Oral Protein Therapeutics Against C. difficile Associated Colitis

点击翻译按钮获取中文摘要

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.