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

> **NIH NIH R21** · BOSTON UNIVERSITY (CHARLES RIVER CAMPUS) · 2020 · $247,500

## Abstract

Abstract
Neutrophil extracellular traps (NETs) are extracellular fibrillary structures of chromatin filaments coated with
histones, proteases and granular and cytosolic proteins released by neutrophils as an antimicrobial mechanism
that `traps' and kills bacteria. Cumulative research reveals that NETs' antimicrobial killing properties can also
induce tissue injury when dysregulated. Hence, NETs are increasingly recognized as a culprit-driver in the
pathogenesis of multiple major diseases – acute respiratory distress syndromes (ARDS), acute coronary
syndromes (ACS), multi-organ failure (MOF) in ARDS, and sepsis – where durable breakthrough therapies are
lacking, despite significant research. Regardless of the disease, the fact that NETs are the common culprit in
diverse and pathogenically disparate diseases argues the importance and high-value priority of targeting
NETs. 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 distress
syndrome (ARDS). To overcome the concomitant biological and biophysical barriers to dismantling NETs and
neutralizing NET-driven tissue injury, a multi-pronged therapeutic is needed. We will therefore develop a novel
therapeutic that comprises: 1) a highly specific, humanized hinge-stabilized S228P IgG4 antibody that targets
the 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 the
DNase1 by cathepsin G cleavage at the NET site will facilitate NET dismantlement and serve as a substrate
decoy for cathepsin G reactivity, thus minimizing its direct endothelial injury activity. This proposed targeted
enzymatic 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-year
R21 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 in
basal 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 without
complement activation, and/or serves as substrate-decoys to attenuate NETs' cathepsin G-induced injury of
human endothelial cells ex vivo. This R21 will develop a prototype bio[nano]conjugate as a breakthrough
therapeutic to dismantle NETs and stop the vicious cycle of endothelial injury in ARDS. Notably, efficacy in
ARDS will open the door to potential applications in ACS and other indications.

## Key facts

- **NIH application ID:** 9843733
- **Project number:** 5R21HL144253-02
- **Recipient organization:** BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
- **Principal Investigator:** MARK W. GRINSTAFF
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $247,500
- **Award type:** 5
- **Project period:** 2019-01-01 → 2020-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9843733

## Citation

> US National Institutes of Health, RePORTER application 9843733, Multi-functional targeted bio-conjugate platform to dismantle neutrophil extracellular traps (NETs) (5R21HL144253-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9843733. Licensed CC0.

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