Intracellular targeting Hsp70 for pulmonary cytoprotection after toxin inhalation

2003年，美国环保署确定了全国123家化工厂，恐怖袭击或事故可能使100多万人暴露在有毒气体中。暴露于某些化学物质后，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，需要将过量的物质输送到肺部，可能导致危险的副作用。我们开发的Fv片段的细胞穿透抗体，单克隆抗体3E 10，作为一种细胞内转运蛋白提供热休克蛋白70（Fv-Hsp 70）进入细胞，我们证明了对氧化损伤的细胞保护作用在体外和体内。我们的长期目标是确定一种便携式的Fv-Hsp 70气雾剂制剂的临床有效性，如果怀疑暴露于有毒吸入剂，则可以自行给药。在追求这一目标的过程中，目的是建立针对光气暴露的有效性的原理证明，鉴于光气在化学工业中的普遍存在，这是一种未满足的医疗需求。我们的中心假设是，Fv-Hsp 70将在治疗上有效地保护肺损伤，由于暴露于光气气体。拟议研究的基本原理是，细胞穿透抗体3E 10是一种细胞内转运蛋白， 可以将Hsp 70直接递送到细胞中，在那里它最小化由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，这些靶标在有受损细胞的地方是非常容易接近的，并且它通过平衡的核苷补救途径穿透仍然存活的细胞。3E 10的独特之处在于它可以穿透细胞而没有明显的伤害，并且已经被施用给人类而没有毒性的证据。Fv-Hsp 70已经被创造出来，并被证明是一种有效的体内细胞保护剂，当给予中风后的大鼠时，脑组织中的梗死体积最小化了68%。我们正在申请资金以实现以下具体目标：1）评估Fv-Hsp 70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，作为基于细胞的效价测定的一部分。2)建立Fv-Hsp 70气雾剂的产品稳定性特征，并确定大鼠的最大耐受剂量（MTD）。3)评价Fv-Hsp 70雾化吸入对光气染毒大鼠的体内治疗效果。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-Hsp 70气雾剂。这项拟议中的研究意义重大，因为它为未满足的需求开发了一种关键的治疗方法。这项研究具有创新性，因为它利用了一种独特的抗体介导的、能量不依赖的细胞内蛋白质治疗递送系统。在体内诱导热休克蛋白的产生可能需要时间，而我们的产品的影响是将Hsp 70快速递送到受损细胞中以防止细胞死亡。

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.