Rapid selection of high-affinity DARPins using click display.

Abstract

Rapid and scalable protein binder discovery platforms are essential for accelerating therapeutic and diagnostic development. Click display is a cell-free protein display technology that covalently links nascent proteins to their encoding cDNA in a one-pot reaction within 2 hours, enabling rapid in vitro selection. Although previously demonstrated to support directed evolution, enrichment efficiency during biopanning was slower than conventional phage display. Here, we report two modifications that significantly enhance click display selection efficiency: (i) selective elution of target-bound complexes using a cleavable biotin linker, and (ii) competitive displacement of non-specific bead binders using a biotinylated blocking protein. These improvements dramatically reduce amplification of non-specific binders and enable efficient enrichment of target-specific binders within three to four rounds of panning. Using a naïve DARPin library, we rapidly enriched binders to interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), identifying clones with low nanomolar binding affinities and receptor-blocking activity. We further applied the optimized protocol to affinity maturation of a PD-L1-binding DARPin, achieving >400-fold improved binding affinity. The best candidate, 1D10, exhibits sub-nanomolar PD-1 inhibition in competition ELISA. These results demonstrate that the optimized click display workflow enables rapid enrichment and affinity maturation of high-affinity DARPins across diverse targets. The platform provides a streamlined, transformation-free approach for accelerating protein engineering and directed evolution.