Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting

Stefan, Nikolas; Zimmermann, Martina; Simon, Manuel; Zangemeister-Wittke, Uwe; Plückthun, Andreas (2014). Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting. Bioconjugate chemistry, 25(12), pp. 2144-2156. American Chemical Society 10.1021/bc500468s

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Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA″), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA″, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA″ was much better tolerated than Ec1-ETA″; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.

Item Type:

Journal Article (Original Article)


04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Pharmacology

UniBE Contributor:

Stefan, Nikolas; Simon, Manuel and Zangemeister-Wittke, Uwe


600 Technology > 610 Medicine & health




American Chemical Society




Anita Dähler

Date Deposited:

19 Jan 2015 14:28

Last Modified:

11 Nov 2015 10:09

Publisher DOI:


PubMed ID:





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