title: Construction, expression, and characterization of a recombinant immunotoxin targeting EpCAM.
abstract: 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.
authors.affiliation: Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Minghua
authors.initials: M
authors.lastname: Lv
authors.affiliation: Department of Neurology, Chinese Navy General Hospital, Beijing 100048, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Qiu
authors.affiliation: Department of Geriatric Gastroenterology, Chinese People's Liberation Army General Hospital, Beijing 100853, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Yuanjie
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Chunmei
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
authors.affiliation: Department of Neurology, Chinese Navy General Hospital, Beijing 100048, China.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Qi
authors.affiliation: Department of Geriatric Gastroenterology, Chinese People's Liberation Army General Hospital, Beijing 100853, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.affiliation: Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China ; Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Kui
authors.initials: K
authors.lastname: Zhang
mesh_terms: D000941:Antigens / Q000737:chemistry; D000951:Antigens, Neoplasm / Q000737:chemistry*; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay; D000071858:Epithelial Cell Adhesion Molecule; D004848:Epithelium / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology / Q000628:therapy*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry
chemical_list: D000941:Antigens; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D017931:DNA Primers; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D000922:Immunotoxins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies