Development of Miniprotein Binders to Inhibit VEGF-Induced Corneal Neovascularization

Purpose: Corneal neovascularization (CNV) disrupts corneal transparency and impairs vision and is driven primarily by vascular endothelial growth factor (VEGF-A165). Current therapies rely on off-label anti-VEGF agents, which are limited by poor corneal penetration and the need for repeated dosing. Herein, we aimed to rationally design and validate novel hyperstable VEGF-A165 miniprotein inhibitors against VEGF-A165 for preventing CNV. Methods: VEGF-A165 miniprotein binders were generated using a de novo diffusion-based protein design protocol and expressed in Escherichia coli (E. coli). Selected candidates were characterized for binding activity via biophysical assays. Cytotoxicity was assessed in immortalized human corneal epithelial cells and primary human corneal fibroblasts. Anti-angiogenic efficacy was screened using in vitro angiogenic assays and validated in vivo using an alkali burn mouse model of CNV. Results: Computational design yielded several VEGF-A165 minibinders, with Vmb-9 displaying the highest affinity (KD ≈ 6 nM). The minibinders were thermally stable (Tm > 85°C) and non-cytotoxic to corneal cells. The lead binder Vmb-9 reduced in vitro tube formation, achieving efficacy comparable to bevacizumab, and suppressed corneal hemangiogenesis and lymphangiogenesis in vivo following alkali burn (P < 0.05). Conclusions: Vmb-9, a novel hyperstable VEGF-A165 minibinder, exhibited potent anti-angiogenic activity and effectively inhibited corneal neovascularization in vivo with topical application. Their small size, reduced immunogenicity, and scalable production highlight their potential as a next-generation therapeutic strategy that overcomes the limitations of conventional anti-VEGF treatments.