Novel Anti–VEGF Molecules for Inhibition of Angiogenesis in the Eye

Abstract

Purpose: : Vascular endothelial growth factor (VEGF) plays a critical role in pathological neovascularization which is a key component of ocular diseases like wet age–related macular degeneration (AMD) or proliferative diabetic retinopathy (PDR). There are several reports of preclinical and clinical studies that demonstrate that antagonizing VEGF is a potentially useful strategy for treating such disorders. One of the most potent binders of VEGF is the VEGF receptor, Flt–1. It has been reported in several publications that Flt–1 domains 2 and 3 are both required for efficient binding and neutralization of VEGF. In the current study we have engineered soluble hybrid forms of Flt–1 that represent novel high–affinity VEGF binders with binding affinities comparable to other known high affinity VEGF binders.

Methods: : Starting with the full–length Flt–1 receptor, we engineered several soluble hybrid versions. The biological activity of these constructs was determined by the HUVECs proliferation and migration assays, VEGF binding assay and BIAcore analysis. Since adeno–associated virus 2 (AAV2) mediated gene delivery offers a means to achieve local, sustained delivery of proteins into the eye we have inserted these novel anti–VEGF molecules into AAV2 vectors. The oxygen–induced retinopathy (OIR) model in mice was used to determine in vivo efficacy of these hybrid molecules.

Results: : Several molecules were identified that have VEGF binding affinities equal to or greater than full–length receptor. Transgene expression was observed for 13 months in the mouse eye following AAV2 mediated delivery and no gross transgene–related toxicities were observed. Preliminary data from the oxygen–induced retinopathy (OIR) model in mice indicates that these novel anti–VEGF molecules are potent inhibitors of retinal neovascularization.

Conclusions: : We have developed several novel hybrid molecules that potently inhibit VEGF in vitro and in vivo and are being currently evaluated in multiple ocular models of pathological neovascularization.

Keywords: growth factors/growth factor receptors • gene transfer/gene therapy • retinal neovascularization