Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling

ABSTRACT The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 (DLL4) with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, Delta MAX , engages human and murine Notch receptors with 500- to 1000-fold increased affinity compared to wild-type human DLL4. Delta MAX also potently activates human Notch in plate-bound, bead-bound, and cellular formats. When administered as a soluble decoy, Delta MAX inhibits Notch activation in response to either Delta-like (DLL) or Jagged (Jag) ligands, highlighting its utility as both an agonist and antagonist. Finally, we demonstrate that Delta MAX stimulates increased proliferation and expression of effector mediators in primary activated human T cells. Taken together, our data defines Delta MAX as a versatile biotechnological tool for broad-spectrum activation or inhibition of Notch signaling.