Details

Autor(en) / Beteiligte

• Schilling, Johannes
• Schöppe, Jendrik
• Plückthun, Andreas

Titel

From DARPins to LoopDARPins: Novel LoopDARPin Design Allows the Selection of Low Picomolar Binders in a Single Round of Ribosome Display

Ist Teil von

• Journal of molecular biology, 2014-02, Vol.426 (3), p.691-721

Ort / Verlag

England: Elsevier Ltd

Erscheinungsjahr

2014

Link zum Volltext

Quelle

ScienceDirect Journals (5 years ago - present)

Beschreibungen/Notizen

• Antibodies are the most versatile binding proteins in nature with six loops creating a flexible continuous interaction surface. However, in some molecular formats, antibodies are aggregation prone. Designed ankyrin repeat proteins (DARPins) were successfully created as alternative design solutions. Nevertheless, their concave shape, rigidity and incompletely randomized binding surface may limit the epitopes that can be targeted by this extremely stable scaffold. Combining conformational diversity and a continuous convex paratope found in many antibodies with the beneficial biophysical properties of DARPins, we created LoopDARPins, a next generation of DARPins with extended epitope binding properties. We employed X-ray structure determination of a LoopDARPin for design validation. Biophysical characterizations show that the introduction of an elongated loop through consensus design does not decrease the stability of the scaffold, consistent with molecular dynamics simulations. Ribosome-display selections against extracellular signal-regulated kinase 2 (ERK2) and four members of the BCL-2 family (BCL-2, BCL-XL, BCL-W and MCL-1) of anti-apoptotic regulators yielded LoopDARPins with affinities in the mid-picomolar to low nanomolar range against all targets. The BCL-2 family binders block the interaction with their natural interaction partner and will be valuable reagents to test the apoptotic response in functional assays. With the LoopDARPin scaffold, binders for BCL-2 with an affinity of 30pM were isolated with only a single round of ribosome display, an enrichment that has not been described for any scaffold. Identical stringent one-round selections with conventional DARPins without loop yielded no binders. The LoopDARPin scaffold may become a highly valuable tool for biotechnological high-throughput applications. [Display omitted] •DARPins are versatile alternative binding proteins, but with constant paratope shapes.•CDR-H3-like loops that retain scaffold stability and increase surface were designed.•Libraries with tailored diversity allow reaching additional epitopes on the target.•X-ray structures confirm design and intended loop conformation and flexibility.•Picomolar binders were obtained in a single round of ribosome display.