Project Summary Abstract Evimero NCATS P1 OPE Project In the proposed work, we examine the feasibility of using target-specific polyclonal antibodies to intercept and neutralize bioactive molecules in the gut microbiome milieu of humans with microbiome-driven inflammation and loss of gut barrier function. Such molecules are the “bridge” between a taxonomically-disordered, or dysbiotic microbiome, and the deleterious effects such microbiomes can have on human tissue. Upregulated inflammatory signaling and loss of barrier integrity are two general, systems biology-level changes common to multiple microbiome-associated disorders (MADS); by demonstrating our ability to neutralize these core mediators of pathology, we will establish the feasibility, and prove the concept, of our approach. The present project is enabled by work done by our collaborators at Oak Ridge National Laboratory, who identified six protein classes as overabundant in metaproteomic surveys of fecal microbiomes of those with inflammatory disorders of the gut (the “ORNL Protein Examples,” or “OPEs.”) Each of these proteins has known impacts on colonic inflammation, loss of barrier function, or both, from prior studies. Here, we explore neutralization of their impacts by polyclonal antibodies extracted from the eggs of laying hens immunized with the target proteins of interest. Known as IgY (immunoglobulin Y), these antibodies have cost and performance advantages superior to either monoclonal antibodies or mammalian polyclonals. In this study’s Specific Aims we 1) Determine the impact of each OPE in a sophisticated human stem-cell-derived in vitro model of human colonic epithelia and discover optimal exposure levels generating inflammatory or barrier-loss signals, and then 2) Determine the ability of each IgY antibody to neutralize those physiological impacts in the same model. Information gleaned from this project will support our broad, long-term objectives, modulating microbiome milieus to restore those protective pathways by mitigating damaging ones. Once the principle of selective, polyclonal antibody-mediated functional modulation has been demonstrated here, we will be well-positioned to obtain support for more ambitious – and costly – primary studies in multi-omics (metagenomics, metabolomics, metaproteomics). These studies, assisted by artificial intelligence/machine learning, will generate detailed microbiome molecular “maps,” revealing critical microbiome pathways (CMPs) that mediate final impact of dysbiosis on the host. Those pathways will identify specific “nodes” at which a pathway can take a health- promoting or health-impairing direction. Those nodes will represent specific proteins whose inhibition will tip the prevailing balance in a favorable direction – and become the final, precise targets for our antibody-mediated neutralization. We expect to combine multiple individually-targeted IgY antibodies into a new family of drugs called critical microbiome pathway inhibitors (CMPIs), rationally designed to block multiple critical pathways in a coordinated fashion that will ultimately be preventive or curative of many different MADs.

Project Narrative Evimero NCATS P1 OPE Project Microbiomes – the vast ecosystems of bacteria, viruses, and other microorganisms that line our intestines, our skin, and in fact everywhere our bodies encounter the outside world – are turning out to be crucial players in maintaining our health. Microbiomes get imbalanced, or “dysbiotic,” when they are exposed to poor diet, too many antibiotics, or from other diseases – and that can contribute to a wide variety of diseases, including some of our most challenging, long-term chronic disorders like inflammatory bowel disease, some cancers, and even Parkinson's or Alzheimer's diseases relevant to hundreds of thousands of people. The technology we’re working on here will systematically eliminate, or neutralize, biologically active proteins and other molecules that connect the imbalanced microbiome to human disease; we expect this to revolutionize discovery of new drugs that can heal broken microbiomes and prevent or treat diseases for which we have no current treatment strategy.