Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy.

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. • Tumor antigen encounter triggers CAR ubiquitination and downmodulation • Tumor antigen-induced CAR ubiquitination targets CARs for lysosomal degradation • CAR signaling in CAR-T cells expressing 4-1BB can be propagated in endosomes • Blocking CAR ubiquitination enhances endosomal CAR signaling and CAR-T persistence Chimeric antigen receptor-T cell therapy offers a promising approach for cancer immunotherapy, but poor CAR-T persistence in patients limits therapeutic efficacy. Li et al. investigated the trafficking of CARs during CAR-T cell activation and designed a recyclable CAR with enhanced persistence and sustained antitumor function. [ABSTRACT FROM AUTHOR]