Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling

Abstract

Angiogenesis denotes the formation of new blood vessels from pre-existing vasculature. Progression of diseases such as cancer and several ophthalmological disorders may be promoted by excess angiogenesis. Novel therapeutics to inhibit angiogenesis and diagnostic tools for monitoring angiogenesis during therapy, hold great potential for improving treatment of such diseases. We have previously generated so-called biparatopic Affibody constructs with high affinity for the vascular endothelial growth factor receptor-2 (VEGFR2), which recognize two non-overlapping epitopes in the ligand-binding site on the receptor. Affibody molecules have previously been demonstrated suitable for imaging purposes. Their small size also makes them attractive for applications where an alternative route of administration is beneficial, such as topical delivery using eye drops. In this study, we show that decreasing linker length between the two Affibody domains resulted in even slower dissociation from the receptor. The new variants of the biparatopic Affibody bound to VEGFR2-expressing cells, blocked VEGFA binding, and inhibited VEGFA-induced signaling of VEGFR2 over expressing cells. Moreover, the biparatopic Affibody inhibited sprout formation of endothelial cells in an in vitro angiogenesis assay with similar potency as the bivalent monoclonal antibody ramucirumab. This study demonstrates that the biparatopic Affibody constructs show promise for future therapeutic as well as in vivo imaging applications.

Radionuclide imaging of VEGFR2 in glioma vasculature using biparatopic affibody conjugate: proof-of-principle in a murine model

“…That selection effort yielded two variants that bound human VEGFR2 and were also cross-reactive for murine VEGFR2. After affinity maturation and construction of the biparatopic high-affinity binder (Z VEGFR2 -Bp2), we again confirmed cross-reactivity between human VEGR2 and murine VEGFR2 29 , 30 . The Z VEGFR2 -Bp2 hence binds both orthologs with similar affinity and the affinity measurements presented here are in agreement with the previously published results, indicating a strong position for future clinical translation.…”

“…The interaction between NODAGA-Z VEGFR2 -Bp 2 and mVEGFR2 was analyzed using a surface plasmon resonance (SPR)-based biosensor assay to verify that conjugation of the NODAGA chelator had no negative influence on the binding to the receptor. The previously studied Z VEGFR2 -Bp 2 -ABD 30 was included in the assay for comparison. The results from the assay showed similar binding to mVEGFR2 for NODAGA-Z VEGFR2 -Bp 2 compared with Z VEGFR2 -Bp 2 -ABD, which suggested that the NODAGA conjugation did not appreciably affect association/dissociation of the interaction ( Figure S1 ).…”

“…A flow cytometry-based assay was performed to investigate the ability of Z VEGFR2 -Bp 2 to bind to the murine cell lines, MS1 (SV-40 transformed endothelial), PC-3 (prostate cancer) and GL261 (glioblastoma). Z VEGFR2 -Bp 2 was produced and purified in fusion with an albumin-binding domain (ABD) to allow detection 30 . Ramucirumab (anti-human VEGFR2 monoclonal antibody) and DC101 (anti-mouse VEGFR2 monoclonal antibody) were included for comparison.…”

“…Although all selected affibody molecules blocked VEGFA-binding to VEGFR2 in vitro , they recognized nonoverlapping epitopes on the receptor and could bind simultaneously 29 . Furthermore, they inhibited VEGFA-induced phosphorylation and cell proliferation 30 .…”

Affibody molecules as engineered protein drugs

“…58 In addition, the resulting biparatopic molecule blocked ligand receptor interaction and inhibited angiogenic sprout formation of endothelial cells as efficiently as the antibody ramucirumab, which is approved for treatment of metastasized lung, colorectal and gastric cancer. 59 This suggests that Affibody molecules could be used for therapeutic intervention, and will be discussed below.…”