title: 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 1.5×107individual 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 EC50of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC50of57nM. 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.
authors.affiliation: Department of Medical Biotechnology and Molecular Science, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran; Department of Molecular Medicine, Pasture Institute of Iran, Tehran, Iran.
authors.forename: Roghaye
authors.initials: R
authors.lastname: Arezumand
authors.affiliation: Department of Molecular Medicine, Pasture Institute of Iran, Tehran, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Mahdian
authors.affiliation: Department of Molecular Medicine, Pasture Institute of Iran, Tehran, Iran. Electronic address: sirouszeinali.pastuer@gmail.com.
authors.forename: Sirous
authors.initials: S
authors.lastname: Zeinali
authors.affiliation: Vrije University Brussel, Research group Cellular & Molecular Immunology, Brussels, Belgium; VIB, Nanobody Service Facility, Vrije University Brussel, Brussels, Belgium.
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Hassanzadeh-Ghassabeh
authors.affiliation: Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.
authors.forename: Kamran
authors.initials: K
authors.lastname: Mansouri
authors.affiliation: Department of Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Khanahmad
authors.affiliation: Laboratory of Animal Sciences, Production and Research Complex for Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Nabiollah
authors.initials: N
authors.lastname: Namvar-Asl
authors.affiliation: Department of Molecular Medicine, Pasture Institute of Iran, Tehran, Iran.
authors.forename: Hamzeh
authors.initials: H
authors.lastname: Rahimi
authors.affiliation: Department of Molecular Medicine, Pasture Institute of Iran, Tehran, Iran.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Behdani
authors.affiliation: Department of Pilot Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Reza Ahangari
authors.initials: RA
authors.lastname: Cohan
authors.affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Eavazalipour
authors.affiliation: Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Ramazani
authors.affiliation: Vrije University Brussel, Research group Cellular & Molecular Immunology, Brussels, Belgium; VIB, Department of Structural Biology, Vrije University Brussel, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
mesh_terms: D020533:Angiogenesis Inhibitors / Q000276:immunology / Q000494:pharmacology*; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D002642:Chick Embryo; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008958:Models, Molecular; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D019151:Peptide Library; D000072483:Placenta Growth Factor / Q000037:antagonists & inhibitors*; D061905:Single-Domain Antibodies / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D019151:Peptide Library; D061905:Single-Domain Antibodies; D000072483:Placenta Growth Factor
keywords: Angiogenesis; Computational analysis; Nanobody; Phage display; PlGF