Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in
Cathrine Elisabeth Olsen1, Lawrence H Cheung2, Anette Weyergang1
1Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, N-0310 Oslo, Norway.
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.
Insights
A novel CD133-targeting immunotoxin (scFvCD133/rGelonin) combined with photochemical internalization (PCI) shows potent cancer cell killing. This combination therapy effectively targets CD133-expressing cancer cells, even those with low CD133 levels.
Area of Science:
- Oncology
- Biotechnology
- Molecular Biology
Background:
- CD133 (prominin-1) is a cancer stem cell marker implicated in tumor progression and therapy resistance.
- Immunotoxins (ITs) offer targeted cancer therapy, but their efficacy can be limited by intracellular delivery.
- Photochemical internalization (PCI) is an endosomal escape technology that enhances the delivery of macromolecules into the cell cytoplasm.
Purpose of the Study:
- To develop and evaluate a novel CD133-targeting immunotoxin, scFvCD133/rGelonin.
- To investigate the combination of scFvCD133/rGelonin with PCI for enhanced cancer cell killing.
- To assess the efficacy of this combination therapy in CD133-expressing and non-expressing cancer cells.
Main Methods:
- Recombinant construction of scFvCD133/rGelonin by fusing CD133-targeting scFv to gelonin ribosome-inactivating protein.
- Assessment of ribosome-inactivating protein (RIP) activity in cell-free assays.
- Evaluation of scFvCD133/rGelonin binding and intracellular accumulation using flow cytometry and fluorescence microscopy.
- Cytotoxicity assessment via MTT assay following PCI treatment in CD133-high, CD133-low, and CD133-negative cell lines.
Main Results:
- scFvCD133/rGelonin demonstrated specific binding and accumulation in CD133-high cells.
- The immunotoxin showed minimal intrinsic cytotoxicity at concentrations below 100 nM.
- PCI significantly enhanced the cytotoxic activity of scFvCD133/rGelonin, leading to log-fold reductions in cancer cell viability.
- PCI of scFvCD133/rGelonin was effective even in CD133-low cancer cells, outperforming PCI of free rGelonin.
Conclusions:
- PCI effectively promotes the cytotoxic activity of the otherwise non-toxic scFvCD133/rGelonin.
- The combination of CD133-targeting immunotoxin and PCI demonstrates significant potential for treating CD133-expressing and CD133-low cancers.
- This novel therapeutic strategy offers a promising approach for overcoming drug delivery challenges in cancer therapy.
