Oral Protein Therapeutics Against C. difficile Associated Colitis

摘要 每年，艰难梭菌（C.艰难梭菌），一种革兰氏阳性，孢子形成的厌氧杆菌， 25万例感染，约15，000例死亡和超过10亿美元的治疗相关费用。的症状 C.艰难梭菌感染（CDI）的范围从轻度腹泻到致命的假膜性结肠炎。虽然 原发性CDI通常可以用抗生素治疗，在过去的几十年里，CDI的复发率 由于出现了耐药性和所谓的高毒力菌株， 复发）。C.艰难梭菌分泌的毒素A（TcdA）和毒素B（TcdB）是引起艰难梭菌感染的关键毒力因子。 一系列疾病统称为CDI。最近FDA批准的CDI治疗剂- ZINPLAVA（bezloxumab，一种静脉给药的抗TcdB单克隆抗体，同时使用 与抗生素）-被发现可以降低复发率，但既不能减轻严重程度，也不能缩短 CDI的持续时间。因此，仍然迫切需要针对CDI的更有效疗法。自从C.艰难及其 分泌的毒素驻留在胃肠道（GI）内，这是通过i. v.施用 抗体，我们假设口服毒素中和剂应该更有效地预防CDI 发病机制以前，抗毒素超免疫牛初乳（HBC）已被证明是一种 用于治疗和/或预防各种病毒和细菌GI感染的有效口服治疗剂， 这是针对CDI的口服抗毒素蛋白治疗剂的先例。最近，我们的实验室设计了一组 锚蛋白重复序列蛋白（DARPin），其具有针对TcdB的有效中和活性。DARPin蛋白支架 进一步工程化以使其对GI驻留蛋白酶的消化具有高度抗性，同时保留其 毒素中和能力在这个项目中，我们打算进一步评估DARPins的治疗潜力， 反对CDI具体地，在目的1中，为了促进抗毒素DARPin更有效地原位递送至细胞， 结肠，将产生用于DARPin分泌的先导益生菌菌株。在目标2中，对于严重 免疫系统受损，不适合接受活微生物，一种替代盲肠/结肠蛋白 将探讨执行战略。与此同时，更多的DARPin将被设计成高度针对 TcdA和TcdB上的保守结构域（目的3）。成功完成拟议的研究将产生反- 毒素作为对抗CDI的潜在下一代口服治疗剂。此外，这项研究可能会建立一个新的 其他肠道疾病的口服治疗范例。

Abstract Each year, Clostridium difficile (C. difficile), a Gram-positive, spore-forming anaerobic bacillus, causes over a quarter million infections, ~15,000 deaths and over $1 billion in treatment-associated costs. The symptoms of C. difficile infection (CDI) ranges from mild cases of diarrhea to fatal pseudomembranous colitis. Although primary CDI can generally be treated with antibiotics, over the past decades, the rate of CDI recurrence has greatly increased due to the emergence of antibiotic-resistant and so-called hypervirulent strains (20-25% relapse). C. difficile secreted toxin A (TcdA) and toxin B (TcdB) are the critical virulence factors that cause a range of diseases collectively designated as CDI. The most recently FDA approved CDI therapeutic – ZINPLAVA (bezloxumab, an intravenously administered anti-TcdB monoclonal antibody to be used concurrent with antibiotics) – was found to reduce the rate of recurrence but neither lessen the severity nor shorten the duration of CDI. Thus, more effective therapies against CDI are still urgently needed. Since C. difficile and its secreted toxins reside within the gastrointestinal (GI) tract, a location not easily accessible by i.v.-administered antibodies, we hypothesize that an oral toxin-neutralizer should be more effective at preventing CDI pathogenesis. Previously, anti-toxin hyperimmune bovine colostrum (HBC) has been demonstrated as an effective oral therapeutic for treating and/or preventing various viral and bacterial GI infections, setting a precedent for oral anti-toxin protein therapeutics against CDI. Recently, our lab engineered a panel of designed ankyrin repeat protein (DARPin) with potent neutralization activity against TcdB. The DARPin protein scaffold was further engineered to render it highly resistant to digestion by GI-resident proteases while retaining its toxin-neutralization ability. In this project, we intend to further evaluate the therapeutic potential of DARPins against CDI. Specifically, in Aim 1, to facilitate more effective in situ delivery of anti-toxin DARPins to the colon, lead probiotic strains will be created for DARPin secretion. In Aim 2, for patients with severely compromised immune system and unfit for receiving live microorganisms, an alternative cecum/colon protein delivery strategy will be explored. Concurrently, additional DARPins will be engineered to target highly conserved domains on TcdA and TcdB (Aim 3). Successful completion of the proposed study will yield anti- toxins as potential next-generation oral therapeutics against CDI. In addition, this study may establish a new oral therapeutic paradigm for other enteric diseases.