Development of Novel Bioaffinity Reagents for the Detection of Emerging IBD Biomarker Keratin 7

Abstract

Inflammatory bowel diseases (IBD) are chronic autoimmune conditions of the gastrointestinal tract, with ulcerative colitis and Crohn’s disease as the primary subtypes. Accurate diagnosis is mainly based on endoscopic imaging, since the most used inflammatory markers, faecal calprotectin and C-reactive protein, are not IBD specific. The increase in IBD incidence throughout recent decades and the difficulty of diagnosis denote the need for new diagnostic methods and biomarkers. Recently, increased intestinal keratin 7 (K7) expression has been linked to IBD. Since K7 is not expressed in healthy intestinal tissue, it could be utilized as an IBD-specific biomarker. The aim of this thesis project was the generation and validation of novel K7 binder molecules that could be utilized to detect and research K7 in IBD and to improve IBD diagnostics.

In this project, K7 binders were developed with phage display. Phage libraries expressing antibody fragments (Fab) and designed ankyrin repeat proteins (DARPin) were biopanned against recombinant K7 in search of novel binder molecules. After the discovery and characterization of K7 binders, the best candidates were expressed as alkaline phosphatase and SpyCatcher3 fusions. A novel method for assembling IgG-like molecules with SpyTag3-SpyCatcher3-technology on protein A column was established. Binder performance was assessed with immunoassays with recombinant K7 and K7 positive IBD patient stool, immunofluorescence microscopy and Western blot. As a result, several binder molecules that recognize recombinant K7 were isolated, of which, Fab molecules had slightly higher binding affinity and DARPin molecules were easier to produce. The isolated novel binders show promise for further development and optimization to properly recognize native K7 in different screening matrices and be used in in vitro diagnostic assays to support IBD diagnostics.