Background Aberrant Neutrophil Extracellular Trap (NET) formation contributes to the induction and propagation of inflammation and plays a key role in causing tissue damage in conditions like sepsis, SLE, RA and vasculitis. Citrullination of proteins is involved in the formation of NETs, autoimmunity, and the breaking of tolerance in NET-driven autoimmune diseases. In SLE and RA, neutrophils undergo enhanced NETosis, and NET components are observed in blood, inflamed tissues and joints.

Objectives Our objective is to develop a novel first in class NET-inhibiting therapeutic anti-citrullinated protein antibody (tACPA) targeting citrullinated histones 2A and 4, for the treatment of human diseases in which aberrant NET formation add to the severity of the pathology with an initial focus on autoimmune diseases. Here, we demonstrate the utility of tACPA for different NET-based diseases beyond RA, including SLE, vasculitis, gout and idiopathic pulmonary fibrosis (IPF).

Methods Previously, using two RA animal models, the therapeutic properties of tACPA have been demonstrated.^{Chirivi et al., 2013} In the current studies, neutrophils from RA and SLE donors, as well as biological NET-inducing stimuli, such as RA synovial fluid (SF), gout SF and activated platelets, have been used to demonstrate the NETosis-inhibiting properties of tACPA in different human disease contexts. We have further expanded tACPA’s therapeutic utility by testing it in a surrogate model for NET-mediated organ damage (sepsis) and IPF.

Results NETosis in human RA and SLE neutrophils have been induced with a calcium ionophore and could be inhibited by tACPA treatment (40%–100% reduction). Similar results were obtained using RA and gout SF or activated platelets as NETosis inducers in combination with neutrophils from healthy donors. These observations have been confirmed with multiple NET readouts such as MPO activity, MPO/DNA ELISA, DNA quantification as well as imaging readouts. In addition, we demonstrated that in an LPS-induced sepsis model 30% of tACPA-treated mice survived (compared to 0% in placebo controls), showing protection against organ failure. In a bleomycin-induced IPF mouse model, tACPA protected mice from the development of lung fibrosis (compared to placebo controls). When determining neutrophil counts in bronchoalveolar lavage samples, we found that in tACPA-treated mice, neutrophil levels were normal, while levels in placebo-treated mice were elevated.

Conclusions In a sepsis and IPF mouse model, tACPA prevented NET-mediated organ damage, providing evidence that tACPA could be a promising therapeutic strategy for diseases where NET-mediated endothelial toxicity causes organ damage like SLE, vasculitis and IPF. Central to our strategy for generating a preclinical data package supporting clinical testing, is to demonstrate that patient NETosis can be significantly inhibited ex vivo. We will present data that confirm that tACPA can block human SLE NETosis as well as human NETosis induced by activated platelets or gout SF.

Disclosure of Interest R. Chirivi Shareholder of: Citryll BV, Employee of: ModiQuest BV, J. van Rosmalen Employee of: ModiQuest BV, K. Kambas: None declared, G. Schmets: None declared, H. Kalisvaart: None declared, G. Bogatkevich: None declared, T. Shaw: None declared, H. van Es Shareholder of: Citryll BV, J. Raats Shareholder of: Citryll BV