Abstract Enteric diseases, including both acute and chronic diarrhea and constipation, are among the most common ailments afflicting humans. Many enteric diseases, such C. difficile infection (CDI) and inflammatory bowel disease (IBD), are currently treated with systemically delivered biologics (e.g. bezlotoxumab for CDI and anti- TNF antibodies for IBD). However, since the GI lumen is also home to trillions of microbes that may cause sepsis if present in the blood, the GI tract has evolved over millions of years to strictly prohibit the diffusion of large molecules, such as proteins and peptides, between the GI lumen and the blood circulation. Consequently, the GI lumen is not readily accessible to systemically delivered biologics. In addition to reduced potency due to poor access, the treatment of enteric diseases with systemically delivered biologics also suffers from additional drug side effects due to the systemic exposure. We posit that orally delivered biologics, whose bailiwick is mostly confined within the GI lumen, would likely be much better suited for treating enteric diseases due to both increased local drug concentration and reduced systemic side effects. Unfortunately, since the primary function of the GI tract is food digestion, conventional biologics are unlikely to be fully active in the protease-rich environment of the GI lumen. In this project, we propose to develop both a protease-stable protein scaffold and a delivery platform custom-made for biologics-mediated treatment of enteric diseases. These studies will exploit an in vitro protein display technology recently developed in our lab that covalently links the displayed protein to its coding cDNA in a one-pot reaction. We will first employ directed evolution to enhance the protease stability of a designed ankyrin repeat protein (DARPin) (Aim 1). Concurrently, we will develop a probiotic yeast platform for in situ delivery of protease-stable DARPins to the GI tract (Aim 2). Finally, using CDI as a model enteric disease, we will perform preclinical development of a toxin-neutralizing and protease-stable DARPin and evaluate its efficacy in vivo (Aim 3). Successful completion of this project should bring forth both a novel protease stable protein scaffold and an efficient in situ protein delivery platform, and may greatly accelerate the development of biologics against diverse enteric diseases.