Design and construction of scFv-PE35KDEL as a novel immunotoxin against human epidermal growth factor receptor 2 for cancer therapy
© 2023. The Author(s), under exclusive licence to Springer Nature B.V..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:45 |
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| Enthalten in: |
Biotechnology letters - 45(2023), 4 vom: 29. Apr., Seite 537-550 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Shariaty Vaziri, Zahra [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 28.03.2023 Date Revised 28.03.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1007/s10529-023-03360-4 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM353140910 |
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| 245 | 1 | 0 | |a Design and construction of scFv-PE35KDEL as a novel immunotoxin against human epidermal growth factor receptor 2 for cancer therapy |
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| 500 | |a Date Revised 28.03.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2023. The Author(s), under exclusive licence to Springer Nature B.V. | ||
| 520 | |a 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) | ||
| 520 | |a 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 | ||
| 520 | |a 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) | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Breast cancer | |
| 650 | 4 | |a Human epidermal growth factor receptor 2 (HER2) | |
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| 700 | 1 | |a Shafiee, Fatemeh |e verfasserin |4 aut | |
| 700 | 1 | |a Akbari, Vajihe |e verfasserin |4 aut | |
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