Engineering Affinity, Specificity and Stability in Protein Therapeutics

Mesotrypsin is a serine protease that is upregulated with tumor progression and associated with poor prognosis in many human cancers. In cancer models, mesotrypsin promotes tumor growth, invasion, and metastasis, making it an attractive target for therapeutic intervention. To date, no selective inhibitors against mesotrypsin, either natural or synthetic, have been reported. Developing selective inhibitors for mesotrypsin presents special challenges, as mesotrypsin shares high sequence homology and structural similarity with other serine proteases, and is resistant to inhibition by many polypeptide inhibitors. The human amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) offers an attractive scaffold for engineering mesotrypsin inhibitors, but has the inherent disadvantage of rapid cleavage by the enzyme. In preliminary studies, we have used directed evolution to generate a novel prototype mesotrypsin inhibitor, based on the APPI scaffold, possessing picomolar affinity and improved proteolytic resistance to mesotrypsin. Recently, we were able to identify highly selective novel mesotrypsin inhibitors based on this prototype for clinical translation as imaging and therapeutic agents. Specifically, we have used a yeast surface display platform and novel competitive screening strategy to identify selective mesotrypsin antagonists from inhibitor libraries. We have evaluated candidate inhibitors for mesotrypsin selectivity, proteolytic stability, and anticancer efficacy in cell culture models. Finally, we have performed preclinical evaluation of the best candidate as a targeting agent for tumor imaging and as a therapeutic. The proposed strategy is likely to produce mesotrypsin inhibitors of low toxicity and immunogenicity, with substantial translational potential as imaging agents and therapeutics.