Abstract
Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is the main mediator of angiogenic signaling in endothelial cells and a primary responder to VEGF. VEGF dependent VEGFR-2 activation regulates endothelial cell migration and proliferation, as well as vessel permeability. VEGF is presented as an antiparallel homodimer, and its binding to VEGFR-2 brings two receptors in close proximity. Downstream signaling is triggered by receptor dimerization, kinase activation, and receptor internalization. Our aim was to further investigate allosteric inhibition using binders targeting extracellular subdomains 4–7 of VEGFR-2 as an alternative to existing anti-angiogenic therapies, which rely on neutralizing VEGF or blocking of the ligand-binding site on the receptor. We applied phage display technology to produce single chain antibody fragments (scFvs) targeting VEGFR-2. Selected antibody fragments were characterized using biophysical and biological assays. We characterized several antibody fragments, which exert their inhibitory effect of VEGFR-2 independent of ligand binding. These reagents led to rapid clearance of VEGFR-2 from the cell surface without kinase activation, followed by an increase in intracellular receptor-positive vesicles, suggesting receptor internalization. Our highly specific VEGFR-2 binders thus represent novel tools for anti-angiogenic therapy and diagnostic applications.
Highlights
Angiogenesis is the process of creation of new blood capillaries from pre-existing vasculature
The synthetic ETH-2 Gold library was used for three rounds of selection of scFvs against hVEGFR-2
As signal output of Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), we examined expression and phosphorylation of PLCγ, AKT, and p38 mitogen-activated protein kinase (MAPK)
Summary
Angiogenesis is the process of creation of new blood capillaries from pre-existing vasculature. It occurs throughout the life of higher organisms, in both healthy and pathological conditions. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are required for vascular and lymphatic homeostasis, but their aberrant signaling can give rise to pathological angiogenesis. Different members of the VEGF protein family bind VEGFR-1, -2, and -3, respectively, thereby regulating endothelial cell survival, proliferation, differentiation, and migration. VEGFR-2 is the major mediator of angiogenic signaling in endothelial cells, various tactics to target VEGF/VEGFR-2 signaling have been developed. Inhibition of VEGF signaling by ligand sequestering agents (bevacizumab) [1] or VEGFR-2 blocking antibodies (ramucirumab) [2,3] can be bypassed by high ligand concentration
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