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 (CAR^KR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CAR^KR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CAR^KR-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.

临床证据表明，患者中嵌合抗原受体T细胞（CAR-T）的持久性差会限制治疗效果。在这里，我们设计了一种具有可回收能力的CAR，以促进体内持久性并维持抗肿瘤活性。我们表明，肿瘤抗原的参与诱导了CAR的快速泛素化，导致CAR的下调，随后是溶酶体降解。通过突变CAR胞质域（CAR ^KR）中的所有赖氨酸来阻止CAR泛素化，通过抑制溶酶体降解同时增强内在化的CAR返回细胞表面的循环，显着抑制了CAR下调。遇到肿瘤抗原后，CAR ^KR-T细胞改善了表面CAR的丧失，提高了其长期杀伤能力。此外，含有4-1BB信号域的CAR ^KR- T细胞显示出内体4-1BB信号升高，从而增强了氧化磷酸化并促进了记忆T细胞分化，从而在体内具有卓越的持久性。总体而言，我们的研究提供了一种直接的策略，可以通过重定向CAR贩运来优化CAR-T抗肿瘤功效。