Background Neutrophils together with aberrant Neutrophil Extracellular Trap (NET) formation contribute to the induction and propagation of inflammation. A growing number of studies indicate that Peptidyl Arginine Deiminases (PADs) mediated conversion of arginine to citrulline residues in proteins are essential for NETosis, generation of auto-antigens, autoimmunity, and the breaking of tolerance in rheumatoid arthritis (RA). In RA patients, enhanced NETosis is observed in circulating and synovial neutrophils, and NET components are observed in blood and joints.
Objectives Our objective is to develop a novel first in class NET-inhibiting therapeutic anti-citrullinated protein antibody (tACPA) treatment for RA and other auto-immune diseases in which aberrant NET formation adds to the severity of the disease.
Methods Human neutrophils from blood donors have been used in order to visualize the NETosis inhibiting properties of tACPA. Furthermore, collagen antibody-induced (CAIA) as well as collagen-induced (CIA) arthritis mouse models have been used in order to test the therapeutic properties of tACPA.
Results In human neutrophils, NETosis induction with calcium ionophore A-23187 or physiological stimuli like human synovial fluid is strongly inhibited by tACPA treatment (40–90% NET reduction compared to non-treated cells (n>40 different donors)). This observation has been confirmed by Myeloperoxidase and Neutrophil Elastase activity as well as immunohistochemistry read-outs. In both CAIA and CIA mouse models, NET-inhibiting tACPAs are able to prevent the onset and/or exacerbation of inflammation, and prevent or strongly reduce joint damage. Histological analysis of tACPA-treated inflamed mouse joints revealed a significant decrease in neutrophil influx. We identified the epitopes recognized by tACPA to be citrullinated domains of histones 2A and 4.
Conclusions We have identified antibodies directed against a citrullinated epitope in murine and human histones 2A and 4. In RA mouse models, we demonstrate that tACPAs strongly prevents the occurrence of swelling and joint damage. We propose that the therapeutic effect of tACPA acts through the inhibition of NET and auto-antigen formation, clearance of formed NETs and toxic histones. In RA patients extinguishing auto-antigen production offers an orthogonal approach for treating this destructive autoimmune disease, potentially without impacting systemic immunity.
Citryll and ModiQuest are developing tACPAs as treatment for diseases where NETs and NETosis are driving or contributing to disease progression.
Disclosure of Interest R. Chirivi Shareholder of: Citryll BV, Employee of: ModiQuest BV, J. van Rosmalen Employee of: ModiQuest BV, G. Schmets Employee of: ModiQuest BV, H. van Es Shareholder of: Citryll BV, Employee of: Citryll BV, J. Raats Shareholder of: Citryll BV and ModiQuest BV