Engineered ligand-based VEGFR antagonists with increased receptor binding affinity more effectively inhibit...

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• Pathologic angiogenesis involves the vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) axis and αvβ3 integrin crosstalk.
• Previous VEGF antagonists had limited potency due to disrupted bivalent binding.
• Engineering VEGF point mutations can create antagonists but lack avidity effects.

Purpose of the Study:

• To engineer a high-affinity VEGFR2 antagonist by enhancing binding affinity.
• To develop a dual-specific antagonist targeting both VEGFR2 and αvβ3 integrin with improved affinity.
• To evaluate the anti-angiogenic potential of the engineered dual-specific protein.

Main Methods:

• Yeast surface display was used to engineer a VEGF variant with increased VEGFR2 binding affinity.
• VEGFR2 phosphorylation and Matrigel implantation assays assessed in vitro and in vivo inhibition of VEGF signaling.
• The high-affinity mutations were incorporated into a previously designed dual-specific antagonist; binding affinities and efficacy in a murine corneal neovascularization model were evaluated.

Main Results:

• Engineered VEGF variant showed ~40-fold higher VEGFR2 binding affinity than the parental antagonist and 14-fold higher than wild-type VEGF.
• The dual-specific protein exhibited high affinity for human (120 ± 10 pM) and murine (360 ± 50 pM) endothelial cells, significantly tighter than wild-type VEGF.
• The engineered high-affinity dual-specific protein successfully inhibited angiogenesis in a murine corneal neovascularization model.

Conclusions:

• Protein engineering strategies can generate potent anti-angiogenic agents.
• The developed high-affinity dual-specific antagonist targeting VEGFR2 and αvβ3 integrin shows promise for clinical development.
• This approach offers a novel strategy for creating targeted anti-angiogenic therapies.