Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein 
overexpressed in human epithelioma but with relatively low expression in normal 
epithelial tissues. To exploit this differential expression pattern for targeted 
cancer therapy, an EpCAM-targeted immunotoxin was developed and its antitumor 
activity was investigated in vitro. An immunotoxin (scFv2A9-PE or APE) was 
constructed by genetically fusing a truncated form (PE38KDEL) of Pseudomonas 
aeruginosa exotoxin with an anti-EpCAM single-chain variable fragment (scFv). 
ELISA and flow cytometry were performed to verify immunotoxin (scFv2A9-PE or 
APE) antigen-binding activity with EpCAM. Cytotoxicity was measured by MTT 
assay. Confocal microscopy was used to observe its cellular localization. The 
results of ELISA and flow cytometry revealed that the immunotoxin efficiently 
recognized recombinant and natural EpCAM. Its antigen-binding activity was 
relatively lower than 2A9. MTT assay confirmed potent reduction in 
EpCAM-positive HHCC (human hepatocellular carcinoma) cell viability (IC50 
50 pM). Immunofluorescence revealed that the immunotoxin localized to 
endoplasmic reticulum 24 h later. In conclusion, we described the development of 
an EpCAM-targeted immunotoxin with potent activity against tumor cells, which 
may lay the foundation for future development of therapeutic antibody for the 
treatment of EpCAM-positive tumors.