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：
9293991
负责人：
Zhilei Chen
金额：
$ 22.51万
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 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9293991
关键词：
Accounting Affinity Ankyrin Repeat Antibiotics Antibodies Antibody Therapy Antitoxins 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 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 alpha Toxin base clinically relevant combat cost design engineering 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，估计对美国的年成本在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，两个抗C。艰难的毒素抗体，阿acoxumab和与艰难梭菌毒素A和B分别结合并中和CDI取得了成功的贝兹洛沙蛋白单抗在II期临床试验中进行治疗。但是，只有贝佐沙蛋白在随后的阶段表现出功效 III临床试验。抗体的中和效力密切取决于表位。由于两个actoxumab 甲氯妥昔单抗是使用实验室艰难梭菌菌株VPI 10463的毒素进行设计的，均显示针对一些最近出现的C.的过度毒性菌株显着降低了中和效力。艰难的。因此，迫切需要开发广泛有效的艰难梭菌毒素中和。我们建议工程师一种非抗体蛋白设计的弯蛋白重复蛋白（DARPIN），能够结合并从广泛的艰难梭菌菌株中中和毒素A和B。 darpins代表多功能类已设计的结合蛋白结合了多种靶标与皮摩尔亲和力结合。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％），并且很容易由于其高温稳定性而纯化。在这个项目中，我们将首先使用噬菌体显示器来隔离darpins 能够与艰难梭菌不同菌株的毒素结合（AIM 1），然后对选定 DARPINS到培养细胞中的抗毒素功能筛选，以鉴定毒素中和darpins（AIM 2）。这选定的DARPINS针对新兴和临床相关的艰难梭菌菌株的效力将是分析并通过定向进化进一步优化（AIM 3）。成功完成这项研究将产生高功率毒素中和darpins的武器库。这些广泛中和的抗毒素darpins可以有可能融合到FC并用作CDI的抗体疗法，或者以口服给予直接中和肠道中的艰难梭菌毒素。用中和细菌毒力因子与 DARPINS还应为其他细菌感染提供新的治疗范式。