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

Abstract：

:Placental growth factor (PlGF), 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. PlGF-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 PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on 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 the AMBER 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 proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×107 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 -461kcal/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 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC50 of57nM. 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.

journal_name

Mol Immunol

journal_title

Molecular immunology

authors

Arezumand R,Mahdian R,Zeinali S,Hassanzadeh-Ghassabeh G,Mansouri K,Khanahmad H,Namvar-Asl N,Rahimi H,Behdani M,Cohan RA,Eavazalipour M,Ramazani A,Muyldermans S

doi

10.1016/j.molimm.2016.09.012

subject

Has Abstract

pub_date

2016-10-01 00:00:00

pages

183-192

eissn

0161-5890

issn

1872-9142

pii

S0161-5890(16)30185-7

journal_volume

78

pub_type

杂志文章

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