Using a CD19 CAR with a CD3ζ signaling domain, Li et al. showed reduced CAR expression by transfected primary T cells encountering CD19+ tumor cells in vitro or in xenografts. T cell antigen recognition led to rapid CAR ubiquitination and trafficking to and degradation in lysosomes. T cells transfected with CARs including a 4-1BB costimulation domain and a cytoplasmic domain mutated to prevent CAR ubiquitination and downmodulation had more CAR-TRAF2-signaling endosomes, sustained NF-κB/mTOR signaling, greater mitochondrial function, and enhanced in vivo TCM differentiation, T cell persistence, antitumor activity, and host survival.
Contributed by Paula Hochman
ABSTRACT: Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.