Background/Purpose: Anti-citrullinated protein antibodies (ACPA) are present in two-thirds of patients with rheumatoid arthritis (RA), and target proteins that have been post-translationally modified by the calcium-dependent peptidylarginine deiminase (PAD) family of enzymes. In addition, anti-PAD4 autoantibodies are found in about 30% of RA patients and are correlated with disease severity. Anti-PAD4 autoantibodies are thought to enhance the calcium (Ca⁺⁺) sensitivity of PAD4, thereby increasing its activity at sub-optimal Ca⁺⁺ concentrations present in the synovial tissues. In this study, we aim to identify, isolate, and functionally characterize anti-PAD4 monoclonal antibodies (mAbs) derived from RA patients’ plasmablasts to investigate their pathophysiological role in disease.

Methods: We sequenced the paired heavy and light chain immunoglobulin genes of individual plasmablasts from RA patients’ blood using DNA cell barcode-enabled sequencing. We recombinantly expressed 72 mAbs representing clonal families identified in the plasmablast antibody repertoires of 6 RA patients, and evaluated their capacity to bind PAD4 by ELISA and immunoprecipitation/Western blotting (IP/WB) assays. We further evaluated the effects of anti-PAD4 mAbs and Ca⁺⁺ concentration on PAD4 enzymatic activity by co-incubating each mAb with recombinant PAD4 and the substrate histone H3. We extended these experiments to assays using freshly isolated human neutrophils.

Results: 7/72 (9.7%) recombinant mAbs recognized PAD4 by ELISA and/or IP/WB. Most of the anti-PAD4 mAbs (5 of 7) belonged to clonal families that included additional ACPA members, and 2 anti-PAD4 mAbs were polyreactive against multiple citrullinated antigens. When the anti-PAD4 mAbs and related clonal family members were tested for their effects on PAD4 activity, we found that the majority enhanced enzymatic activity, resulting in increased citrullination of H3 (citH3), both in the recombinant PAD4 and neutrophil in vitro assays. Interestingly, in the latter assay, increased citrullinated H3 was observed both at 0.5 (suboptimal) and 2 (optimal) mM Ca⁺⁺. We also observed that combinations of anti-PAD4 mAbs were more effective at enhancing enzyme activity, suggesting additive or synergistic effects of anti-PAD4 mAbs on potentiating PAD4 activity.

Conclusion: We identified affinity-matured and class-switched RA blood plasmablast clonal families that encode PAD4-reactive antibodies, suggesting that they arise from antigen- and T-cell-driven mechanisms. Most of the anti-PAD4 mAbs identified enhanced PAD4 activity in vitro, particularly at sub-optimal Ca⁺⁺ concentrations. Experiments to characterize the specific binding epitopes of anti-PAD4 mAbs and to evaluate their pathogenic effects in vivo are ongoing.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterization-of-monoclonal-anti-pad4-autoantibodies-from-rheumatoid-arthritis-patients-functional-implications-for-citrullination-and-disease-progression/