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Published on: June 14, 2018
Production and introduction of a novel immunotoxin based on engineered RNase A for inducing death to Her1-positive
Ali Forouharmehr1,2, Mohammadreza Nassiri3, Shahrokh Ghovvati Roudsari4
1Department of Animal Science, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
Abstract:
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.
Insights
Researchers engineered Ribonuclease A (RNase A) to evade inhibitors and combined it with Cetuximab
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapy
Background:
- Ribonuclease A (RNase A) is a potential anti-cancer agent but is inhibited in the cytoplasm.
- Cetuximab targets the Her1 receptor, often overexpressed in cancers.
Purpose of the Study:
- To design and evaluate an immunotoxin comprising engineered RNase A and Cetuximab scFv for targeted cancer therapy.
- To enhance RNase A's stability and inhibitor evasion for improved therapeutic potential.
Main Methods:
- Site-directed mutagenesis to engineer RNase A, evading its inhibitor.
- Protein-protein docking to assess engineered RNase A interaction with ribonuclease inhibitor.
- Conjugation of engineered RNase A with Cetuximab single-chain variable fragment (scFv) via a cleavable linker.
- Expression and purification of wild-type RNase A, engineered RNase A, and the immunotoxin in E. coli.
- Western blot analysis for protein confirmation.
- RNA fragmentation assay to assess enzymatic activity.
- Cytotoxicity assays on Her1-positive and Her1-negative cell lines.
Main Results:
- Engineered RNase A maintained structural integrity and disulfide bonds, evading its inhibitor.
- Successful expression and purification of all protein constructs.
- Both wild-type and engineered RNase A demonstrated RNA degradation activity.
- The scFv-engineered RNase A immunotoxin exhibited potent cytotoxicity against Her1-positive cell lines (IC50 = 50 nM).
Conclusions:
- The engineered RNase A successfully evaded its inhibitor while retaining enzymatic activity.
- The Cetuximab scFv-engineered RNase A immunotoxin demonstrates targeted cytotoxicity against Her1-positive cancer cells.
- This engineered immunotoxin represents a promising therapeutic candidate for Her1-positive cancers.
