Optimization of velocity receptor transduction in CAR T cells

Abstract

Limited infiltration capacity significantly limits the effectiveness of chimeric antigen receptor (CAR) T cells for solid tumors. We have recently developed a large family of highly modular synthetic cytokine receptors termed velocity receptors (VRs), capable of binding key inflammatory cytokines, such as IL5, IL8, and TNF, which drive CAR T cells into an elevated motility state. These new CAR T cells sense and amplify these autocrine secreted cytokines, thereby maintaining a self-propelled, high migratory state, facilitating penetration into dense tumor cores. In this study, we systematically evaluated key factors influencing VR transduction in order to improve their stable integration and expression. We established a dual-fluorescence reporter system to allow simultaneous monitoring of both VR and CAR constructs, and while evaluating modifications to the vector construct and generating standardized infectious unit (IFU) curves under various conditions. Our results demonstrate that the attempt to reduce overall vector size by eliminating non coding sections upstream of the central polypurine tract (cPPT) do not yield better transduction efficiency, though it is unclear if the effect is due to viral production or integration impairment. We also observed a log-linear relationship between viral dose and transduction efficiency for a subset of VRs previously tested in various mouse models of human cancer, with VR5IL8 and VR5TNF VRs consistently outperforming VR5IL5 and V5 (full length native IL5 receptor). Overall, these findings establish an optimized and reproducible framework that offers valuable guidance for the future development and functional study of VR-CAR T cells in cellular therapies for solid tumors.