title: Development and Evaluation of a Cetuximab-based Humanized Single Chain Antibody Against EGFR-overexpressing Tumors.
abstract: Production of humanized single chain antibodies (hscFv) can potentially be a powerful solution to limitations imposed by large size and murine nature of cetuximab. The present study describes generation of a cetuximab-based hscFv using CDR-grafting method. Cetuximab CDRs were grafted on frameworks selected from human germline antibody sequence repertoire. The strategy employed in selecting human sequences was the highest sequence similarity of variable domains between human and parental antibodies as well as similarity in the CDRs canonical structures. To maintain the binding affinity, the parental vernier zone residues were retained murine in hscFv. Recombinant hscFv was expressed in E. coli and affinity purified by Ni-NTA column. The potency of hscFv in targeting EGFR was evaluated using A431, a cell line over-expressing EGFR. Dot blot and ELISA tests were used to assess the reactivity and MTT assay to evaluate the growth inhibition of hscFv on A431 cell line. The humanization of cetuximab variable regions resulted in 22.2% increase in humanness of hscFv. Reactivity analyses of hscFv on A431 cells showed better binding affinity and higher growth inhibition effect (2.6 times) comparing to murine counterpart. In conclusion, hscFv produced in this study displayed reduced potential immunogenicity as well as enhanced cytotoxic effect on EGFR- overexpressing tumor cells.
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: K
authors.initials: K
authors.lastname: Veisi
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: S
authors.initials: S
authors.lastname: Farajnia
authors.affiliation: Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: N
authors.initials: N
authors.lastname: Zarghami
authors.affiliation: University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
authors.forename: H R K
authors.initials: HR
authors.lastname: Khorshid
authors.affiliation: Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: N
authors.initials: N
authors.lastname: Samadi
authors.affiliation: Department of Biotechnology, Faculty of Advanced Medical Science Technologies, Golestan University of Medical Sciences, Gorgan, Iran.
authors.forename: Y
authors.initials: Y
authors.lastname: Safdari
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: V
authors.initials: V
authors.lastname: Ahmadzadeh
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000068818:Cetuximab / Q000276:immunology* / Q000494:pharmacology / Q000627:therapeutic use*; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000276:immunology*; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000494:pharmacology / Q000627:therapeutic use*
chemical_list: D000970:Antineoplastic Agents; D057127:Single-Chain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors; D000068818:Cetuximab