Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis

Placental growth factor (PIGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PIGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PIGF-specific Nb. An Nb library was constructed against human recombinant PIGF (rPIGF). After, panning on immobilized rPIGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PEELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PIGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5 x 10(7) individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461 kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p < 0.05) with an EC50 of 35 nM, 42 nM and 24 nM and invasion of MDA-MB231 was significantly suppressed (p < 0.05) with an EC50 of57 nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future. (C) 2016 Elsevier Ltd. All rights reserved.