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Published on: September 17, 2019
A Novel Immunotoxin Targeting Epithelial Cell Adhesion Molecule Using Single Domain Antibody Fused to Diphtheria
Reyhaneh Roshan1, Shamsi Naderi1, Mahdi Behdani1
1Venom and Biotherapeutics Molecules Laboratory, Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
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.
Insights
A novel anti-Epithelial Cell Adhesion Molecule immunotoxin (α-EpCAM IT) effectively targets EpCAM-overexpressing cancer cells, inducing apoptosis and reducing tumor growth. This targeted therapy shows potential for treating various adenocarcinomas.
Area of Science:
- Oncology
- Molecular Biology
- Immunotherapy
Background:
- Epithelial Cell Adhesion Molecule (EpCAM) is highly expressed in several adenocarcinomas, making it a promising therapeutic target.
- Targeting EpCAM offers a potential strategy for developing novel cancer treatments.
Purpose of the Study:
- To develop and evaluate an anti-EpCAM immunotoxin (α-EpCAM IT) for cancer therapy.
- To assess the efficacy of α-EpCAM IT against EpCAM-expressing cancer cells both in vitro and in vivo.
Main Methods:
- Genetic fusion of an anti-EpCAM nanobody (α-EpCAM Nb) to a truncated diphtheria toxin to create α-EpCAM IT.
- Recombinant protein expression, purification, and refolding.
- In vitro cytotoxicity and apoptosis assays (MTT, Annexin V-FITC-PI) on EpCAM-expressing (MCF7) and non-expressing (HEK293, HUVEC) cells.
- In vivo anti-tumor activity evaluation in a mouse xenograft model.
Main Results:
- Successful expression, purification, and refolding of α-EpCAM IT.
- α-EpCAM IT demonstrated dose- and time-dependent anti-proliferative effects on MCF-7 cells.
- No significant anti-proliferative activity was observed on HEK293 and HUVEC cells.
- α-EpCAM IT significantly increased the apoptotic rate in MCF-7 cells.
- In vivo studies showed significant tumor size reduction with α-EpCAM IT treatment.
Conclusions:
- The developed α-EpCAM IT exhibits specific cytotoxicity and apoptosis-inducing effects on EpCAM-overexpressing cancer cells.
- α-EpCAM IT demonstrates significant anti-tumor activity in vivo, supporting its potential as a novel cancer therapeutic.
- Targeted inhibition of EpCAM using immunotoxins represents a promising strategy for adenocarcinoma treatment.
