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，具有 据估计，美国每年的成本在7.5亿至32亿美元之间。CDI的病理是 由细菌分泌的毒素引起。最近，两名抗C.艰难梭菌毒素抗体、阿克索单抗和 分别与艰难梭菌毒素A和B结合并中和的贝洛昔单抗在CDI中取得了成功 II期临床试验中的治疗。然而，只有贝洛昔单抗在随后的阶段显示出疗效。 III临床试验。抗体的中和效力强烈依赖于表位。因为两个Actoxumab 和贝洛昔单抗都是使用艰难梭菌实验室菌株vpi 10463的毒素进行改造的，两者都显示 显著降低了对最近出现的一些超强毒力菌株的中和能力。 艰难抉择。因此，迫切需要开发广谱有效的艰难梭菌毒素中和剂。我们建议 设计了一种非抗体蛋白，设计了锚蛋白重复蛋白(DARPin)，能够结合和 中和各种艰难梭菌菌株的毒素A和B。DARPins代表了一类用途广泛的 结合蛋白，已被设计为以高达皮摩尔亲和力结合不同的靶标。此外， Darpin可以在大肠杆菌中非常高效地表达(占所有大肠杆菌蛋白质的50%)，而且非常容易 由于其高热稳定性而提纯。在这个项目中，我们将首先使用噬菌体展示来分离DARPins 能够与艰难梭菌不同菌株的毒素结合(目标1)，然后让选定的 将DARPins与培养细胞中的抗毒素功能筛选相结合，以鉴定中和毒素的DARPins(目标2)。这个 选定的DARPins对一组新出现的和临床相关的艰难梭菌的效力将是 通过定向进化进行分析并进一步优化(目标3)。这项研究的成功完成将产生 一个高效毒素中和DARPins的武器库。这些广泛中和的抗毒素DARPins可以 可能与Fc融合，用于CDI的抗体治疗，或配制为口服给药 直接中和肠道中的艰难梭菌毒素(S)。中和细菌毒力因子的方法 DARPins还应该为其他细菌感染提供一种新的治疗范例。

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.