A Novel Immunologically Enhanced Probiotic for Treating Rheumatoid Arthritis

项目摘要 我们的目标是将R-2487商业化，作为一种新的口服疗法，用于长期缓解 类风湿关节炎(RA)。RA是一种慢性系统性炎症性疾病，影响美国高达1%的地区 人口1，2.虽然类风湿性关节炎的许多器官都受到影响，但滑膜关节主要是虚弱的 发炎。随着疾病的发展，大约一半的类风湿关节炎患者会致残。 类风湿性关节炎的治疗仍然是一个重要的未得到满足的医疗需求。尽管靶向治疗非常有效 细胞因子和T-B的相互作用，没有任何治疗方法可以导致疾病的长期缓解34。肿瘤坏死因子-α拮抗剂可以 有效消炎，减轻软骨、骨质破坏7-10。然而，一些患者 要么对抗肿瘤坏死因子治疗无效，要么复发。长期治疗会使患者变得易感。 癌症和机会性感染11-14。此外，目前许多治疗类风湿性关节炎的方法，无论多么有效， 让患者接受终生昂贵的生物治疗，伴随着医源性并发症的风险。为 出于这些原因，我们正在开发一种口服免疫疗法，它可以调节自身抗原特异的调节性T细胞 (Tregs)提供旁观者容忍度。这种方法的两个主要目标是：1)“关闭”免疫系统 以持久的方式对宿主自己的组织做出反应，2)最大限度地平衡关系 不同血统的效应细胞之间的作用，以防止慢性炎症复发。这样的方法将 为患者提供长期缓解和增加安全性，而无需全身免疫抑制。 最初被认为是人类腹泻疫苗，我们发现定居因子抗原I(CFA/I)来自 产肠毒素大肠杆菌(ETEC)能有效诱导自身抗原特异性T调节细胞18、22、54、55、56。 CFA/I作为纯化蛋白口服或通过乳酸乳杆菌或沙门氏菌细菌载体传递。 有效预防和治疗关节炎的多种实验模型16、18、33、57，以及 干燥综合征、糖尿病和多发性硬化症17、58、59。以避免与生产相关的挑战 足量重组CFA/I及改善CFA/I口服后粘膜PK/PD特性的研究 为了给药，我们成功地培育出一种新的乳酸乳杆菌菌株(简称R-2487)，它表达 来自基因组整合表达操纵子的功能性CFA/I。我们还验证了R-2487具有 一种可行的生物治疗候选药物的重要关键设计特征，包括对人类细胞的功能。 现在我们已经在IND前会议的背景下与FDA会面，以最终确定我们的产品开发 战略，这项第二阶段SBIR应用旨在建立在我们的成功和推动R-2487朝着 IND备案阶段。目标是：1)生产足够数量的R-2487以支持体内发育， 2)建立测定R-2487和CFA/I蛋白肠道暴露的PK法；3)确定最佳口服 R-2487在小鼠体内的剂量和PK/PD；4)制备GMP主细胞库，制造用于GLP的R-2487 药物/毒性研究，以及5)使用R-2487进行GLP毒理学研究，以实现IND备案。

Project Summary Our goal is to commercialize R-2487 as a novel, orally administered therapy for long-term disease remission of rheumatoid arthritis (RA). RA is a chronic, systemic inflammatory disorder affecting up to 1% of the US population1, 2. While many organs are affected in RA, the synovial joints are primarily afflicted with debilitating inflammation. Approximately half of all RA patients become disabled as the disease progresses. Treatment of RA remains a significant unmet medical need. Despite highly effective therapies targeting cytokines and T-B interactions, no therapy can induce long-term disease remission34. TNF-α antagonists can effectively diminish inflammation and attenuate destruction of cartilage and bone7-10. However, some patients either fail to respond to, or relapse with, anti-TNF therapy. Prolonged treatment can make patients susceptible to cancer and opportunistic infections11-14. Moreover, many current treatments for RA, no matter how effective, consign patients to a lifetime of costly biologic therapies with attendant risks for iatrogenic complications. For these reasons, we are developing an oral immunotherapeutic that regulates auto-Ag-specific regulatory T cells (Tregs) to provide bystander tolerance. The two main goals of this approach are: 1) to “switch off” the immune response against the host’s own tissues in an enduring manner and 2) maximally balance the relationships between effector cells of different lineages to prevent relapses of chronic inflammation. Such an approach will provide patients long-term remission and increased safety without systemic immunosuppression. Originally conceived as a human diarrheal vaccine, we found that colonization factor antigen I (CFA/I) from human enterotoxigenic E. coli (ETEC) is effective at inducing auto-Ag-specific T regulatory cells18, 22, 54, 55, 56. CFA/I administered orally as purified protein or delivered via an L. lactis or Salmonella bacterial vector was effective at preventing and treating multiple experimental models for arthritis16, 18, 33, 57, as well as models for Sjogren’s syndrome, diabetes, and multiple sclerosis17, 58, 59. To avoid challenges associated with producing sufficient quantities of recombinant CFA/I and to improve the mucosal PK/PD properties of CFA/I following oral administration, we successfully developed a new L. lactis strain (referred to as R-2487) that expresses functional CFA/I from a genome-integrated expression operon. We also validated that R-2487 possesses the important key design features for a viable biotherapeutic candidate including functionality on human cells. Now that we have met with the FDA in the context of a pre-IND meeting to finalize our product development strategy, this Phase II SBIR application is intended to build upon our success and advance R-2487 towards the IND filing stage. The objectives are: 1) produce sufficient quantities of R-2487 to support in vivo development, 2) develop PK assays to measure R-2487 and CFA/I protein intestinal exposure, 3) determine the optimal oral dose and PK/PD for R-2487 in mice, 4) prepare a GMP master cell bank and manufacture R-2487 for GLP pharm/tox studies, and 5) perform GLP toxicology studies with R-2487 to enable IND filing.