Background/Purpose:

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. We developed a novel first in class NET-inhibiting anti-citrullinated protein antibody (tACPA) treatment for RA.

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 the CAIA mouse model, tACPA prevents the onset and/or exacerbation of inflammation, and prevents or strongly reduces joint damage. Therapeutic administration of tACPA resulted in the arrest of inflammation and prevented a further increase of the inflammatory response. Histological analysis of inflamed joints from tACPA-treated mice revealed a significant decrease in neutrophil influx as well as a decrease in joint damage, as compared to control animals. We identified the epitopes recognized by tACPA to be citrullinated domains of histone-2A, 3 and 4. Since neutrophils together with aberrant NET formation contribute to the induction and propagation of inflammation, we investigated whether tACPA’s cellular mode of action could be through blocking NET formation or its activity by binding to citrullinated histone-2A, 3 and 4. Other groups have already demonstrated that PAD activity and citrullinated epitopes are essential for NET formation. For this purpose, we isolated neutrophils from blood of healthy human donors and induced NETosis with PMA or calcium ionophore A-23187. Treating neutrophils that undergo NETosis with tACPA led to a 40-60% NET reduction if compared to non-treated cells (n>34 different donors). This observation has been confirmed by Myeloperoxidase activity as well as immunohistochemistry read-outs.

Conclusion:

We have identified antibodies directed against a citrullinated epitope in murine and human histone-2A, 3 and 4. In RA mouse models, we demonstrate that tACPAs exhibit a strong anti-inflammatory activity and prevent 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 is developing tACPAs for first in man PoC trials for diseases where dysregulated NETs and NETosis are driving or contributing to disease progression. A particular suited RA patient subpopulation for tACPA therapy are those patients with activated PAD4 enzymes and increased NETosis.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/mode-of-action-of-therapeutic-anti-citrullinated-protein-antibodies-revealed-inflammation-reduction-due-to-inhibition-of-netosis/