Engineering synthetic agonists for targeted activation of Notch signaling

Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although there are several classes of Notch inhibitors, the mechanical force requirement for Notch activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonists (SNAGs) by tethering affinity-matured Notch ligands to proteins that internalize their targets. This bispecific format enables SNAGs to 'pull' on mechanosensitive Notch receptors, triggering their activation in the presence of desired biomarkers. We successfully developed SNAGs targeting six independent surface markers, including the tumor antigens PDL1, CD19 and HER2 and the immunostimulatory receptor CD40. HER2-SNAGs and CD19-SNAGs increased the expression of T cell activation markers and Notch target genes in cocultures with tumor cells, highlighting their potential for immunotherapeutic applications. These insights have broad implications for the pharmacological activation of mechanoreceptors and will expand our ability to modulate Notch signaling in biotechnology.