Design and construction a novel humanized biparatopic nanobody-based immunotoxin against epidermal growth factor receptor (EGFR)

Abstract

Targeted therapy has been developed for increasing the efficacy of cancer treatment. The first step in targeted therapy is to select the appropriate cell surface marker as targeting. Epidermal growth factor receptor (EGFR) is one of the tumor cell surface markers that overexpressed in many epithelial cancers and makes it a good target for targeted therapies. The unique properties of nanobodies made them an effective tool for targeted therapy. However, the immunogenicity of nanobodies limited them to the treatment of human disease. Humanization is a capable strategy to overcome this problem. In this study, a novel humanized anti-EGFR immunotoxin developed consisted of humanized biparatopic nanobody and PE24. The recombinant IT (RIT) was cloned in pET-22b vector and expressed in Escherichia coli BL21 Plys S, and then the protein was purified. The cell-based ELISA was used to determine the binding activity of RIT to the EGFR and cytotoxicity evaluated by using MTT assay. The results showed that cytotoxicity occurred in EGFR-positive cell lines in a dose-dependent manner but not in the CHO cell line. The humanized RIT with potency against EGFR-positive cancers could be developed and proposed as a promising tool to target EGFR-positive tumors.