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

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

doi:10.3390/jcm9010068

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 CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low 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 CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Highlights

Cancer stem cells (CSC), or tumor initiating cells, are highly aggressive malignant cells that have gained stem cell biology including capacity to both self-renew and differentiate, making them responsible for maintaining tumor heterogeneity [1,2]
Since scFvCD133/rGelonin is independent of the glycosylation status of the receptor we may bypass potential problems with detecting different post-translational versions of CD133/prominin-1 [37]
This study is the first report on the development and production of a CD133-targeting recombinant fusion toxin aimed for the specific delivery by Photochemical Internalization (PCI)

Summary

Introduction

Cancer stem cells (CSC), or tumor initiating cells, are highly aggressive malignant cells that have gained stem cell biology including capacity to both self-renew and differentiate, making them responsible for maintaining tumor heterogeneity [1,2]. As differentiated cancer cells can dedifferentiate and gain stem cell biology after hypoxia-mediated changes in the tumor microenvironment (TME) [7] or as a response to. Med. 2020, 9, 68 therapy-mediated alteration of the TME [1], it is important to establish new therapeutic interventions that simultaneously target and kill both CSCs and differentiated cancer cells [8]. Such strategies may involve multimodality treatments including immunotoxins (IT) targeting e.g., CD133-expressing [9,10]

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Conclusion