Broadly neutralizing non-antibody protein for treating clostridium difficile infection

摘要 艰难梭菌感染（CDI）是住院患者感染性腹泻的主要原因， 估计美国每年的成本在7.5亿美元到32亿美元之间。CDI的病理是 是由细菌分泌的毒素引起的最近，两个抗C。艰难梭菌毒素抗体、阿克妥尤单抗和 bezloxumab，结合并中和C.艰难梭菌毒素A和B分别在CDI中取得了成功 在II期临床试验中进行治疗。然而，只有贝珠单抗在后续阶段显示出疗效 III临床试验。抗体的中和效力具有强烈的表位依赖性。由于阿克妥尤单抗 和贝兹洛木单抗使用来自实验室C的毒素进行工程改造。艰难梭菌菌株VPI 10463，均显示 显著降低了对最近出现的一些C. 很难因此，迫切需要开发广泛有效的C。艰难毒素中和剂。我们建议 工程化非抗体蛋白，设计的锚蛋白重复蛋白（DARPin），其能够结合并 中和来自广泛范围的毒素A和B。艰难菌株DARPins代表了一种多功能的 结合蛋白已经被改造成以高达皮摩尔的亲和力结合不同的靶标。此外，委员会认为， DARPin可以在大肠杆菌中非常有效地表达。大肠杆菌（占大肠杆菌总数的50%以上）。大肠杆菌蛋白质）， 由于其高的热稳定性而纯化。在这个项目中，我们将首先使用噬菌体展示来分离DARPins 能够与不同的C.艰难（目标1），然后将选定的 DARPins用于在培养的细胞中进行抗毒素功能筛选以鉴定毒素中和DARPins（Aim 2）。的 所选DARPin对一组新出现的和临床相关的C.困难将是 分析并通过定向进化（Aim 3）进一步优化。成功完成这项研究将产生 一个高效的毒素中和DARPin的军火库这些广泛中和的抗毒素DARPin可以 可能与Fc融合并用作CDI的抗体治疗，或配制用于口服给药， 直接中和C.肠内艰难梭菌毒素。细菌毒力因子的中和途径 DARPins还应该为其他细菌感染提供新的治疗模式。

Abstract Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea in hospitalized patients, with an estimated annual cost to the United States of between $750 million and $3.2 billion. The pathology of CDI is caused by the toxins secreted by the bacteria. Recently, two anti-C. difficile toxin antibodies, actoxumab and bezloxumab, that bind to and neutralize C. difficile toxins A and B, respectively, have achieved success for CDI treatment in a phase II clinical trial. However, only bezloxumab demonstrated efficacy in the subsequent phase III clinical trial. The neutralization potency of antibody is strongly epitope-dependent. Since both actoxumab and bezlotoxumab were engineered using toxins from laboratory C. difficile strain VPI 10463, both showed significantly reduced neutralization potencies against some recently emerged hypervirulent strains of C. difficile. Thus, there is an urgent need to develop broadly effective C. difficile toxins neutralizer. We propose to engineer a non-antibody protein, designed ankyrin repeat protein (DARPin), that is able to bind to and neutralize toxins A and B from a broad range of C. difficile strains. DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity. Furthermore, DARPin can be very efficiently expressed in E. coli (accounting for >50% of all E. coli proteins) and very easily purified due to its high thermostability. In this project, we will first use bacteriophage display to isolate DARPins that are able to bind to toxins from different strains of the C. difficile (Aim 1) and then subject the selected DARPins to an anti-toxin functional screen in cultured cells to identify toxin-neutralizing DARPins (Aim 2). The potency of the selected DARPins against a panel of emerging and clinically relevant strains of C. difficile will be analyzed and be further optimized by directed evolution (Aim 3). Successful completion of this study will yield an arsenal of high-potency toxin-neutralizing DARPins. These broadly neutralizing anti-toxin DARPins can potentially be fused to Fc and used as antibody therapy for CDI, or be formulated for oral administration to directly neutralize C. difficile toxin(s) in the gut. The approach of neutralizing bacterial virulence factors with DARPins should also offer a new treatment paradigm for other bacterial infection.