Multi-functional targeted bio-conjugate platform to dismantle neutrophil extracellular traps (NETs)

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.