Selection of high-affinity Centyrin FN3 domains from a simple library diversified at a combination of strand and loop positions

Alternative scaffold molecules represent a class of proteins important to the study of protein design and mechanisms of protein-protein interactions, as well as for the development of therapeutic proteins. Here, we describe the generation of a library built upon the framework of a consensus FN3 domain sequence resulting in binding proteins we call Centyrins. This new library employs diversified positions within the C-strand, CD-loop, F-strand and FG-loop of the FN3 domain. CIS display was used to select high-affinity Centyrin variants against three targets; c-MET, murine IL-17A and rat TNFα and scanning mutagenesis studies were used to define the positions of the library most important for target binding. Contributions from both the strand and loop positions were noted, although the pattern was different for each molecule. In addition, an affinity maturation scheme is described that resulted in a significant improvement in the affinity of one selected Centyrin variant. Together, this work provides important data contributing to our understanding of potential FN3 binding interfaces and a new tool for generating high-affinity scaffold molecules.