Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy

Neutrophils, the most abundant white blood cells in the circulation, are closely related to cancer development and progression. Primary neutrophils from healthy donors present potent cytotoxicity against different human cancer cell lines through direct contact and via the generation of reactive oxygen species (ROS). However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with widely used chimeric antigen receptors (CARs) to enhance their anti-tumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells (hPSCs) with different synthetic CARs and successfully differentiated them into functional neutrophils by implementing a novel chemically-defined differentiation platform. Neutrophils expressing the chlorotoxin (CLTX)-T-CAR presented specific cytotoxicity against glioblastoma (GBM) cells both in monolayer and 3D cultures. In a GBM xenograft mouse model, systematically-administered CLTX-T-CAR neutrophils also displayed enhanced anti-tumor activity and prolonged animal survival compared with peripheral blood-neutrophils, hPSC-neutrophils and CLTX-NK-CAR natural killer (NK) cells. Collectively, we established a new platform for production of CAR-neutrophils, paving the way to myeloid cell-based therapeutic strategies that would complement and boost current cancer treatment approaches.