Project Summary The goal of this project is to develop a novel immunologically designed probiotic therapy to inhibit gut inflammatory processes for the treatment of inflammatory bowel disease (IBD). Over 3 million adults in the U.S. suffer from IBD, an umbrella term encompassing two chronic inflammatory diseases of the gastrointestinal tract: Crohn’s disease (CD) and ulcerative colitis (UC)6. IBD is typically diagnosed in the second or third decades of life; it is life-long, and there is no cure. Current IBD treatments are systemic and can have serious side-effects. Novel therapies that are safe and effective, particularly restoring the natural interaction between the immune system and gut microbiome, are needed and would be life-changing for patients. Intestinal immune regulatory signals tightly govern healthy gut homeostasis, and their breakdown may result in IBD38. The human microbiome, harboring trillions of bacteria, is a critical regulator of these mechanisms. Commensal bacteria function to maintain intestinal epithelial barrier integrity and regulate innate and adaptive immune cell function39. Lactobacillus (L.) acidophilus, a common bacterial species sold as a probiotic that ‘promotes immune health’, contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA)40-43. These Slps interact with pattern recognition receptors (PRR; e.g., C-type lectin receptors) expressed on innate intestinal cells to fine-tune immunity in steady state and disease conditions42. Recently, our research team demonstrated that SlpA binding to the C-type lectin Specific Intracellular adhesion molecule-3 Grabbing Non-integrin homolog-Related 3 (SIGNR3) receptor expressed on dendritic cells lining the gut prevents experimentally induced colitis in multiple models3. Oral delivery of SlpA reduced inflammatory cytokines, strengthened the mucosal membrane barrier, and supported a healthier microbiota make-up in animal models of gut inflammation. In contrast, the effects and protection were not observed in Signr3-/- mice, suggesting that SlpA interaction with SIGNR3 plays a key protective role in regulating the disease condition3. Our goal is to develop R-3750, a SlpA-expressing, thymidine-dependent L. lactis strain, as a novel, orally administered probiotic that functions in IBD to reduce gut inflammation, improve gastrointestinal mucosal barrier function, and restore the natural microbiome make-up. L. lactis provides two key advantages as a delivery vehicle for conveying SlpA to the gut; namely, it has already been safely used in human clinical trials in a genetically manipulated form1, 4 and it does not express any native Slps but can be engineered to selectively overexpress SlpA. This Phase II SBIR application is intended to build upon success of the Phase I and advance R-3750 towards clinical testing. The specific Aims are: 1) optimize R-3750 upstream process development to support manufacturing, 2) create GMP master cell bank, 3) manufacture R-3750 for animal dosing/biomarker and GLP toxicology studies, and 4) complete R-3750 dose optimization and biomarker- based PD studies in mice.

Project Narrative Over 3 million adults in the U.S. suffer from inflammatory bowel disease (IBD). This project aims to develop a tailored probiotic therapy, R-3750, which delivers SlpA to induce local colonic regulatory T cells for the treatment of IBD. Oral delivery of SlpA reduces gut inflammation, reestablishes gut mucosal barrier function, induces positive maintenance of the gastrointestinal microbiome, and eliminates symptoms of experimental colitis. Based upon these properties, commercialization of R-3750 could provide an important new IBD treatment that is oral-based, safer than current drug options, and potentially lower cost.