Selection and Characterization of Her2 Binding-designed Ankyrin Repeat Proteins

Zahnd, Christian; Pecorari, Frédéric; Straumann, Nadine; Wyler, Emanuel; Plückthun, Andreas

doi:10.1074/jbc.m602547200

Cited by 100 publications

(86 citation statements)

References 30 publications

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“…12 and cloned into pDST67 (20). DARPin pools were screened by a crude extract ELISA according to previous studies (12,47). Protein sequences of DARPins subjected to detailed analysis in this study are listed in Fig.…”

Section: Methodsmentioning

confidence: 99%

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Kummer

Parizek

Rube

et al. 2012

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

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126

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We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state-specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.intrabodies | X-ray crystallography

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Section: Methodsmentioning

confidence: 99%

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Kummer

Parizek

Rube

et al. 2012

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

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126

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“…The resulting DARPin was sequenced and designated Off7DM. The anti-EpCAM DARPin Ec1 (12) has no internal methionine codons.…”

Section: Site-directed Mutagenesis Of Darpinsmentioning

confidence: 99%

“…We previously generated high-affinity DARPins targeting various tumor-associated antigens, including members of the EGF receptor family (11,12) and the epithelial cell adhesion molecule (EpCAM; ref. 13).…”

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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“…Derived from naturally occurring ankyrin repeat proteins, they have been engineered by a consensus design approach. DARPins have been selected from libraries via ribosome or phage display to bind to a wide range of different protein targets (18,(21)(22)(23)(24)(25)(26). DARPins are very well expressed in the cytoplasm of Escherichia coli, monomeric in solution, highly soluble, and very stable; moreover, they can easily be fused to other protein domains to generate multispecific or multivalent DARPins or be chemically conjugated to other molecules (27)(28)(29).…”

Section: The Egf Receptor (Egfr) Has Been Implicated In the Developmentioning

confidence: 99%

Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling

Boersma

Chao

Steiner

et al. 2011

Journal of Biological Chemistry

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Background:The EGF receptor (EGFR) is an important therapeutic target. Results: Bispecific anti-EGFR designed ankyrin repeat proteins (DARPins), alternative targeting molecules efficiently produced in bacteria, were shown to inhibit A431 cell proliferation and receptor recycling. Conclusion: One bispecific construct containing four DARPins showed a biological activity superior to that of the registered antibody cetuximab. Significance: Bispecific DARPins may form building blocks for tomorrow's cancer therapeutics.

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Selection and Characterization of Her2 Binding-designed Ankyrin Repeat Proteins

Cited by 100 publications

References 30 publications

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

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

Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling

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