Evolution of an Interloop Disulfide Bond in High-Affinity Antibody Mimics Based on Fibronectin Type III Domain and Selected by Yeast Surface Display: Molecular Convergence with Single-Domain Camelid and Shark Antibodies

The 10th human fibronectin type III domain (10Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity 10Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the 10Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (1012 unique sequences). Here we describe the selection and affinity maturation of 10Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (107–109 different sequences), which were constructed by randomizing only 14 10Fn3 residues. The finding that two adjacent loops in human 10Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type 10Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the 10Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from libraries accessible to phage and yeast surface display methods.