Abstract Introduction: Effective anti-tumor immunity requires the presence of abundant effector T cells in the tumor microenvironment with both cancer cell specificity and cytotoxic potency. Chimeric antigen receptor (CAR) T cells engineered ex vivo to fulfill such quintessence are effective in both experimental and clinical settings with curative potential. However, the efficacy of CAR T cells can be limited by trogocytosis whereby CAR T cells extract cognate antigens from target cells thereby eliciting antigen escape and then express the target antigen on the CAR triggering fratricide that reduces the CAR persistence and efficacy. Methods: Epidermal growth factor variant III (EGFRvIII) specific CAR T cells were generated from various donors and analyzed for EGFRvIII cytotoxicity, trogocytosis, and in vivo therapeutic activity which was modulated with verteporfin, an inducer of autophagy and bafilomycin A1, an inhibitor of autophagy. A three-way co-culture system modeling the glioblastoma microenvironment of the CAR T cell interaction with targets (U87-EGFRvIII) and bystanders (U87-EGFR) was used to evaluate phagocytic and autophagy functions of the CAR T cells. Results: Glioblastoma-specific EGFRvIII CARs induce trogocytosis of the cognate tumor antigen upon engaging target tumor cells within 2 hours. Trogocytic activity varies in a donor-dependent manner and correlates with impaired CAR performance in murine models of orthotopic gliomas expressing EGFRvIII. Trogocytosis is blocked with verteporfin which restores cytolytic capacity of the CAR. In the three-way co-culture system, therapeutically effective in vivo CARs demonstrate superior phagocytic clearance of U87-EGFRvIII released mitochondria that is blocked by autophagy inhibition. The induction of immune checkpoints such as PD-L1 during CAR-target interaction is enhanced when autophagy is blocked. Furthermore, autophagy blockade prevents phagocytic clearance of cytolytic target release of mitochondria. We further demonstrate that pharmacological facilitation of autophagy with verteporfin inhibits trogocytic expression of target tumor antigen on CARs and thereby markedly increases CAR persistence in the glioma microenvironment in mice. Conclusions: Our data establish autophagy as an antagonizing mechanism counteracting CAR-induced trogocytosis and immune checkpoint activation, providing evidence for a new strategy to innovate high-performance CARs. Citation Format: Jiyong Liang, Dexing Fang, Amy B. Heimberger. Chimeric antigen receptor (CAR) T cell donor dependent dysfunction modulation with activation of autophagy and inhibition of trogocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1492.