ABSTRACT
Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although several Notch inhibitors are under development as cancer therapies, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins that mimic the tension-generating mechanism of endogenous ligands. SNAGs were designed by fusing a high-affinity variant of the Notch ligand Delta-like 4 (DLL4) to antibody fragments that induce target internalization. This bispecific format enables the SNAG-bound biomarkers to “pull” on Notch receptors, triggering Notch activation in mixed populations of biomarker-expressing and non-expressing cells. SNAGs targeting the immune checkpoint PDL1 potently activated Notch in co-cultures of Notch1-and PDL1-expressing cells, but not in monocultures of Notch1-expressing cells alone. Additional SNAGs targeting the tumor antigens CD19 and HER2 also activated Notch in mixed cell populations, indicating that the SNAG design concept is adaptable to multiple biomarkers. SNAG-mediated Notch activation was blocked by a dynamin inhibitor, and efficacy increased dramatically when SNAGs were dimerized via fusion to antibody Fc domains, suggesting that endocytosis and multimerization are important for optimal SNAG function. These insights will greatly expand our ability to modulate Notch signaling for applications in immunotherapy and regenerative medicine.
Competing Interest Statement
V.C.L. is a consultant on unrelated projects for Cellestia Biotech, Remunix, and Curie.Bio. The remaining authors have no competing interests. V.C.L. and D.H.P. have filed provisional patents (serial numbers 63/548,615 and 63/663,744) based on the described technology.
Footnotes
Figure 5 now includes panels showing that CD19-SNAGs are endocytosed following binding to CD19-overexpressing 3T3 cells. The text, figure legend, and methods were updated to reflect this change.