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

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 with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, Delta, binds human and murine Notch receptors with 500- to 1,000-fold increased affinity compared with wild-type human Delta-like 4. Delta also potently activates Notch in plate-bound, bead-bound and cellular formats. When administered as a soluble decoy, Delta inhibits Notch in reporter and neuronal differentiation assays, highlighting its dual utility as an agonist or antagonist. Finally, we demonstrate that Delta stimulates increased proliferation and expression of effector mediators in T cells. Taken together, our data define Delta as a versatile tool for broad-spectrum activation or inhibition of Notch signaling.