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

Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is con-sidered as an important antigen associated with pathological conditions such as cancer cell growth, andmetastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these patholo-gies. In this work, phage-display and computational approach was employed to develop a high affinityPlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panningon immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using theAMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions.Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by prolif-eration, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM)model. An immune library of 1.5 × 107individual Nb clones was constructed. By PE-ELISA 12 clones withstrong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed highbinding scores ranging between −378.7 and −461 kcal/mol. Compared to a control Nb, Nb-C18 signifi-cantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p < 0.05) with anEC50of 35 nM, 42 nM and 24 nM and invasion of MDA-MB231was significantly suppressed (p < 0.05) withan EC50of57 nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation inthe chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future.

Publication year:2016

CSS-citation score:1

Authors:International