(467f) Evolving a 45-Amino Acid Ligand Scaffold for Enhanced Stability and Tumor Targeting

Protein scaffolds provide a platform for engineering ligands for targeted delivery. Relative to antibodies, small scaffolds can provide superior stability, production, conjugation, and distribution – via enhanced permeability and diffusion as well as rapid clearance for imaging applications. Through a systematic computational analysis of domains within the Protein Data Bank, we identified the 45-amino acid Gp2 domain as a candidate protein scaffold for molecular recognition. We diversified twelve amino acids in two adjacent loops within this scaffold in a yeast surface display library and screened for binders to several protein targets. Novel ligand discovery and directed evolution yielded binders with affinities as strong as 200±100 pM and no observable nonspecific binding. Circular dichroism demonstrated that evolved ligands have secondary structure comparable to the wild-type protein. Midpoints of thermal denaturation range from 70±4ºC to 80±1ºC for evolved ligands relative to 67±4º for wild-type. Further exploration of this unexpected stabilization upon diversification and selection revealed that, unlike evolved binders, a substantial fraction of the unselected naïve library is not readily produced in the soluble fraction in E. coli. Thus, we are performing both fundamental studies and directed evolution to improve the stability and solubility of the domain and the combinatorial library to further enhance the utility of this scaffold. Nevertheless, the original library has yielded binders to all attempted targets. We have evolved binders to epidermal growth factor receptor with nanomolar affinity. We will discuss clone characterization and application to tumor targeting including in vivo biodistribution via molecular imaging and excised tissue activity analysis of tumor xenografted mice.