Intracellular targeting Hsp70 for pulmonary cytoprotection after toxin inhalation

细胞内靶向 Hsp70 用于毒素吸入后的肺细胞保护

基本信息

批准号：
8741964
负责人：
Robert Nishimura
金额：
$ 30.02万
依托单位：
RUBICON BIOTECHNOLOGY, INC.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-25 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8741964
关键词：
Accidents Address Adverse effects Aerosols Air Alveolar Alveolar Cell Antibodies Binding Biological Assay Breathing Bronchiolo-Alveolar Adenocarcinoma Buffers Cell Death Cell Survival Cells Cerebral Infarction Cessation of life Chemical Industry Chemicals Chimeric Proteins Chromatography Clinical Trials Clinical effectiveness Cytoprotection Cytoprotective Agent DNA Data Analyses Development Dose Drug Formulations Effectiveness Electrophoresis Event Exposure to FDA approved Funding Gases Glutathione Goals Heat shock proteins Heat-Shock Proteins 70 Histology Human Hydrogen Peroxide In Situ In Vitro Industrial Accidents Industry Infarction Inhalant dose form Injury Intracellular Transport Lung Mass Spectrum Analysis Maximum Tolerated Dose Measures Mediating Medical Membrane Mission Modeling Molecular Nucleosides Oxidation-Reduction Oxidative Stress Oxygen Pathway interactions Phase III Clinical Trials Phosgene Plants Preparation Prevalence Primates Production Protein Denaturation Pulmonary Edema Radioimmunoconjugate Rattus Reactive Oxygen Species Reagent Research Research Personnel Research Proposals Self Administration Self-Administered Source Specificity Staging Stroke Structure of parenchyma of lung Surface System Testing Therapeutic Time Tissues Toxic effect Toxin Training Treatment Efficacy Weight Weight Gain Work aerosolized base brain tissue cell injury efficacy testing emergency service responder extracellular in vivo innovation liquid formulation novel oxidative damage pre-clinical prevent protein aggregation public health relevance respiratory small molecule stability testing therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): In 2003, the EPA identified 123 chemical plants in the nation where a terrorist attack or accident could potentially expose more than 1 million people to a cloud of toxic gas. Upon exposure to certain chemicals, a major cause of cell death is from oxidative stress. Current strategies to prevent oxidative stress include the use of small molecule reagents, but they lack specificity and require excessive material to be delivered to the lungs possibly causing dangerous side effects. We developed the Fv fragment of a cell-penetrating antibody, mAb 3E10, as an intracellular transporter to deliver heat-shock protein 70 (Fv-Hsp70) into cells, and we demonstrated cytoprotection against oxidative damage in vitro and in vivo. Our long-term goal is to determine the clinical effectiveness of an aerosol formulation of Fv-Hsp70 that is portable and can be self-administered if exposure to toxic inhalants is suspected. The objective here in the pursuit of this goal is to establish proof-of- principle for effectivenes against phosgene exposure, an unmet medical need given the prevalence of phosgene in the chemical industry. Our central hypothesis is that Fv-Hsp70 will be therapeutically effective in protecting against lung damage as a result of exposure to phosgene gas. The rationale for the proposed research is that the cell penetrating antibody, 3E10, is an intracellular transporter that can deliver Hsp70 directly into cells where it minimizes protein denaturation and aggregation caused by oxidative stress. The antibody binds extracellular DNA and nucleosides, targets that are quite accessible where there are damaged cells, and it penetrates still viable cells through an equilibrative nucleoside salvage pathway. 3E10 is unique in that it penetrates cells without apparent harm and has been administered to humans without evidence of toxicity. Fv-Hsp70 has already been created and shown to be an effective cytoprotectant in vivo, minimizing by 68% of the infarct volume in brain tissue when administered to rats after a stroke. We are requesting funding to achieve the following specific aims: 1) Evaluate the effect of Fv-Hsp70 on survival and barrier integrity in vitro of lung cells exposed to triphosgene (a phosgene simulant) as part of a cell-based potency assay. 2) Establish a product stability profile for the Fv-Hsp70 aerosol and determine the maximum tolerated dose (MTD) in rats. 3) Evaluate the therapeutic efficacy of aerosolized Fv-Hsp70 in vivo with rats exposed to phosgene. A single- dose of Fv-Hsp70 aerosol will be provided either pre-exposure, 30 minutes post-exposure, or 60 minutes post- exposure. The proposed research is significant because it develops a critical therapeutic for an unmet need. The proposed research is innovative because it utilizes a unique antibody-mediated, energy-independent intracellular delivery system for protein therapeutics. Inducing heat shock protein production in vivo can take time, whereas the impact of our product is the rapid delivery of Hsp70 into damaged cells to prevent cell death.

描述（由申请人提供）：2003 年，美国环保局 (EPA) 确定了全国 123 家化工厂，这些工厂的恐怖袭击或事故可能导致超过 100 万人暴露在有毒气体云中。接触某些化学物质后，细胞死亡的主要原因是氧化应激。目前预防氧化应激的策略包括使用小分子试剂，但它们缺乏特异性，并且需要将过量的物质输送到肺部，可能会导致危险的副作用。我们开发了细胞穿透抗体 mAb 3E10 的 Fv 片段作为细胞内转运蛋白，将热休克蛋白 70 (Fv-Hsp70) 递送至细胞内，并在体外和体内证明了细胞保护作用以抵抗氧化损伤。我们的长期目标是确定 Fv-Hsp70 气雾剂制剂的临床有效性，该气雾剂制剂是便携式的，并且在怀疑接触有毒吸入剂时可以自行给药。追求这一目标的目的是建立针对光气暴露有效性的原理验证，鉴于光气在化学工业中的普遍存在，这一医疗需求尚未得到满足。我们的中心假设是，Fv-Hsp70 在预防因暴露于光气而造成的肺损伤方面具有治疗效果。拟议研究的基本原理是细胞穿透抗体 3E10 是一种细胞内转运蛋白，可以将 Hsp70 直接输送到细胞中，最大限度地减少氧化应激引起的蛋白质变性和聚集。该抗体结合细胞外 DNA 和核苷，这些目标在受损细胞中很容易接近，并且它通过平衡核苷挽救途径渗透仍然存活的细胞。 3E10 的独特之处在于它能穿透细胞而没有明显的伤害，并且已用于人体且没有毒性证据。 Fv-Hsp70 已被创建并被证明是一种有效的体内细胞保护剂，当对中风后的大鼠施用时，可将脑组织中的梗塞体积减少 68%。我们正在申请资金以实现以下具体目标：1) 作为基于细胞的效力测定的一部分，评估 Fv-Hsp70 对暴露于三光气（一种光气模拟物）的肺细胞体外存活和屏障完整性的影响。 2) 建立 Fv-Hsp70 气雾剂的产品稳定性曲线并确定大鼠的最大耐受剂量 (MTD)。 3) 评价雾化Fv-Hsp70对暴露于光气的大鼠体内的治疗效果。将在暴露前、暴露后30分钟或暴露后60分钟提供单剂量的Fv-Hsp70气雾剂。拟议的研究意义重大，因为它为未满足的需求开发了一种关键的治疗方法。拟议的研究具有创新性，因为它利用独特的抗体介导的、不依赖于能量的细胞内递送系统进行蛋白质治疗。诱导体内热休克蛋白的产生需要时间，而我们产品的作用是将 Hsp70 快速递送到受损细胞中以防止细胞死亡。