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A Novel Immunotoxin Targeting Epithelial Cell Adhesion Molecule Using Single Domain Antibody Fused to Diphtheria Toxin

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Abstract

Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in a variety of cancers such as colon, stomach, pancreas, and prostate adenocarcinomas. Inhibition of EpCAM is considered as a potential target for cancer therapy. In current study, anti-EpCAM immunotoxin (α-EpCAM IT) was developed using genetic fusion of α-EpCAM single domain antibody (nanobody) (α-EpCAM Nb) to truncated form of diphtheria toxin. The expression of recombinant α-EpCAM IT was induced by Isopropyl β-d-1-thiogalactopyranoside (IPTG) and confirmed by SDS-PAGE and western blot. Recombinant α-EpCAM IT was purified from the inclusion bodies and refolded using urea gradient procedure. The cytotoxicity and apoptosis activity of α-EpCAM IT on EpCAM over-expressing (MCF7), low-expressing (HEK293), and no-expressing (HUVEC) cells were evaluated by 3–4,5-Dimethylthiazol-2-yl (MTT) assay and annexin V-FITC-PI assay as well. In addition, anti-tumor activity of α-EpCAM IT was evaluated on nude mice bearing MCF7 tumor cells. Results showed success expression and purification of α-EpCAM IT. The α-EpCAM IT showed time and dose-dependent anti-proliferative activity on MCF-7 cells. However, α-EpCAM IT did not show any anti-proliferative activity on HEK293 and HUVEC cells as well. In addition, the annexin V-FITC-PI assay results showed that α-EpCAM IT significantly increased apoptotic rate in MCF-7 cells with no effect on HEK293 and HUVEC as well. Moreover, α-EpCAM IT significantly reduced tumor size in vivo study. The achieved results indicate the potential of designing α-EpCAM IT as a novel therapeutic for cancer therapy.

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Acknowledgements

This study was financially supported by Pasteur Institute of Iran

Funding

This study was funded by Pasteur Institute of Iran (Grant Number BD-9470).

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Authors and Affiliations

  1. Venom and Biotherapeutics Molecules Laboratory, Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Reyhaneh Roshan, Shamsi Naderi, Mahdi Behdani & Fatemeh Kazemi-Lomedasht

  2. Department of Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran

    Reza Ahangari Cohan

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  1. Reyhaneh Roshan
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  2. Shamsi Naderi
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  4. Reza Ahangari Cohan
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  5. Fatemeh Kazemi-Lomedasht
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Correspondence to Reza Ahangari Cohan or Fatemeh Kazemi-Lomedasht.

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Roshan, R., Naderi, S., Behdani, M. et al. A Novel Immunotoxin Targeting Epithelial Cell Adhesion Molecule Using Single Domain Antibody Fused to Diphtheria Toxin. Mol Biotechnol 65, 637–644 (2023). https://doi.org/10.1007/s12033-022-00565-2

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  • DOI: https://doi.org/10.1007/s12033-022-00565-2

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