Project Summary Systemically administered soluble native ACE2 is currently tested as a viral decoy to prevent SARS-CoV-2 cell entry in COVID-19 patients. This soluble ACE2 protein, however, has a short half-life and limited bioavailability in the lung, especially at the target sites for SARS-CoV-2 entry, namely nasal and pulmonary type II alveolar cells. To overcome this problem, we developed AGT001, a novel variant of soluble human ACE2 (ACE2 1-618) and fused with an albumin binding domain (ABD). This protein, that we termed AGT001 has an increased protein half-life and prolonged ACE2 activity in vivo. More recently, we introduced a dodecapeptide (DDC) motif to AGT001, that leads to dimerization. The resulting protein that we have termed AGT002 and propose to use in this resubmission application has 20-30 times increased binding affinity for SARS-CoV-2 as compared to AGT001. We will use intranasal delivery of AGT002 to take advantage of these properties to effectively increase respiratory tract luminal surface concentrations of ACE2 activity and increase its capacity to act as a decoy to intercept SARS- CoV-2 from binding to its main receptor, the membrane-bound FL-ACE2. In addition, AGT002 will supplement ACE2 enzymatic activity potentially reducing inflammatory processes associated with excess of Angiotensin II and des-Arg9 Bradykinin, substrates of ACE2 driven degradation. Even in an era of SARS-CoV-2 vaccinations benefactors of a commercially marketed AGT002 could be unvaccinated and/or vaccination refractory COVID-19 patients owing to immunosuppression as in transplant and dialysis patients. AGT002, moreover, would be available for new SARS-CoV-2 variants that escape the vaccine or other future coronavirus that also use FL-ACE2 as main receptor. Thus, the objective of this program is to 1) test proof of concept efficacy of pulmonary-delivered AGT002 in a permissive mouse model (k18-hACE2), 2) assess AGT002’s aerosol development potential, and 3) assess AGT002’s initial safety and pharmacokinetic profile in wild-type mice. We have assembled a first-class team of experts to facilitate the performance of the project to include the 1) co-inventors of AGT002 at Northwestern, 2) state of the art BSL-3 facility at the University of Chicago to be able to infect permissive mice with SARS-CoV-2 and test the efficacy of AGT002 and 3) aerosol, toxicology and pharmacokinetic expertise at Lovelace Biomedical. The results of this program will be a decision gate for pursuing an Investigational New Drug (IND) application. If successful, we will move AGT002 through traditional chemical manufacturing and controls (CMC), GLP toxicology, IND application, and human safety and proof of concept studies utilizing the Phase II SBIR and/or traditional financing.