Directed Evolution of High-Affinity Antibody Mimics Using mRNA Display Original Research Article

We constructed a library of >1012 unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-α were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-α with dissociation constants (Kd) between 1 and 24 nM. After affinity maturation, the lowest Kd measured was 20 pM. Selected antibody mimics were shown to capture TNF-α when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.