Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases

Renato G S Chirivi,Jos W G Van Rosmalen,Konstantinos Kambas,Oliver Soehnlein,Maximilien Euler,Galina Bogatkevich,Markus H Hoffmann,Jonas Hahn,Jos M H Raats,Quinte Braster,Helmuth H G Van Es,Gonny Schmets,Maarten Van Der Linden

doi:10.1038/s41423-020-0381-3

Abstract

Excessive release of neutrophil extracellular traps (NETs) is associated with disease severity and contributes to tissue injury, followed by severe organ damage. Pharmacological or genetic inhibition of NET release reduces pathology in multiple inflammatory disease models, indicating that NETs are potential therapeutic targets. Here, we demonstrate using a preclinical basket approach that our therapeutic anti-citrullinated protein antibody (tACPA) has broad therapeutic potential. Treatment with tACPA prevents disease symptoms in various mouse models with plausible NET-mediated pathology, including inflammatory arthritis (IA), pulmonary fibrosis, inflammatory bowel disease and sepsis. We show that citrulline residues in the N-termini of histones 2A and 4 are specific targets for therapeutic intervention, whereas antibodies against other N-terminal post-translational histone modifications have no therapeutic effects. Because citrullinated histones are generated during NET release, we investigated the ability of tACPA to inhibit NET formation. tACPA suppressed NET release from human neutrophils triggered with physiologically relevant human disease-related stimuli. Moreover, tACPA diminished NET release and potentially initiated NET uptake by macrophages in vivo, which was associated with reduced tissue damage in the joints of a chronic arthritis mouse model of IA. To our knowledge, we are the first to describe an antibody with NET-inhibiting properties and thereby propose tACPA as a drug candidate for NET-mediated inflammatory diseases, as it eliminates the noxious triggers that lead to continued inflammation and tissue damage in a multidimensional manner.

Highlights

Neutrophils are the most abundant type of leukocytes in human blood, contribute to the first line of defense and use their extensive armory to protect the host against infection
The development of these antibodies occurred as follows: (1) m-therapeutic anti-citrullinated protein antibody (tACPA) was derived from a hybridoma screen of mice that were immunized with a citrullinated Nterminal H2A peptide; (2) a chimera was generated from m-tACPA that contained the mouse variable domains and human constant domains; (3) a humanized version was generated from ch-tACPA by CDR grafting and germlining toward a human germline; (4) an isomerization site was removed from the light chain CDR1, which resulted in a fully optimized development candidate and backup tACPA molecules that are suitable for large-scale production of clinical grade batches, which can be tested in patients
Here, we showed that tACPAs, antibodies that recognize citrullinated H2A (citH2A) and citrullinated H4 (citH4), have therapeutic efficacy in murine models of neutrophil-mediated inflammatory diseases, including inflammatory arthritis (IA), pulmonary fibrosis (PF), inflammatory bowel disease (IBD), and sepsis

Summary

Introduction

Neutrophils are the most abundant type of leukocytes in human blood, contribute to the first line of defense and use their extensive armory to protect the host against infection. A more recently described antimicrobial function of neutrophils is neutrophil extracellular trap (NET) formation.[1] NETs confine and efficiently eliminate pathogens and have been shown to protect mice and humans against bacterial[2,3] and fungal[4] infections. Despite their importance in host defense, aberrant and prolonged NET release is associated with the pathophysiology of many acute and chronic inflammatory disorders NETs are a source of autoantigens and trigger autoimmunity, which is associated with the production of autoantibodies against various NET components in rheumatoid arthritis (RA),[17] small-vessel vasculitis (SVV),[18] antiphospholipid syndrome (APS),[19] and SLE.[19,20]

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Conclusion