Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells
| Autor: | Mahdieh Pourafshar, Yaghoub Safdari, Anvarsadat Kianmehr, Arsham Banisadr |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Immunoblotting Immunology Cetuximab chemical and pharmacologic phenomena Antibodies Monoclonal Humanized Germline law.invention 03 medical and health sciences 0302 clinical medicine Affinity chromatography law Cell Line Tumor Neoplasms Escherichia coli medicine Humans Immunology and Allergy Epidermal growth factor receptor Pharmacology biology Chemistry respiratory system Molecular biology ErbB Receptors 030104 developmental biology 030220 oncology & carcinogenesis Cancer cell MCF-7 Cells biology.protein Recombinant DNA Antibody Linker Research Paper medicine.drug |
| Zdroj: | Human Vaccines & Immunotherapeutics. 14:856-863 |
| ISSN: | 2164-554X 2164-5515 |
| DOI: | 10.1080/21645515.2017.1407482 |
| Popis: | The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly4Ser)3] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells. |
| Databáze: | OpenAIRE |
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