title: Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.
abstract: Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.
authors.affiliation: Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Hossien
authors.initials: H
authors.lastname: Hosseini
authors.affiliation: Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Electronic address: rajabi_m@sbmu.ac.ir.
authors.forename: Masoumeh
authors.initials: M
authors.lastname: Rajabibazl
authors.affiliation: Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Walead
authors.initials: W
authors.lastname: Ebrahimizadeh
authors.affiliation: Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Gholamreza Rafiei
authors.initials: GR
authors.lastname: Dehbidi
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000918:Antibody Specificity; D049109:Cell Proliferation / Q000187:drug effects; D061505:Cell Surface Display Techniques; D002478:Cells, Cultured; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D057127:Single-Chain Antibodies; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A
keywords: Angiogenesis; Cancer; Chorioallantoic membrane assay; Phage display; Single-chain variable fragment; Vascular endothelial growth factor