Engineering Pan-Reactive VEGF Antagonists to Treat Neovascular Eye Diseases

Ocular neovascularization drives the pathogenesis of neovascular age-related macular degeneration (NVAMD) and diabetic retinopathy (DR), two of the leading causes of blindness in adults. Vascular endothelial growth factor (VEGF) ligands are major stimulants of neovascularization, and blocking the binding of these ligands to their cognate receptors (VEGFRs) has shown clinical benefit in the treatment of both NVAMD and DR. Most VEGF-targeted therapies that have received FDA approval for use in treating NVAMD and DR block the activity of VEGF-A ligands, which are the most prominent regulators of blood vessel formation, by competitively inhibiting their signaling through VEGFRs on the surface of epithelial cells. However, VEGF-C is increasingly implicated in pathological neovascularization, and this ligand is found to be upregulated following anti-VEGF-A treatment, leading to therapeutic resistance. Moreover, VEGF-C is just as potent as VEGF-A in driving retinal neovascularization under hypoxic conditions. No current therapies bind both VEGF-A and VEGF-C, which would provide a more complete blockade of neovascularization. We designed an error-prone mutagenic DNA library templated on domains 2&3 of VEGFR-2 (VEGFR-2 D23), which binds both VEGF-A and VEGF-C. We then used the yeast surface display directed evolution platform to isolate and characterize high-affinity clones that bind both VEGF-A and VEGF-C. We further designed novel dual decoy receptor antagonists by fusing the binding domains of VEGFR-2 and VEGFR-1 to a human antibody Fc domain for a potent inhibition of both VEGF-A and VEGF-C activity. These promising proteins could act as superior therapeutics to treat neovascular eye diseases.