Potent and selective eradication of tumor cells by an EpCAM-targeted Ras-degrading enzyme

Valentina Palacio-Castañeda; Bas van de Crommert; Elke Verploegen; Mike Overeem; Jenny van Oostrum; Wouter P R Verdurmen

doi:10.1016/j.omto.2023.06.002

Potent and selective eradication of tumor cells by an EpCAM-targeted Ras-degrading enzyme

Mol Ther Oncolytics. 2023 Jun 27:30:16-26. doi: 10.1016/j.omto.2023.06.002. eCollection 2023 Sep 21.

Authors

Valentina Palacio-Castañeda¹, Bas van de Crommert¹, Elke Verploegen¹, Mike Overeem¹, Jenny van Oostrum¹, Wouter P R Verdurmen¹

Affiliation

¹ Department of Medical BioSciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.

Abstract

Despite decades of efforts, an urgent need remains to develop tumor cell-selective rat sarcoma (Ras)-targeting therapies that can treat patients with Ras-driven tumors. Here we report modular engineered proteins that degrade Ras selectively in tumor cells that overexpress the tumor cell marker epithelial cell adhesion molecule (EpCAM) by fusing the Ras degrader Ras-Rap1-specific endopeptidase with the translocation domain of the Pseudomonas aeruginosa exotoxin A (ETA) or diphtheria toxin (DT). Redirection to EpCAM is achieved by a designed ankyrin repeat protein. In two-dimensional tumor cell cultures, complete degradation of Ras proteins after 24 h was observed with EpCAM-targeted Ras degraders fused to ETA or DT in EpCAM-overexpressing MCF7 and HCT116 cells, with median inhibition concentration values at sub-nanomolar levels. The viability of EpCAM-low non-cancerous fibroblasts remained unaffected. In a three-dimensional (3D) tumor-on-a-chip system that mimics the natural tumor microenvironment, effective Ras degradation and selective toxicity toward tumor cells, particularly with the ETA-fused constructs, was determined on-chip. To conclude, we demonstrate the potential of modular engineered proteins to kill tumor cells highly selectively by simultaneously exploiting EpCAM as a tumor-specific cell surface molecule as well as Ras as an intracellular oncotarget in a 3D system mimicking the natural tumor microenvironment.

Keywords: Pseudomonas aeruginosa exotoxin A; Ras-Rap1-specific endopeptidase; designed ankyrin repeat protein; diphtheria toxin; epithelial cell adhesion molecule; tumor-specific targeting.