(585a) Structure-Guided Molecular Engineering of a VEGF Antagonist to Treat Retinal Eye Diseases

The repertoire of naturally occurring proteins is limited and many molecules induce multiple conflicting functional responses. Powerful advances in biomolecular engineering enable the enhancement of existing proteins and the evolution of novel proteins with desirable therapeutic activities. Whereas most protein engineering efforts adopt a random approach to directed evolution, valuable insights from structural biology can be applied to inform molecular design and, in turn, crystallographic characterization of engineered proteins enables elucidation of their mechanistic activities. Herein, we fuse molecular engineering with structural biology to design superior disease-relevant therapeutics.

Neovalscularization in the eye leads to a host of retinal diseases including age-related macular degeneration and exacerbates the pathogenesis of other eye diseases such as diabetic retinopathy. Obstruction of vascular endothelial growth factor (VEGF) interaction with its receptor (VEGFR) shows clinical benefit in the treatment of retinal diseases, but effective suppression of VEGF signaling presents a major unmet medical challenge. The FDA-approved drug aflibercept, an Fc-fused chimera of VEGFR that serves as a VEGF decoy protein, provides a good starting point for VEGF neutralization, but further improvements are needed. The VEGF affinity of aflibercept is equivalent to that of membrane-tethered VEGFR and, moreover, aflibercept interacts with VEGF-A but not VEGF-C isoforms, which can lead to secondary drug resistance through amplification of VEGF-C signaling. Using recent crystallographic data from VEGF/VEGFR complexes, we displayed a site-directed mutagenic library of aflibercept on the surface of yeast and developed a customized directed evolution scheme to isolate a high-affinity drug variant that cross-reacts with both VEGF-A and VEGF-C isoforms as a superior therapeutic for retinal eye diseases. Moreover, our structure-guided approach to molecular engineering focuses the library selection workflow to catalyze the discovery of novel targeted protein drugs, presenting a general strategy that can be used for a vast array of therapeutic design applications.