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Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

Hum Vaccin Immunother. 2018; 
BanisadrArsham,SafdariYaghoub,KianmehrAnvarsadat,PourafsharMah
Products/Services Used Details Operation
HRPProtein L (GenScript, Cat. Number: M00098) and DAB (Diaminobenzidine) tablets (Cat. Number: D4293) were purchased from GenScript (NJ, USA) and Sigma Aldrich, respectively. Cetuximab (Erbitux) (concentration of 5mg/mL, total volume of 20 mL) was obtained from Red Cross Pharmacy of Gorgan city (Golestan Province, Northern Iran).

摘要

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 [(GlySer)] 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�... More

关键词

VH3,VL3 kappa,cetuximab,germline humanization,