Background/Purpose: Neutrophil extracellular traps (NETs) contribute to the pathophysiology of multiple inflammatory and autoimmune diseases (Chirivi et al., 2021; DOI: 10.1038/s41423-020-0381-3). Targeting the NETosis pathway has demonstrated significant therapeutic potency in various disease models. Here we describe a first in class monoclonal antibody (CIT-013) with high affinity for citrullinated histones H2A and H4 which inhibits NET formation and reduces tissue NET burden in vivo with significant anti-inflammatory consequences. CIT-013 is currently in phase 1 clinical trials with phase 2a studies in RA due to commence in 2024.

The objective of the current study was to further unravel CIT-013’s mode of action. Questions which we wanted to answer are: 1) How and when does CIT-013 interfere with the NETosis pathway?; and 2) what is the faith of CIT-013 opsonized NETs and NETting neutrophils? Furthermore, we investigated whether CIT-013’s epitope is expressed in rheumatoid arthritis (RA) synovial tissue.

Methods: In vitro life imaging studies using neutrophils and macrophages in combination with monovalent as well as bivalent CIT-013 have been performed to investigate CIT-013’s mode of action. An in vivo lung inflammation mouse model was used to investigate CIT-013’s effect on NET burden as well as phagocytic macrophages. Finally, the presence of CIT-013’s epitope in human RA synovial tissues have been investigated using immunohistochemistry techniques.

Results: Detection of CIT-013 epitopes in RA synovium provides evidence that RA is an autoimmune disease with excessive citrullinated-NETs that can be targeted by CIT-013. We show that CIT-013 acts upon the final stage of NET formation, binding to its chromatin epitopes when plasma membrane integrity is compromised to prevent NET release. Bivalency of CIT-013 is necessary for NETosis inhibition. In addition, we show that CIT-013 binding to NETs and netting neutrophils enhances their phagocytosis by macrophages. Furthermore, we demonstrate that a mouse variant of CIT-013 reduces tissue NET burden in vivo at least in part through enhanced macrophage phagocytosis.

Conclusion: Since NETs contribute to the pathophysiology of many immune mediated inflammatory diseases, including autoimmune diseases such as RA, CIT-013’s unique ability to both inhibit NET release and enhance NET clearance indicates the importance of this first in class therapeutic antibody as a new emerging therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-citrullinated-histone-antibody-cit-013-a-dual-action-therapeutic-for-neutrophil-extracellular-trap-associated-autoimmune-disease/