AbstractNeutrophil extracellular traps (NETs) are extracellular fibrillary structures of chromatin filaments coated withhistones, proteases and granular and cytosolic proteins released by neutrophils as an antimicrobial mechanismthat `traps' and kills bacteria. Cumulative research reveals that NETs' antimicrobial killing properties can alsoinduce tissue injury when dysregulated. Hence, NETs are increasingly recognized as a culprit-driver in thepathogenesis of multiple major diseases ? acute respiratory distress syndromes (ARDS), acute coronarysyndromes (ACS), multi-organ failure (MOF) in ARDS, and sepsis ? where durable breakthrough therapies arelacking, despite significant research. Regardless of the disease, the fact that NETs are the common culprit indiverse and pathogenically disparate diseases argues the importance and high-value priority of targetingNETs. We hypothesize that successful neutralization and dismantlement of intravascular NETs will stop NET-driven endothelial injury at pulmonary vascular-alveolar barrier injury sites in acute respiratory distresssyndrome (ARDS). To overcome the concomitant biological and biophysical barriers to dismantling NETs andneutralizing NET-driven tissue injury, a multi-pronged therapeutic is needed. We will therefore develop a noveltherapeutic that comprises: 1) a highly specific, humanized hinge-stabilized S228P IgG4 antibody that targetsthe dual endothelin1/signal peptideVEGF receptor (DEspR) detected on NETosing neutrophils ? anti-DEspR-humab; and, 2) DNase1 conjugated to a tripeptide linker that is cleaved by cathepsin G (cg). Release of theDNase1 by cathepsin G cleavage at the NET site will facilitate NET dismantlement and serve as a substratedecoy for cathepsin G reactivity, thus minimizing its direct endothelial injury activity. This proposed targetedenzymatic bioconjugate is enabled by a novel method of stoichiometric, site-specific conjugation to antibodies? i.e., the NanoZip which utilizes the supramolecular assembly of coiled coils (SMACC) to achieve selective,specific coupling of two DNase1 enzymes to the C-terminus of an antibody. The specific aims of this two-yearR21 proposal are: Aim 1. Prepare the antiDEspR-humab-cg-peptide-DNase1 therapeutic prototype(DESPRnase1) and evaluate structural stability and dose-dependent release of DNase1 by cathepsin G inbasal plasma conditions, and in the presence of low pH and high ROS milieus present in ARDS. Aim 2.Determine whether DESPRnase1 targets and binds to DEspR+ NETs, dismantles DEspR+ NETs withoutcomplement activation, and/or serves as substrate-decoys to attenuate NETs' cathepsin G-induced injury ofhuman endothelial cells ex vivo. This R21 will develop a prototype bio[nano]conjugate as a breakthroughtherapeutic to dismantle NETs and stop the vicious cycle of endothelial injury in ARDS. Notably, efficacy inARDS will open the door to potential applications in ACS and other indications.