title: Novel prodrug-like fusion toxin with protease-sensitive bioorthogonal PEGylation for tumor targeting.
abstract: 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.
authors.affiliation: Department of Biochemistry, Winterthurerstrasse 190, University of Zurich , CH-8057 Zurich, Switzerland.
authors.forename: Nikolas
authors.initials: N
authors.lastname: Stefan
authors.forename: Martina
authors.initials: M
authors.lastname: Zimmermann
authors.forename: Manuel
authors.initials: M
authors.lastname: Simon
authors.forename: Uwe
authors.initials: U
authors.lastname: Zangemeister-Wittke
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000086042:3C Viral Proteases; D036002:ADP Ribose Transferases / Q000737:chemistry*; D000818:Animals; D017089:Ankyrin Repeat; D000951:Antigens, Neoplasm / Q000737:chemistry; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D019540:Area Under Curve; D001427:Bacterial Toxins / Q000737:chemistry*; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000737:chemistry; D045744:Cell Line, Tumor; D057930:Click Chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D015195:Drug Design; D000071858:Epithelial Cell Adhesion Molecule; D005098:Exotoxins / Q000737:chemistry*; D005260:Female; D006207:Half-Life; D006801:Humans; D008810:Mice, Inbred C57BL; D011092:Polyethylene Glycols / Q000737:chemistry*; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism; D037521:Virulence Factors / Q000737:chemistry*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D000086042:3C Viral Proteases; D036002:ADP Ribose Transferases; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D015815:Cell Adhesion Molecules; D003546:Cysteine Endopeptidases; D000071858:Epithelial Cell Adhesion Molecule; D005098:Exotoxins; D011092:Polyethylene Glycols; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C499379:EPCAM protein, human