title: Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells.
abstract: Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Lv
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China. sunaiyou@ecust.edu.cn.
authors.forename: Aiyou
authors.initials: A
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Yaling
authors.initials: Y
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003064:Cnidarian Venoms / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000473:pathology*; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D003064:Cnidarian Venoms; D000922:Immunotoxins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Cancer cell targeting; Gigantoxin-4; HER2/neu; Immunotoxins; Ovarian carcinoma SK-OV-3 cell