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 effectiveness 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% 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.
The research proposal is relevant to Counteract because the development of pulmonary cytoprotectants is an unmet medical need in battlefield and industrial accident scenarios. Thus, the proposed research is relevant to the mission of Counteract to develop novel, easily accessible, inhaled cytoprotectants that prevent injury to lungs from phosgene inhalation. Ultimately, the Fv-Hsp70 treatment will be available as a self-administered aerosol for phosgene exposure not only for warfighters, first responders, and industrial workers, but also available for supportive treatment in medical facilities.