Broadly neutralizing non-antibody protein for treating clostridium difficile infection

用于治疗艰难梭菌感染的广泛中和非抗体蛋白

基本信息

批准号：
9167525
负责人：
Zhilei Chen
金额：
$ 19.94万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-10 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9167525
关键词：
Accounting Affinity Ankyrin Repeat Antibiotics Antibodies Antibody Therapy Asians Bacteria Bacterial Infections Bacteriophages Binding Binding Proteins Biological Assay Blood Cell Culture Techniques Cessation of life Clostridium difficile Computer Analysis Conceptions Cultured Cells Data Diarrhea Directed Molecular Evolution Engineering Ensure Epidemic Epitopes Escherichia coli Escherichia coli Proteins Exhibits Exotoxins Goals Human Individual Infection Intestines Laboratories Libraries Link London Oral Administration Pathogenesis Pathology Patients Peptide Hydrolases Phase Phase II Clinical Trials Phase III Clinical Trials Placebos Protein Engineering Proteins Recurrence Resistance Stream TNFRSF6B gene Therapeutic Toxin Treatment Efficacy United States Vero Cells Virulence Factors abstracting base clinically relevant combat cost design improved intravenous injection member nanomolar neutralizing antibody novel strategies pathogen prevent scaffold standard of care success therapeutic protein thermostability

项目摘要

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.

抽象的艰难梭菌感染（CDI）是住院患者感染性腹泻的主要原因，据估计，美国每年的成本在 7.5 亿至 32 亿美元之间。 CDI的病理是是由细菌分泌的毒素引起的。最近，有两个反C.艰难梭菌毒素抗体、actoxumab 和 bezloxumab 分别结合并中和艰难梭菌毒素 A 和 B，在 CDI 方面取得了成功 II 期临床试验中的治疗。然而，只有 Bezloxumab 在后续阶段显示出疗效三期临床试验。抗体的中和效力强烈依赖于表位。由于阿妥昔单抗和 bezlotoxumab 是使用来自实验室艰难梭菌菌株 VPI 10463 的毒素进行设计的，两者均显示对一些最近出现的高毒力菌株的中和效力显着降低。艰难梭菌。因此，迫切需要开发广泛有效的艰难梭菌毒素中和剂。我们建议设计一种非抗体蛋白，即设计的锚蛋白重复蛋白 (DARPin)，它能够结合并中和多种艰难梭菌菌株中的毒素 A 和 B。 DARPins 代表一类多功能的结合蛋白经过工程设计，可以以高达皮摩尔的亲和力结合不同的靶标。此外， DARPin 可以在大肠杆菌中非常有效地表达（占所有大肠杆菌蛋白的 50% 以上）并且非常容易由于其高热稳定性而纯化。在这个项目中，我们将首先使用噬菌体展示来分离DARPins 能够与不同艰难梭菌菌株的毒素结合（目标 1），然后对选定的 DARPins 在培养细胞中进行抗毒素功能筛选，以鉴定毒素中和 DARPins（目标 2）。这选定的 DARPins 对一组新出现的和临床相关的艰难梭菌菌株的效力将是通过定向进化进行分析和进一步优化（目标 3）。成功完成这项研究将产生高效毒素中和 DARPins 库。这些广泛中和抗毒素 DARPins 可以可能与 Fc 融合并用作 CDI 抗体疗法，或配制为口服给药直接中和肠道中的艰难梭菌毒素。中和细菌毒力因子的方法 DARPins 还应该为其他细菌感染提供新的治疗范例。