Design, Expression, Purification and Characterization of the Recombinant Immunotoxin 4D5 scFv-TRAIL

Immunotoxins are chimeric proteins that combine antibodies and toxins in targeted cancer therapy. Despite the promising application prospects, immunogenicity and nonspecific cytotoxicity of immunotoxins with toxins from bacterial or plant origin have hindered their clinical application. In this study, an anti-HER2 single-chain antibody fragment 4D5, was fused to a human-origin antitumor drug, TRAIL to produce a humanized immunotoxin in Escherichia coli BL21 (DE3). An appropriate linker between the domains was selected using the software Antheprot 5.0. The 4D5 scFv-TRAIL was obtained using a SUMO tag for solubility-enhancing and purified by Ni–NTA affinity chromatography after tag removing. The purity and yield of 4D5 scFv-TRAIL reached 92% and 11.6 mg/L in shake flasks, respectively. Additionally, MTT assays showed 4D5 scFv-TRAIL exhibited selective in vitro cytotoxicity on the breast cancer cell line MDA-MB-231 without cytotoxic effect on the normal cell FBHE. Therefore, 4D5 scFv-TRAIL is a potential humanized immunotoxin for treatment of HER2-overexpressing tumor cells.