Internal Toxin Neutralizer for Treating STEC-infection

用于治疗 STEC 感染的内毒素中和剂

• 批准号: 9886184
• 负责人: Zhilei Chen
• 金额: $ 23.1万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-05 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9886184
• 关键词: Adsorption; Affect; Affinity; Ankyrin Repeat; Antibiotic Therapy; Antibiotics; Antibodies; Apoptosis; Bacteria; Bacterial Infections; Bacteriophages; Binding; Binding Proteins; Biological Assay; Blood Circulation; Cell Survival; Cell membrane; Cell surface; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Child; Cytosol; Developed Countries; Disease Outbreaks; Elderly; Endosomes; Engineering; Enzyme-Linked Immunosorbent Assay; Escherichia coli EHEC; Escherichia coli Infections; Estrogen receptor positive; Exhibits; Exotoxins; Fluorescence; Food; Germany; Glycosphingolipids; Goals; Golgi Apparatus; Hemolytic Anemia; Hemolytic-Uremic Syndrome; Hemorrhagic colitis; In Vitro; Incubated; Infection; Injections; Intracellular Transport; Kidney Failure; Liquid substance; Morbidity - disease rate; Mus; Oral; Pain management; Pathology; Patients; Peptide Hydrolases; Peptides; Phase III Clinical Trials; Protein Biosynthesis; Protein Family; Proteins; Reporter; Ribosomes; Risk; Series; Shiga Toxin; Shiga-Like Toxin I; Shiga-Like Toxin II; Spinach - dietary; Supportive care; Therapeutic; Thrombocytopenia; Toxic effect; Toxin; Treatment Efficacy; United States; Vero Cells; alpha Toxin; antitoxin; base; bean; clinical application; clinical development; combat; design; effective therapy; experimental study; extracellular; high throughput screening; immunogenicity; in vivo; mortality; nanomolar; neutralizing monoclonal antibodies; novel therapeutics; receptor; receptor binding; receptor mediated endocytosis; reconstitution; retrograde transport; scaffold; screening; shiga toxin 2 B subunit; therapeutic protein; time interval

Abstract The Shiga toxin-producing E. coli (STEC) is the most common cause of bloody diarrhea and afflicts an estimated 73,000 people in the US annually, causing significant morbidity. The most recent and largest STEC outbreak occurred in Germany in 2011, affecting >3,800 people, including 54 deaths. Currently there is no effective treatment for STEC infection. The pathology of STEC infection derives from two exotoxins – Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) – that are secreted by STEC in the gut. Although antibiotic treatment can reduce the load of STEC, it also augments Shiga toxin release, leading to increased risk of developing the more serious hemolytic uremic syndrome (HUS) and kidney failure (up to 25%). Consequently, the CDC recommends that antibiotics not be used in STEC patients and that only supportive therapy (e.g. oral and i.v. fluid, pain control) be used. Although anti-toxin antibodies have been identified, the inability of antibodies to cross the cell membrane renders them powerless against toxins already absorbed by the host cells, limiting their clinical application. We hypothesize that a cytosol-accessible anti-toxin should be able to neutralize both extracellular and intracellular Shiga toxin, leading to a much-prolonged therapeutic window and better therapeutic efficacy. The overall goal of this study is to engineer a panel of intracellular toxin neutralizers (ITNs) against Shiga toxin 2 (Stx2). As a scaffold for the proposed ITN, we will use a designed ankyrin repeat protein (DARPin). DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity and possess low immunogenicity. In this project, we will first isolate DARPins that bind and neutralize Stx2 (Aim 1). Concurrently, we will screen a panel of cell-penetrating peptides (CPPs) for their ability to transport ITNs into cells (Aim 2). In Aim 3, we will assemble anti-Stx2 ITNs using the best anti-Stx2 DARPin and CPP and evaluate the therapeutic potential of these anti-Stx2 ITNs in vitro and in vivo. The approach of using ITN to combat toxins in circulation offers a new paradigm for the treatment of both STEC and non-STEC bacterial infections.

抽象的 产生志贺毒素的大肠杆菌 (STEC) 是导致血性腹泻的最常见原因，并困扰着人们。 据估计，美国每年有 73,000 人罹患严重的 STEC。 2011年德国爆发疫情，影响超过3,800人，其中54人死亡。 STEC 感染的有效治疗 STEC 感染的病理源于两种外毒素——志贺毒素。 毒素 1 (Stx1) 和志贺毒素 2 (Stx2) – 由 STEC 在肠道中分泌，尽管抗生素治疗可以。 减少 STEC 的负荷，它还会增加志贺毒素的释放，导致患上更多志贺氏菌的风险增加 CDC 建议进行严重溶血性尿毒症综合征 (HUS) 和肾衰竭（高达 25%）的检测。 STEC 患者不能使用抗生素，只能进行支持性治疗（例如口服和静脉输液、止痛） 尽管已鉴定出抗毒素抗体，但抗体无法穿过细胞。 膜使它们无力抵抗宿主细胞已经吸收的毒素，从而限制了它们的临床 我们追求细胞质可及的抗毒素应该能够中和细胞外的两种物质。 和细胞内志贺毒素，从而延长治疗窗和更好的治疗效果。 本研究的总体目标是设计一组针对志贺毒素的细胞内毒素中和剂 (ITN) 2 (Stx2) 作为所提出的 ITN 的支架，我们将使用设计的锚蛋白重复蛋白 (DARPin)。 代表了一类多功能的结合蛋白，这些蛋白被设计用于结合不同的靶标，最多可达 皮摩尔亲和力并具有低免疫原性 在这个项目中，我们将首先分离结合并具有低免疫原性的 DARPins。 同时，我们将筛选一组细胞穿透肽 (CPP) 的能力。 将 ITN 转运到细胞中（目标 2），在目标 3 中，我们将使用最好的抗 Stx2 DARPin 组装抗 Stx2 ITN。 和 CPP 并评估这些抗 Stx2 ITN 的体外和体内治疗潜力。 使用 ITN 对抗循环中的毒素为 STEC 和非 STEC 的治疗提供了新的范例 细菌感染。

项目成果

A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.

多特异性 DARPin 通过同时调节两个毒素亚基来有效中和志贺毒素 2。

• 发表时间: 2022-09-27
• 作者: Zeng, Yu;Jiang, Mengqiu;Robinson, Sally;Peng, Zeyu;Chonira, Vikas;Simeon, Rudo;Tzipori, Saul;Zhang, Junjie;Chen, Zhilei
• 通讯作者: Chen, Zhilei