Background: Anti-citrullinated protein antibodies (ACPAs) have high diagnostic value for rheumatoid arthritis (RA) and serve as predictive biomarkers for disease progression. While the specificity of patient-derived monoclonal ACPAs has been extensively assessed in vitro , in vivo ACPA targets have remained poorly defined. In this study we evaluated the biodistribution of patient-derived monoclonal ACPAs in mice. We analysed the clones 1325:04C03 (C03) and 1325:01B09 (B09), both originally obtained from synovial plasma cells [1]. Previous studies demonstrated that the transfer of C03 and B09 into healthy mice recapitulated very early symptomatology observed in individuals at risk of RA, including pain, bone erosion and tenosynovitis[2–4]. At the same time, both antibodies exerted anti-inflammatory effects in arthritic mice [5].

Objectives: We aimed to determine the distribution of monoclonal ACPA and control antibodies in mice, both at steady-state and during ongoing arthritis.

Methods: Monoclonal ACPA and control antibodies were radiolabelled with the ⁸⁹ Zr isotope and injected into healthy mice or into animals developing collagen antibody-induced arthritis (CAIA). Mice were scanned with positron emission tomography-computed tomography (PET-CT) camera and radioactivity was measured in individual organs at different time-points. Antibody binding to inflamed and non-inflamed joint samples was also analysed using immunohistochemistry.

Results: At steady state, ACPAs transiently accumulated in smaller joints (such as ankles, wrists and feet) following the injection, whereas ACPA and control antibody levels were similar in larger joints. During the first 24 hours, ACPA levels declined in the small joints and remained only slightly elevated compared to the control antibody. In CAIA, inflammation led to increased antibody levels in the joints, however, no differences were observed between ACPA and control antibodies. In ACPA-treated animals, joint radioactivity decreased during the subsequent days, consistent with the reduced overall swelling induced by the injected ACPA clone. Nevertheless, within the joints, ACPA and control antibody densities remained similarly elevated during the follow up period of 6 days. At the same time, immunohistochemical analyses indicated that only C03, but not the control antibody, could bind to targets in the synovial tissue of arthritic mice, predominantly matrix components with low cellularity.

Conclusions: The early accumulation of monoclonal ACPAs in smaller joints suggests specificity-driven joint targeting at steady state. During arthritis, joint influx and overall density of ACPAs and control antibodies were very similar, despite the likely upregulation of protein citrullination in arthritic joints. Nonetheless, microscopic analyses indicated specific binding of ACPAs to structures within inflamed joints.

REFERENCES: [1] Steen J, Forsström B, Sahlström P, Odowd V, Israelsson L, Krishnamurthy A, et al. Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell-Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis. Arthritis Rheumatol. 2019 Feb;71(2):196–209.

[2] Krishnamurthy A, Circiumaru A, Sun J, Kisten Y, Damberg P, Sakuraba K, et al. Combination of Two Monoclonal Anti-Citrullinated Protein Antibodies Induced Tenosynovitis, Pain, and Bone Loss in Mice in a Peptidyl Arginine Deiminase-4-Dependent Manner. Arthritis Rheumatol. 2023 Feb;75(2):164–70.

[3] Jurczak A, Delay L, Barbier J, Simon N, Krock E, Sandor K, et al. Antibody-induced pain-like behavior and bone erosion: links to subclinical inflammation, osteoclast activity, and acid-sensing ion channel 3-dependent sensitization. Pain. 2022 Aug 1;163(8):1542–59.

[4] Jurczak A, Sandor K, Bersellini Farinotti A, Krock E, Hunt MA, Agalave NM, et al. Insights into FcγR involvement in pain-like behavior induced by an RA-derived anti-modified protein autoantibody. Brain Behav Immun. 2023 Oct;113:212–27.

[5] Raposo B, Afonso M, Israelsson L, Wähämaa H, Stålesen R, Wermeling F, et al. Divergent and dominant anti-inflammatory effects of patient-derived anticitrullinated protein antibodies (ACPA) in arthritis development. Ann Rheum Dis. 2023 May;82(5):724–6.

Acknowledgments: NIL.

Disclosure of Interests: None declared.