Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization

Cathrine Elisabeth Olsen; Lawrence H Cheung; Anette Weyergang; Kristian Berg; Daniel A Vallera; Michael G Rosenblum; Pål Kristian Selbo

doi:10.3390/jcm9010068

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization

J Clin Med. 2019 Dec 26;9(1):68. doi: 10.3390/jcm9010068.

Authors

Cathrine Elisabeth Olsen¹, Lawrence H Cheung², Anette Weyergang¹, Kristian Berg¹, Daniel A Vallera³, Michael G Rosenblum², Pål Kristian Selbo¹

Affiliations

¹ Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, N-0310 Oslo, Norway.
² Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
³ Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota, Masonic Cancer Center, Minneapolis, MN 55455, USA.

Abstract

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133^high and CD133^low cell lines and a CD133^neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133^high cells compared to CD133^low cells. No cytotoxic responses were detected in either CD133^high or CD133^low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133^low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133^high and CD133^low cancer cells.

Keywords: CD133; cancer stem cells; drug delivery; immunotoxins; photochemical internalization; photodynamic therapy; prominin-1; ribosome-inactivating proteins.

Abstract

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