Abstract
CAR-NK cells are promising off-the-shelf tumor therapies, yet a scarcity of suitable targets hinders their development. Death receptor 5 (DR5) is an attractive target due to its selective overexpression on tumors and ability to trigger apoptosis. A central challenge, however, is that many tumors exhibit inherent resistance to DR5-mediated cell death, which has stymied the efficacy of existing therapies. Here, we developed a novel DR5-specific chimeric antigen receptor (CAR) from the monoclonal antibody 3E7, which binds human and murine DR5 without cross-reacting to DR4. Primary natural killer (NK) cells transduced with baboon envelope-pseudotyped lentiviral vectors showed stable CAR expression and robust expansion. Remarkably, the culture self-enriched to near 100% CAR positivity, a phenomenon we attribute to the selective elimination of DR5-expressing senescent NK cells that acted as inadvertent feeder cells. In vitro, the engineered NK cells potently killed DR5+ tumor cells through direct cytolysis and DR5-mediated apoptosis, even under exhaustion-like conditions. In vivo, they suppressed tumor growth in xenograft models without observable toxicity to normal tissues, despite their recognition of murine DR5. Furthermore, they exhibited negligible cytotoxicity against human liver organoids, underscoring a favorable safety profile. These results position DR5 CAR-NK cells as a potent, scalable, and safe therapeutic strategy for DR5+ tumors, including those resistant to conventional apoptosis-based therapies.