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 sulfate 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 IC50 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.