Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR).
Various strategies have been attempted for targeting of epidermal growth factor receptor (EGFR), as an essential biomarker in a variety of cancers. Several anti-EGFR antibodies including cetuximab are used in clinics for treatment of EGFR-overexpressing colorectal and head and neck cancers but the efficiency of these antibodies is threatened by their large size and chimeric nature. Humanized single chains antibodies (huscFv) are smaller generation of antibodies with lower immunogenicity may overcome these limitations. This article reports production and evaluation of a novel humanized anti-EGFR scFv. The CDRs of cetuximab heavy and light chains were grafted onto human antibody frameworks as framework donors. To maintain the antigen binding affinity of murine antibody, the murine vernier zone residues were retained in framework regions of huscFv. Additionally, two point mutations in CDR-L1 and CDR-L3 and three point mutations in CDR-H2 and CDR-H3 loops of the humanized scFv (huscFv) were introduced to increase affinity of the huscFv to EGFR. Analysis of results demonstrated that the humanness degree of resultant huscFv was increased as 19%. HuscFv was expressed in BL21 (DE3) and affinity purified via Ni-NTA column. The reactivity of huscFv with EGFR was evaluated by ELISA and dot blot techniques. Analysis by ELISA and dot blot showed that the huscFv was able to recognize and react with EGFR. Toxicity analysis by MTT assay indicated an inhibitory effect on growth of EGFR-overexpressing A431 cells. In conclusion, the huscFv produced in this study revealed decreased immunogenicity while retained growth inhibitory effect on EGFR-overexpressing tumor cells.
Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
Bahman
B
Akbari
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address: farajnias@tbzmed.ac.ir.
Safar
S
Farajnia
Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Nosratollah
N
Zarghami
Genetic Research Lab, Rajaee Heart Center, Tehran, Iran.
Nejat
N
Mahdieh
Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Mohammad
M
Rahmati
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Shiva Ahdi
SA
Khosroshahi
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Leila
L
Rahbarnia
D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D045744:Cell Line, Tumor; D000068818:Cetuximab / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D061067:Antibodies, Monoclonal, Humanized; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors; D000068818:Cetuximab
EGFR; Escherichia coli; Humanized single chain antibody