Abstract
Pathologic angiogenesis is mediated by the coordinated action of the vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling axis, along with crosstalk contributed by other receptors, notably αvβ3 integrin. We build on earlier work demonstrating that point mutations can be introduced into the homodimeric VEGF ligand to convert it into an antagonist through disruption of binding to one copy of VEGFR2. This inhibitor has limited potency, however, due to loss of avidity effects from bivalent VEGFR2 binding. Here, we used yeast surface display to engineer a variant with VEGFR2 binding affinity approximately 40-fold higher than the parental antagonist, and 14-fold higher than the natural bivalent VEGF ligand. Increased VEGFR2 binding affinity correlated with the ability to more effectively inhibit VEGF-mediated signaling, both in vitro and in vivo, as measured using VEGFR2 phosphorylation and Matrigel implantation assays. High affinity mutations found in this variant were then incorporated into a dual-specific antagonist that we previously designed to simultaneously bind to and inhibit VEGFR2 and αvβ3 integrin. The resulting dual-specific protein bound to human and murine endothelial cells with relative affinities of 120 ± 10 pM and 360 ± 50 pM, respectively, which is at least 30-fold tighter than wild-type VEGF (3.8 ± 0.5 nM). Finally, we demonstrated that this engineered high-affinity dual-specific protein could inhibit angiogenesis in a murine corneal neovascularization model. Taken together, these data indicate that protein engineering strategies can be combined to generate unique antiangiogenic candidates for further clinical development.
Original language | English GB |
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Pages (from-to) | 81-91 |
Number of pages | 11 |
Journal | Bioengineering and Translational Medicine |
Volume | 2 |
Issue number | 1 |
DOIs | |
State | Published - Mar 2017 |
Externally published | Yes |