The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein–protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv‐engineered RNase A. Then, wild‐RNase A (14 kD), engineered RNase A (14 kD) and scFv‐engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni‐NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild‐RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv‐engineered RNase A, a dose–response cytotoxicity assay was performed on Her1‐positive and Her1‐negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1‐positive cell lines with an IC50 of 50 nM. It appears that scFv‐engineered RNase A can be a promising molecule for use.

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