부산대학교 도서관

Proteins are a valuable class of therapeutics because proteins, like antibodies or non-antibody scaffolds, can bind their targets with high selectivity and affinity. The upper gastrointestinal tract, however, rapidly inactivates protein-based targeting reagents. Compared to injections, oral administration of protein therapies is favourable for treating gastrointestinal disease. In this thesis, I have developed a protein scaffold termed a gastrobody, that is suited to applications in the gastrointestinal tract. Gastrobodies are derived from Kunitz-type soybean trypsin inhibitor (SBTI). SBTI is resilient to digestion with stomach-like concentrations of pepsin and pH 2. Furthermore, SBTI is stable in the presence of intestinal proteases and bile acids, as well as being resilient to high temperatures. I used the protein modelling software Rosetta to assist design of a binding surface on SBTI. I also established phage display of SBTI to enable directed evolution. As proof of concept, gastrobody binders to domains of two toxins of the enteric pathogen Clostridium difficile were developed. After optimisation of the binding surface and affinity maturation, I identified gastrobodies that bound their target with nanomolar affinity. Gastrobodies bind their target after incubation with physiological concentrations of gastrointestinal proteases, including pepsin at pH 2. Lastly, I explored alternative gastrobody backbones using structural homologues of SBTI. The work presented in this thesis may provide a foundation for generating binding reagents for therapeutic and diagnostic use in the gastrointestinal tract.