Yeast Display of Single-Chain Fc Fragments with Modulated Binding of Effector Molecules

Yeast display platform has proven instrumental for directed evolution of diverse proteins towards altered binding specificity, affinity, or improved biophysical properties. Particularly in antibody display, this method has delivered variants that are featured today as integral parts of approved therapeutic agents. While broadly used for discovery of binding agents, yeast display has also been applied for optimization of properties of Fc fragment. Among the bispecific antibody formats, which come in a variety of different molecular architectures, the single-chain Fc is a less explored alternative. This format offers novel opportunities for positioning of binding sites in a multispecific molecule, however, necessitates special design due to its unique features such as linker. We tested the amenability of yeast system for the efficient presentation of the single-chain IgG Fc and characterized the displayed fragments for binding of structural markers and Fc gamma receptors. Taken together, this chapter sets the base for successful use of yeast display in isolation of single-chain Fc variants with tailored ligand-binding properties.