Key words: Mucosal targeting, pIgR, Phage display, Automation, Amino acid substitution, Mutation analysis, GFP-fusion protein.
Delivery of therapeutic macromolecules, gene therapy vectors and diagnostics to tissue may be hampered by endothelial or epithelial barriers. In an effort to overcome this obstacle, delivery strategies exploiting innate trans-barrier transport mechanisms have been suggested. The polymeric immunoglobulin receptor (pIgR) directs formation and trans-epithelial transportation of secretory immunoglobulins that provides a first line of humoral defence at mucosal surfaces. Consequently, the pIgR-mediated mucosal secretion system potentially represents a route for targeted delivery to epithelial cells and mucosal surfaces. We aimed at identifying novel small peptides that bind to the extracellular domains of pIgR, known as the secretory component (SC), and that are specifically transcytosed across Madin Darby Canine Kidney (MDCK) cells transfected with human pIgR, by affinity selection from phage display libraries. Assuming that one key to identifying peptides, with the sought after properties, is the number of screened phage single clones, semi-automated screening procedures were designed. We have identified 5 novel SC binding phage displayed peptides by affinity selection using two libraries. One of the identified clones, C9-4, bind immobilized SC in a SpsA-like manner and is specifically transcytosed across MDCK cells transfected with human pIgR. Amino acid substitution mutation analysis revealed that all, but three, amino acids were indispensable to the selected peptide’s functionality as an SC binder. Furthermore we constructed and expressed C9-4 as a fusion to green fluorescent protein (GFP). Despite the successful affinity selection, transcytosis, and fusion protein construction and expression, interaction with immobilized SC could not be demonstrated in the new fusion format.