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Updated: Aug 9, 2025
Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo
Published on: January 7, 2019
Design and construction of scFv-PE35KDEL as a novel immunotoxin against human epidermal growth factor receptor 2 for
Zahra Shariaty Vaziri1, Fatemeh Shafiee1, Vajihe Akbari2
1Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
Propose:
Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL).
Methods:
The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni2+ affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay.
Results:
In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25 °C and 1 mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7 mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC50 ~ 95 nM) compared with HER2-negative cells, MDA-MB-23 (IC50 ˃ 200 nM).
Conclusion:
This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein.
Insights
A novel immunotoxin targeting HER2-overexpressing breast cancer cells was developed. This anti-HER2 IT demonstrated significant toxicity against cancer cells in vitro, showing potential for targeted cancer therapy.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- Human epidermal growth factor receptor 2 (HER2) is a key target in breast cancer therapy.
- Immunotoxins offer a targeted approach to cancer treatment by combining antibody-based targeting with cytotoxic payloads.
Purpose of the Study:
- To design and produce a novel immunotoxin (anti-HER2 IT) targeting HER2-overexpressing cancer cells.
- To evaluate the in silico structure, expression, purification, and in vitro cytotoxicity of the novel immunotoxin.
Main Methods:
- A fusion protein was constructed using anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified Pseudomonas exotoxin (PE35KDEL).
- In silico modeling predicted the protein's structure and HER2 interaction.
- Protein expression in E. coli, purification via Ni2+ affinity chromatography, and refolding by dialysis were performed.
- Cytotoxicity was assessed using MTT assays on HER2-positive and HER2-negative breast cancer cell lines.
Main Results:
- In silico studies confirmed high affinity to HER2 and effective linker function.
- The anti-HER2 IT was successfully expressed, purified (45.7 mg/L), and refolded.
- The immunotoxin exhibited significantly higher toxicity against HER2-overexpressing cells (BT-474, IC50 ~95 nM) compared to HER2-negative cells (MDA-MB-23, IC50 >200 nM).
Conclusions:
- The novel anti-HER2 immunotoxin demonstrates potent and targeted cytotoxicity against HER2-overexpressing cancer cells in vitro.
- This engineered immunotoxin holds promise as a therapeutic candidate for HER2-targeted cancer therapy.
- Further in vitro and in vivo studies are warranted to validate its efficacy and safety.
