title: Isolation and characterization of a novel human scFv inhibiting EGFR vIII expressing cancers.
abstract: EGFRvIII, a mutant form of epidermal growth factor receptor is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. This tumor specific antigen has emerged as a promising candidate for antibody based therapy of several cancers. The aim of the present study was isolation and characterization of a human single chain antibody against EGFRvIII as a promising target for cancer therapy. For this, a synthetic peptide corresponding to EGFRvIII protein was used for screening the naive human scFv phage library. Selection was performed using a novel screening strategy for enrichment of rare specific clones. After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, a clone with an amber mutation in VH CDR2 coding sequence showed higher reactivity. The mutation was corrected through site directed mutagenesis and then scFv fragment was expressed after subcloning into the bacterial expression vector. Expression in BL21 pLysS resulted in a highly soluble scFv appeared in soluble fraction of E. coli lysate. Bioinformatic in silico analysis between scFv and EGFRvIII sequences confirmed specific binding of desired scFv to EGFRvIII in CDR regions. The specific reactivity of the purified scFv with native EGFRvIII was confirmed by cell based ELISA and western blot. In conclusion, human anti- EGFRvIII scFv isolated from a scFv phage library displayed high reactivity with EGFRvIII. The scFv isolated in this study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers.
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Student research committee, University of Medical Sciences, Tabriz, Iran.
authors.forename: Leila
authors.initials: L
authors.lastname: Rahbarnia
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address: Farajnia@gmail.com.
authors.forename: Safar
authors.initials: S
authors.lastname: Farajnia
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Babaei
authors.affiliation: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Jafar
authors.initials: J
authors.lastname: Majidi
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences Tabriz, Iran.
authors.forename: Hassan
authors.initials: H
authors.lastname: Dariushnejad
authors.affiliation: Istanbul University, Faculty of Sciences, Molecular Biology and Genetic, Istanbul, Turkey.
authors.forename: Mohammad Kazem
authors.initials: MK
authors.lastname: Hosseini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000517:prevention & control; D019151:Peptide Library; D011485:Protein Binding / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D022801:Complementarity Determining Regions; D019151:Peptide Library; D057127:Single-Chain Antibodies; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors
keywords: Cancer; EGFRvIII; Human single chain antibody; Phage display