DARPin

Abstract: We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.

Abstract: Designed ankyrin repeat proteins (DARPins) can recognize targets with specificities and affinities that equal or surpass those of antibodies, but because of their robustness and extreme stability, they allow a multitude of more advanced formats and applications. This review highlights recent advances in DARPin design, illustrates their properties, and gives some examples of their use. In research, they have been established as intracellular, real-time sensors of protein conformations and as crystallization chaperones. For future therapies, DARPins have been developed by advanced, structure-based protein engineering to selectively induce apoptosis in tumors by uncoupling surface receptors from their signaling cascades. They have also been used successfully for retargeting viruses. In ongoing clinical trials, DARPins have shown good safety and efficacy in macular degeneration diseases. These developments all ultimately exploit the high stability, solubility, and aggregation resistance of these molecules, permitting a wide range of conjugates and fusions to be produced and purified.