The overexpression of epidermal growth factor receptor (EGFR) could result in the development of solid tumors of prostate, breast, gastric, colorectal, ovarian, and head and neck, leading to carcinoma. Antibody therapies are ideal methods to overcome malignant diseases. However, immunoribonucleases are a new generation of antibodies in which an RNase binds to a specific antibody and shows a stronger ability to terminate cancer cells. In this study, we engineered Rana pipiens RNase to bind to the scFv of human antiepidermal growth factor receptor antibody. The molecular dynamic simulations confirmed protein stability and the ability of scFv−ranpirnase (rantoxin) to bind to epidermal growth factor receptor protein. Then, the rantoxin construct was synthesized in a pCDNA 3.1 Neo vector. CHOK1 cells were used as expression hosts and the construct was transfected. Cells were selected by antibiotic therapies using neomycin, 120 mg/ml, and the highyield colony was screened by realtime polymerase chain reaction (PCR) methods. Then, the recombinant protein production was confirmed using the sodium dodecyl sul- fate polyacrylamide gel electrophoresis and western blot analyses. The molecular dynamic simulation (MDS) confirmed that the I467, S468, Q408, and H409 amino acids of EGFR bonded well to rantoxin. As revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analyses, the rantoxin production and PCR analysis showed that the T3 colony can produce rantoxin messenger RNA fourfold higher than the GAPDH gene. The immunotoxin function was assessed in A431 cancer cells and EGFRnegative HEK293 cells, and IC 50 values were estimated to be 22.4 ± 3 and >620.4 ± 5 nM, respectively. The results indicated that the immunotoxins produced in this study had the potential for use as anticancer drugs.

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