Background/Purpose: Anti-citrullinated protein autoantibodies (ACPA) promote inflammation and joint tissue injury and define a poor prognostic group of patients with rheumatoid arthritis (RA). Citrullinated autoantigens that drive this autoimmune response are generated by Peptidyl Arginine Deiminase (PAD) enzymes. PAD2 and PAD4 are increased in the blood and synovial fluid of RA patients, and are found on the surface of neutrophils and monocytes. Previously, we have shown that AZD1163 a bi-specific antibody targeting these enzymes potently inhibits PAD activity in blood, serum and synovial fluid using a histone H3 citrullination assay. To date, a suppressive role for AZD1163 against the diverse set of citrullinated antigens found in RA patients has not been established, and the impact on cell surface PAD activity has not been evaluated. Herein we evaluate the potential of AZD1163 to inhibit the generation of a broad set of RA-associated citrullinated antigens, and its capacity to suppress cell surface PAD activity.

Methods: Recombinant PAD2 and PAD4 enzymes were used to citrullinate discovery protein arrays and bespoke peptide antigens. Serum from seropositive and seronegative RA patients were evaluated to assess reactivity to citrullinated and unmodified antigens in an ELISA-based format. To assess the effect of AZD1163 on the activity of soluble and membrane PADs, cell culture supernatants or immune cells were incubated with substrates and citrullinated proteins were detected by Western Blot or ELISA with specific antibodies.

Results: Citrullinated antigens were preferentially recognized by autoantibodies in RA patients’ serum seropositive for ACPA. Both PAD2 and PAD4 contributed to the generation of a spectrum of citrullinated autoantigens recognized by RA autoantibodies. Importantly, AZD1163 potently inhibited the generation of these citrullinated autoantigens irrespective of the substrate. AZD1163 also inhibited PAD activity and the citrullination of substrates from both soluble and immune cell membrane associated PADs.

Conclusion: AZD1163 is a bi-specific anti-PAD2/4 antibody in phase I clinical trials (NCT06103877) for the treatment of RA. AZD1163 blocks the enzymic activity of soluble and membrane PAD2 and PAD4 preventing protein citrullination. Targeting the generation of the citrullinated autoantigens that drive autoimmunity and chronic inflammatory responses in the joints is a novel approach for the treatment of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/blockade-of-soluble-and-cell-surface-pad-activity-prevents-the-generation-of-citrullinated-autoantigens-recognized-by-ra-patients-serum/