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Identification and Characterization of a Novel Nanobody Against Human Placental Growth Factor to Modulate Angiogenesis Publisher Pubmed



Arezumand R1, 2 ; Mahdian R2 ; Zeinali S2 ; Hassanzadehghassabeh G3, 4 ; Mansouri K5 ; Khanahmad H6 ; Namvarasl N7 ; Rahimi H2 ; Behdani M2 ; Cohan RA8 ; Eavazalipour M9 ; Ramazani A10 ; Muyldermans S3, 11
Authors
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Authors Affiliations
  1. 1. Department of Medical Biotechnology and Molecular Science, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran
  2. 2. Department of Molecular Medicine, Pasture Institute of Iran, Tehran, Iran
  3. 3. Vrije University Brussel, Research group Cellular & Molecular Immunology, Brussels, Belgium
  4. 4. VIB, Nanobody Service Facility, Vrije University Brussel, Brussels, Belgium
  5. 5. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
  6. 6. Department of Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran
  7. 7. Laboratory of Animal Sciences, Production and Research Complex for Pasteur Institute of Iran, Tehran, Iran
  8. 8. Department of Pilot Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran
  9. 9. Department of Pharmaceutical Biotechnology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran
  10. 10. Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
  11. 11. VIB, Department of Structural Biology, Vrije University Brussel, Belgium

Source: Molecular Immunology Published:2016


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 −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-MB231was 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. © 2016 Elsevier Ltd
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