(T1230-05-27) Neutrophil Extracellular Traps (NETs) Targeting Strategies with Monoclonal Antibody (2C5)

Purpose: NETs are extracellular structures that include chromatin and neutrophil granular proteins. NETs major components are deoxyribonucleic acid (DNA) and histones. The primary role of NETs is to fight against pathogens at the site of the ongoing infection, thanks to the interaction of histones with highly bactericidal properties. Pathogens can not only induce NETs but also prevent their formation, thus protecting the pathogen against the antibacterial action of NETs, which in turn causes the development of infection. One of the methods used to brake network integrity is the production of deoxyribonucleases (DNases) by pathogens [1]. Over-generation of NETs, with the simultaneous lack of appropriate mechanisms for removing already formed DNA traps, in acute or chronic inflammatory diseases has shown that NET formation correlates with disease sequelae [2] also, in many human diseases, such as cancer, stroke, myocardial trauma, or pregnancy complications, increased levels of circulating free DNA have been reported [3]. NETs also play a role in the development of periodontitis, cystic fibrosis, type 2 diabetes, COVID-19, or rheumatoid arthritis [4]. In our approach, we are focusing on mimicking one of the natural mechanisms of destroying excessive NETs by delivering deoxyribonuclease I to the specific site of pathological NETs accumulation by modifying the nanoparticles using an anti-nucleosome monoclonal antibody (2C5). The antibody is specific to nucleosomes and can recognize histones in NETs [5].  DNase I is U.S. Food and Drug Administration (FDA)-approved active component and is commonly used in therapeutic methods of modern medicine for cystic fibrosis to clear extracellular DNA fibers in the lungs and systemic lupus erythematosus. Recent findings have also shown the effectiveness of DNase I in the digestion of NETs. However, the low serum stability and fast deactivation by environmental stimuli have been considered as the limiting factors for clinical applications of DNase I [6], which can be overcome by its targeted specific delivery in pharmaceutical nanocarriers.

Methods: In this study, we generate NETs in vitro using neutrophils and HL-60 cells differentiated into granulocyte-like cells. We used interleukin-8, lipopolysaccharide from E.Coli (LPS), phorbol myristate acetate (PMA), and calcium ionophore A23187 (CI) to generate the NETs. We confirmed the specificity of 2C5 toward NETs by ELISA, which showed that it binds to NETs with a specificity like that for purified nucleohistone substrate. We further utilized that feature to create a micellar system to deliver DNAse I enzyme (MDM) to NETs and destroy them, which was confirmed by staining NETs with SYTOX Green dye and followed by flow cytometric measurements.

Results: Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies as shown in Figure 1. Also, we proved that our carrier can successfully deliver DNase to NETs and degrade them as shown in Figure 2.

Conclusion: The monoclonal 2C5 antibody and DNase I constructs, that have the ability to self-organize into micelle-like structures were used as a platform to specifically deliver an enzyme to the NETs and showed higher effectiveness in NETs degradation than free DNase in established in vitro models.

References: 1. Buchanan, J.T., et al., DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol, 2006. 16(4): p. 396-400.
2. Branzk, N. and V. Papayannopoulos, Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol, 2013. 35(4): p. 513-30.
3. Swarup, V. and M.R. Rajeswari, Circulating (cell-free) nucleic acids--a promising, non-invasive tool for early detection of several human diseases. FEBS Lett, 2007. 581(5): p. 795-9.
4. Huang, S.U. and K.M. O'Sullivan, The Expanding Role of Extracellular Traps in Inflammation and Autoimmunity: The New Players in Casting Dark Webs. Int J Mol Sci, 2022. 23(7).
5. Mendes, L.P., et al., Monoclonal antibody 2C5 specifically targets neutrophil extracellular traps. MAbs, 2020. 12(1): p. 1850394.
6. Hosseinnejad, A., et al., DNase I functional microgels for neutrophil extracellular trap disruption. Biomater Sci, 2021. 10(1): p. 85-99.

Figure 1 Immunoreactivity of mAb 2C5 targeted DNase I micelles (2C5 MDM). Indirect ELISA was used to evaluate the binding of 2C5 antibodies to neutrophil-produced NETs. Detection was done using horseradish peroxidase (HRP)-conjugated secondary antibody. Data represent the mean ± SD, n = 3



Figure 2 The figure presents the fluorescent signal coming from NETs induced by A Phorbol myristate acetate (PMA), B calcium ionophore A23187 (CI), C interleukin-8 and D) LPS, in the presence of free DNase and targeted and non-targeted DNase micelles.