Development of Miniprotein Binders to Inhibit VEGF-Induced Corneal Neovascularization.

Area of Science:

• Ophthalmology
• Biotechnology
• Protein Engineering

Background:

• Corneal neovascularization (CNV) impairs vision and is driven by vascular endothelial growth factor (VEGF-A165).
• Current anti-VEGF therapies have limitations including poor corneal penetration and frequent dosing.
• Novel therapeutic strategies are needed to effectively treat CNV.

Purpose of the Study:

• To rationally design and validate novel hyperstable miniprotein inhibitors targeting VEGF-A165.
• To develop a new therapeutic approach for preventing CNV.

Main Methods:

• De novo protein design protocol used to generate VEGF-A165 miniprotein binders.
• Biophysical assays characterized binding affinity and thermal stability.
• In vitro and in vivo models (alkali burn mouse model) assessed anti-angiogenic efficacy and CNV suppression.

Main Results:

• Computational design yielded hyperstable miniprotein binders with high affinity for VEGF-A165 (lead binder Vmb-9: KD ≈ 6 nM).
• Vmb-9 demonstrated thermal stability (>85°C) and was non-cytotoxic to corneal cells.
• Vmb-9 effectively inhibited in vitro tube formation and suppressed corneal hemangiogenesis and lymphangiogenesis in vivo.

Conclusions:

• Vmb-9 is a novel, hyperstable VEGF-A165 minibinder with potent anti-angiogenic activity.
• Topical application of Vmb-9 effectively inhibited corneal neovascularization in vivo.
• These miniproteins represent a promising next-generation therapeutic strategy for CNV, overcoming limitations of current treatments.