A Novel Immunological Probiotic for Treating Inflammatory Bowel Disease

项目摘要 本项目的目标是开发一种新的免疫学设计的益生菌疗法，以抑制肠道 本发明提供了用于治疗炎性肠病（IBD）的炎症过程的药物组合物。在美国有超过300万成年人。 患有IBD，这是一个涵盖两种胃肠道慢性炎症性疾病的总称 道：克罗恩病（CD）和溃疡性结肠炎（UC）6。IBD通常在第二或第三个月被诊断出来。 几十年的生活，它是终身的，没有治愈。目前的IBD治疗是全身性的， 副作用安全有效的新疗法，特别是恢复 免疫系统和肠道微生物组是必要的，将改变患者的生活。 肠道免疫调节信号严格控制健康的肠道稳态，它们的破坏可能导致 IBD 38。人类的微生物组中含有数万亿的细菌，是这些机制的关键调节器。 共生细菌的功能是维持肠上皮屏障的完整性，并调节先天性和适应性 免疫细胞功能39.乳酸杆菌（L.）嗜酸乳杆菌，一种常见的细菌物种作为益生菌出售， “促进免疫健康”，含有独特的表面层蛋白（Slps），包括SlpA，SlpB，SlpX， 脂磷壁酸（LTA）40-43。这些Slp与模式识别受体（PRR;例如，c型凝集素 受体）表达于先天性肠细胞上，以微调稳态和疾病状况下的免疫42。 最近，我们的研究小组证明，SlpA结合C型凝集素特异性细胞内粘附 树突状细胞衬里上表达的非整合素同源物相关3（SIGNR 3）受体 肠道在多种模型中预防实验诱导的结肠炎3。口服递送SlpA减少了炎症反应。 细胞因子，加强粘膜屏障，并支持更健康的微生物群组成， 肠道炎症的动物模型。相反，在Signr 3-/-小鼠中未观察到效果和保护， 这表明SlpA与SIGNR 3的相互作用在调节疾病状况中起关键的保护作用3。 我们的目标是开发R-3750，一种表达SlpA的胸苷依赖性L.乳酸菌菌株，作为一种新的，口服 给予益生菌，在IBD中发挥作用，以减少肠道炎症，改善胃肠道粘膜 屏障功能，并恢复自然微生物组成。L.乳酸菌作为一种 用于将SlpA输送到肠道的递送载体;即，它已经安全地用于人类临床试验 以遗传操作的形式1，4，并且它不表达任何天然Slps，但可以被工程化以 选择性过表达SlpA。第二阶段SBIR应用程序旨在建立在第一阶段成功的基础上， 并将R-3750推向临床测试。具体目的是：1）优化R-3750上游工艺 开发以支持生产，2）创建GMP主细胞库，3）生产动物用R-3750 给药/生物标志物和GLP毒理学研究，以及4）完成R-3750剂量优化和生物标志物- 基于小鼠的PD研究。

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.