A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering

Summary Small protein ligands can provide superior physiological distribution compared with antibodies, and improved stability, production, and specific conjugation. Systematic evaluation of the PDB identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α helix opposite a β sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 10⁸ mutants and directed evolution toward four targets yielded target-specific binders with affinities as strong as 200 ± 100 pM, T_ms from 65°C ± 3°C to 80°C ± 1°C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ± 8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold.