Increasing the antitumor effect of an EpCAM-targeting fusion toxin by facile click PEGylation
Manuel Simon1, Nikolas Stefan, Lubor Borsig
1Corresponding Authors: Andreas Plückthun, Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. plueckthun@bioc.uzh.ch.
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.
Insights
This study enhances cancer therapy fusion toxins by using click chemistry to attach polyethylene glycol (PEG). This improves their circulation time and tumor targeting, boosting anti-cancer efficacy.
Area of Science:
- Bioconjugation Chemistry
- Cancer Therapeutics
- Protein Engineering
Background:
- Fusion toxins for cancer therapy suffer from short circulation half-lives, limiting tumor delivery and effectiveness.
- Improving the pharmacokinetic profile of these toxins is crucial for enhancing their therapeutic potential.
Purpose of the Study:
- To improve the pharmacokinetics and tumor-targeting efficacy of a fusion toxin by bioorthogonal conjugation with polyethylene glycol (PEG).
- To utilize strain-promoted click chemistry for site-specific PEGylation of a designed ankyrin repeat protein (DARPin)-based fusion toxin.
Main Methods:
- Engineered a fusion toxin (Ec1-ETA″) incorporating azidohomoalanine for click PEGylation.
- Characterized the mono-PEGylated fusion toxin using biochemical analyses.
- Assessed in vitro cytotoxicity and in vivo tumor targeting in preclinical cancer models.
Main Results:
- Achieved facile, site-specific mono-PEGylation of the fusion toxin via click chemistry, yielding a homogeneous product.
- The PEGylated toxin maintained high binding affinity and potent cytotoxicity (femtomolar range).
- Demonstrated a significant increase in elimination half-life (approx. 6-fold) and AUC (approx. 7-fold) in vivo, leading to enhanced and prolonged anti-tumor effects.
Conclusions:
- Bioorthogonal click chemistry offers a powerful strategy to enhance the pharmacokinetics and efficacy of engineered fusion toxins.
- This approach overcomes limitations of current fusion toxin designs for improved cancer therapy.
- Combining DARPin technology with click PEGylation presents a promising avenue for developing next-generation cancer therapeutics.
