Engineering Neo-Epitope Antibodies to Investigate Proteolytic Signaling Networks In Cells and Patient Samples

Proteolysis is an essential component of a range of biological processes, including cell proliferation and cell death, and has been implicated in a variety of pathological processes, such as cancer, arthritis, neurodegenerative disorders, and viral infections (e.g. HIV and influenza).  Systems biology efforts have globally mapped complex proteolytic pathways within genetically homogeneous cellular populations.  However, single cell analysis of the proteolytic signaling networks within these populations has elucidated remarkable heterogeneity in response to various stimuli, which can significantly influence the response to therapeutic drugs. These studies were ultimately limited by the lack of molecular tools to monitor key nodes within these signaling networks.  Since very few monoclonal antibodies or other detection reagents have been developed with both high affinity and specificity toward the N- or C-terminal cleavage junctions (neo-epitopes) produced by protease cleavage, there exists a growing need for the development of novel tools to monitor key proteolytic events in a variety of biological contexts.  Conventionally, the development of highly specific monoclonal antibodies by hybridoma technologies is costly ($8000-10,000 per antibody), low throughput, and performed with no guarantee for epitope specificity. Our work aims to apply in vitro phage selection technology and large (>10¹⁰), diverse antibody libraries to develop novel neo-epitope antibodies to detect protein substrates cleaved by the caspase family of proteases during apoptosis.

Using a novel N-terminal enrichment strategy and mass spectrometry-based identification, the Wells lab has mapped the precise location of caspase-mediated cleavage within >1500 proteins.  We have leveraged this knowledge to develop a high-throughput, recombinant platform to generate neo-epitope antigens.  In parallel, we have also developed several novel antibody libraries with design strategies that involve a mixture of structure-based and combinatorial methods to target neo-epitopes and peptides in general.  We have successfully generated several neo-epitope specific antibodies that are currently undergoing biophysical and biological characterization. Future work with this novel tool will include a single cell, systems-level analysis of the apoptotic proteolytic signaling networks and elucidate potential changes to the network in response to various external (e.g. chemotherapeutic drugs) and internal stimuli (e.g. removal of protein nodes).  Furthermore, these tools will aid in the detection and quantification of proteolytic peptides derived from apoptosis within samples from patients undergoing chemotherapy. Ultimately, our high-throughput platform will facilitate the rapid, robust generation of highly specific monoclonal antibodies to detect other therapeutically relevant peptides and post-translational modifications, such as phosphorylation or acetylation.