A novel CD28-mutant CAR with improved IL-2 secretion and enhanced antitumor efficacy in a model of pancreatic cancer 3805

Description CD28-derived co-stimulation modules are frequently included in the signaling domain of chimeric antigen receptors (CAR). These modules contain well characterized tyrosine-containing moieties (YMNM and PYAP) that become phosphorylated upon CAR activation, and result in IL-2 secretion and T cell expansion. We reported that a poorly characterized CD28 tyrosine residue (Y218) was also phosphorylated upon CAR activation. To determine the functional relevance of this phosphorylation event, we generated mutant CARs where this residue was replaced with a non-phosphorylatable amino acid (Y218F). This mutation was associated with reduced IL-2 and it abrogated the therapeutic effect of CAR-T cells in a pancreatic tumor model. We further identified ITK (IL-2-inducible T-cell kinase) as the kinase required for this phosphorylation event. Based on this finding, we designed a novel mutant CAR containing a synthetic ITK binding site surrounding the Y218 residue. This new CAR, called PYRP, showed a 2-fold increase in IL-2 production in vitro, compared to the parental CAR. Importantly, the PYRP CAR showed superior antitumor effect than the parental CAR, in a xenograft model of pancreatic adenocarcinoma. In conclusion, these results show that ITK-mediated phosphorylation of Y218 is critical for IL-2 production and antitumor activity. Both features are enhanced by the introduction of the PYRP mutation, leading to improved CAR-T cell performance.