In silico and experimental methods for designing a potent anticancer arazyme-herceptin fusion protein in HER2-positive breast cancer

Abstract

Breast cancer is the most prevalent type of malignancies among women worldwide and is associated with serious physical and mental consequences. Current chemotherapies may lack successful outcomes; thus, the development of targeted recombinant immunotoxins is plausible. The predicted B cell and T cell epitopes of arazyme of the fusion protein are able to elicit immune response. The results of codon adaptation tool of herceptin-arazyme have improved from 0.4 to 1. The in silico immune simulation results showed significant response for immune cells. In conclusion, our findings show that the known multi-epitope fusion protein may activate humoral and cellular immune responses and maybe a possible candidate for breast cancer treatment. In this study, the selected monoclonal antibody constituting herceptin and the bacterial metalloprotease, arazyme, was used with different peptide linkers to design a novel fusion protein to predict different B cell and T cell epitopes by the means of the relevant databases. Modeler 10.1 and I-TASSER online server were used to predict and validate the 3D structure and then docked to HER2-receptor using HADDOCK2.4 web server. The molecular dynamics (MD) simulations of the arazyme-linker-herceptin-HER2 complex were performed by GROMACS 2019.6 software. The sequence of arazyme-herceptin was optimized for the expression in prokaryotic host using online servers and cloned into pET-28a plasmid. The recombinant pET28a was transferred into the Escherichia coli BL21DE3. Expression and binding affinity of arazyme-herceptin and arazyme to human breast cancer cell lines (SK-BR-3/HER2 + and MDA-MB-468/HER2 -) were validated by the SDS-PAGE and cell‑ELISA, respectively.