Development of high-throughput methods used to identify and characterize novel interactions within the human secretome with focus on the Ig and TNF receptor superfamilies

Abstract

Abstract While it is estimated that roughly 15% of the human genome encodes secreted proteins, many remain “orphans” with no known ligand or are poorly characterized. Even for those with known ligands, new binding partners continue to be identified that fundamentally reshape our understanding of their role in specific biological processes and diseases (i.e. PD-L1:B7-1, ICOS-L:CD28, PD-L2:RGMb). This highlights the need for systematic screening efforts to identify such interactions, broadening our general knowledge while also providing potential new targets for therapeutic intervention. To this end we have developed a set of novel technologies using both cell microarray and high-throughput flow cytometry techniques to screen for novel extracellular protein: protein interactions. Initially we focused on screening for interactions within the Ig and TNF receptor superfamilies, two protein families that are the central underpinning of our immune response but for which much is still unknown. We have generated two expression-validated libraries for cell-based screening, a 366 member Type I expression library of Ig and TNF receptor proteins and a ~3800 member full-length human plasma membrane protein library encompassing much of the human membrane proteome. Through rounds of screening we identified novel interactions for B7-1 (CD80), ICOS-L, TrkA and TrkC. The identified interactions were validated biochemically and are being characterized for their biological significance. Additionally we are using these technologies to characterize the binding interfaces of both known and newly discovered protein complexes as well as to screen for potential off-target interactions of protein-based therapeutics.