+CD62Lhigh phenotype closely similar, but not identical, to NK and cytokine activated killer (CIK) cells. IL12-CAR T cells with specificity for CEA additionally acquired antigen-independent, HLA-E restricted cytotoxicity eliminating both CEA-negative and CEA-positive cancer cells in an antigen-dependent and independent fashion that was in contrast to conventional CEA CAR T cells. Simultaneous signaling through CD3 of the CAR and through IL12 were required and sufficient for inducing NK-like cytotoxicity. Overall, we present a prototype of a new family of CARs that harbor a cytokine integrated into the extracellular module in order to reprogram the functional capacities of CAR T cells towards augmented tumor recognition and elimination. 6 samples were analysed, 3 triplicates (control: T cells engineered with conventional CAR, sample 1, 2 and 3) and 3 samples T cells engineered with IL12-CAR, sample 4, 5 and 6)