DARPins Recognizing the Tumor-Associated Antigen EpCAM Selected by Phage and Ribosome Display and Engineered for Multivalency

Stefan, Nikolas; Martin-Killias, Patricia; Wyss-Stoeckle, Sascha; Honegger, Annemarie; Zangemeister‐Wittke, Uwe; Plückthun, Andreas

doi:10.1016/j.jmb.2011.09.016

Cited by 112 publications

(132 citation statements)

References 32 publications

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“…Also the EpCAM-specific DARPin Ec1 (ref. 20) was efficiently displayed on the particle surface ( Supplementary Fig. 7) and 13).…”

Section: Resultsmentioning

confidence: 98%

“…16) and the EpCAM-specific DARPin Ec1 (ref. 20) were amplified by PCR using the primer pair DARPin-for/DARPin-rev or DARPin-for/DARPin EC1-rev respectively, (Supplementary Table 1) and were inserted into pGFP (green fluorescent protein)-VP2 (ref. 21) by sticky-end ligation using AgeI and BsrGI restriction sites resulting in plasmids pHer2-VP2, pCD4-VP2 and pEpCAM-VP2.…”

Section: Methodsmentioning

confidence: 99%

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Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

et al. 2015

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We describe receptor-targeted adeno-associated viral (AAV) vectors that allow genetic modification of rare cell types ex vivo and in vivo while showing no detectable off-targeting. Displaying designed ankyrin repeat proteins (DARPins) on the viral capsid and carefully depleting DARPin-deficient particles, AAV vectors were made specific for Her2/neu, EpCAM or CD4. A single intravenous administration of vector targeted to the tumour antigen Her2/neu was sufficient to track 75% of all tumour sites and to extend survival longer than the cytostatic antibody Herceptin. CD4-targeted AAVs hit human CD4-positive cells present in spleen of a humanized mouse model, while CD8-positive cells as well as liver or other off-target organs remained unmodified. Mimicking conditions of circulating tumour cells, EpCAM-AAV detected single tumour cells in human blood opening the avenue for tumour stem cell tracking. Thus, the approach developed here delivers genes to target cell types of choice with antibody-like specificity.

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“…Also the EpCAM-specific DARPin Ec1 (ref. 20) was efficiently displayed on the particle surface ( Supplementary Fig. 7) and 13).…”

Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

et al. 2015

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“…3, 4, and 6). Because we can generate such retargeting DARPin modules to essentially any target using ribosome or phage display (37,44,50,51,(58)(59)(60)(61)(62)(63)(64), we can exploit these capabilities [e.g., in tumor therapy in animal models (63,(65)(66)(67)] to use Ad5 to deliver payloads with tumorcontrolling potential.…”

Section: Discussionmentioning

confidence: 99%

“…They previously had been reported to bind EpCAM specifically at different epitopes (51). Adapters containing either retargeting module were used to coat Ad5luc at an adapter-to-knob ratio of 1:1 and their transduction efficiency was investigated (Fig.…”

Section: Shp As N-terminal Fusionsmentioning

confidence: 99%

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Dreier

Honegger

Hess

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.protein design | tumor targeting | viral retargeting | X-ray crystallography | protein engineering

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“…13). EpCAM, also known as CD326, is a 40 kDa type I membrane glycoprotein frequently expressed in human carcinomas, and involved in cell proliferation by linking to components of the Wnt signaling pathway and regulators of the cell cycle (14,15).…”

Section: Introductionmentioning

confidence: 99%

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Simon

Stefan

Borsig

et al. 2014

Molecular Cancer Therapeutics

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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 00 ) 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 00 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 00 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 welldefined 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 00 , 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. Mol Cancer Ther; 13(2); 375-85. Ó2013 AACR.

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DARPins Recognizing the Tumor-Associated Antigen EpCAM Selected by Phage and Ribosome Display and Engineered for Multivalency

Cited by 112 publications

References 32 publications

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

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