(6c) Engineering Picomolar Affinity Into a 5 Kda Scaffold for Tumor Targeting

Engineered protein scaffolds are conserved stable frameworks upon which amino acid diversity can be introduced to develop novel function. Scaffolds serve as strong platforms for ligand discovery towards the multitude of clinically, scientifically, and industrially impactful targets. Of particular interest here are small protein scaffolds capable of improved permeability and distribution for targeting nascent tumors. We aim to develop a platform for engineering a very small (5 kDa), stable, scaffold capable of efficient evolution to picomolar affinity requisite for sustained delivery in vivo. Towards this end, we analyzed the secondary structure, stability, phylogenetic sequence diversity, and the accessible surface area and shape of the potential paratope of proteins throughout characterized proteomes to identify candidate scaffolds for engineering molecular recognition. We identified a 45 amino acid domain consisting of both beta strand and alpha helical content including two exposed loops amenable to mutation. We diversified twelve amino acids in these two loops using complementarity bias and length diversity to generate a combinatorial library for ligand discovery. Directed evolution was performed using yeast surface display and magnetic and fluorescence selections. Binders to three targets were isolated with affinities as strong as 160 pM. Biophysical characterization, including circular dichroism and thermal stability, will be presented. In vivo biodistribution, via molecular imaging and excised tissue activity analysis of tumor xenografted mice, will be shown. Robust, efficient engineering of small (5 kDa), stable ligands has strong potential for both diagnostic and therapeutic tumor targeting.