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A novel strategy for engineering of a smart generation of immune ribonucleases against EGFR+ cells
Nooshin Taghizadegan1, Mohsen Firoozrai1, Mohammadreza Nassiri2
1Department of Biochemistry, Shahrood Branch, Islamic Azad University, Shahrood, Iran.
Abstract:
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.
Insights
Researchers engineered an immunotoxin by combining Rana pipiens RNase with an antibody targeting the epidermal growth factor receptor (EGFR). This novel rantoxin demonstrated potent cancer cell-killing ability, showing promise as an anticancer therapeutic.
Area of Science:
- Biotechnology and Molecular Biology
- Cancer Research
- Immunotherapy
Background:
- Epidermal growth factor receptor (EGFR) overexpression is linked to various solid tumors, including prostate, breast, gastric, colorectal, ovarian, and head and neck cancers.
- Antibody-based therapies offer a promising strategy for combating malignant diseases.
- Immunoribonucleases, a novel class of antibodies, combine RNase activity with specific antibody targeting for enhanced cancer cell elimination.
Purpose of the Study:
- To engineer a novel immunotoxin, rantoxin, by fusing Rana pipiens RNase with a human anti-EGFR single-chain variable fragment (scFv).
- To confirm the stability and binding affinity of the engineered scFv-ranpirnase (rantoxin) to the EGFR protein using molecular dynamic simulations.
- To evaluate the in vitro efficacy of the produced rantoxin against EGFR-expressing cancer cells.
Main Methods:
- Engineered Rana pipiens RNase to bind to the scFv of human anti-EGFR antibody.
- Utilized molecular dynamic simulations to assess protein stability and binding interactions.
- Synthesized the rantoxin construct, transfected it into CHO-K1 cells, and selected positive clones using neomycin and real-time PCR.
- Confirmed recombinant protein production via SDS-PAGE and Western blot analysis.
- Assessed immunotoxin function and determined IC50 values in A431 (cancer) and HEK293 (EGFR-negative) cells.
Main Results:
- Molecular dynamic simulations confirmed the stability of rantoxin and its effective binding to EGFR, specifically involving amino acids I467, S468, Q408, and H409.
- SDS-PAGE and Western blot analyses validated rantoxin production, with PCR indicating a fourfold higher mRNA expression in the T3 colony compared to GAPDH.
- In vitro assays showed potent cytotoxicity against A431 cancer cells with an IC50 of 22.4 ± 3 nM, while exhibiting minimal activity against EGFR-negative HEK293 cells (>620.4 ± 5 nM).
Conclusions:
- The engineered scFv-ranpirnase (rantoxin) is a stable and effective immunotoxin targeting EGFR.
- Rantoxin demonstrates significant cancer cell-killing potential with high specificity for EGFR-expressing tumors.
- These findings suggest that rantoxin holds considerable promise as a novel anticancer therapeutic agent.
