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

Roghaye Arezumand, Reza Mahdian, Sirous Zeinali, Gholamreza Hassanzadeh Ghassabeh, Kamran Mansouri, Hossein Khanahmad, Nabiollah Namvarasl, Hamzeh Rahimi, Mahdi Behdani, Reza Ahangari Cohan, Mehdi Eavazalipour, Ali Ramazani, Serge Muyldermans

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)


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.
Original languageEnglish
Pages (from-to)183-192
Number of pages10
JournalMolecular Immunology
Publication statusPublished - Oct 2016


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