Design and construction of scFv-PE35KDEL as a novel immunotoxin against human epidermal growth factor receptor 2 for cancer therapy.

PROPOSE

Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL).

METHODS

The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay.

RESULTS

In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25 °C and 1 mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7 mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC ~ 95 nM) compared with HER2-negative cells, MDA-MB-23 (IC ˃ 200 nM).

CONCLUSION

This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein.