title: Increasing the antitumor effect of an EpCAM-targeting fusion toxin by facile click PEGylation.
abstract: Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I-truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA″ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA″, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.
authors.affiliation: Corresponding Authors: Andreas Plückthun, Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. plueckthun@bioc.uzh.ch.
authors.forename: Manuel
authors.initials: M
authors.lastname: Simon
authors.forename: Nikolas
authors.initials: N
authors.lastname: Stefan
authors.forename: Lubor
authors.initials: L
authors.lastname: Borsig
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.forename: Uwe
authors.initials: U
authors.lastname: Zangemeister-Wittke
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D057930:Click Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D000071858:Epithelial Cell Adhesion Molecule; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics; D005260:Female; D019073:HT29 Cells; D006801:Humans; D061986:MCF-7 Cells; D008819:Mice, Nude; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D011092:Polyethylene Glycols / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D020349:Surface Plasmon Resonance; D047368:Tumor Burden / Q000187:drug effects; D037521:Virulence Factors / Q000737:chemistry* / Q000235:genetics; D023041:Xenograft Model Antitumor Assays; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D005098:Exotoxins; D011092:Polyethylene Glycols; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C499379:EPCAM protein, human