Background: One third of rheumatoid arthritis (RA) patients do not respond to first-line treatment with classical synthetic disease-modifying anti-rheumatic drugs (csDMARD), such as methotrexate (MTX), and short-term glucocorticoids. We previously identified a panel of 3 antibody biomarkers against University Hasselt (UH)-RA peptides, that were associated with the lack of first-line therapy response. One of these antibodies, anti-UH-RA.329 antibody, showed binding to fibroblast-like synoviocytes (FLS) in RA synovial tissue (Vandormael et al ., 2024).

Objectives: This study aimed to biologically characterize the anti-UHA-RA.329 antibody by determining its pathological effects on therapy response in a mouse model of arthritis

Methods: To establish an MTX-induced remission model, collagen-induced arthritis (CIA) was induced in 8-week-old DBA/1 mice. Three MTX dosing regimens were tested (n=5/group): 2.5 mg/kg subcutaneously (SC) twice/week, 20 mg/kg SC once/week, and 35 mg/kg intraperitoneally (IP) twice/week. Untreated arthritic and healthy control groups were additionally included. Visual arthritis scores (VAS), ankle, and paw thickness were monitored over 70 days. The optimal MTX regimen (35 mg/kg IP) was used in a subsequent experiment evaluating the pathological effects of anti-UH-RA.329 antibody. Thus, DBA/1 mice were divided into four groups (n=7/group): arthritic mice, arthritic mice treated with MTX, arthritic mice treated with MTX and polyclonal rabbit anti-UH-RA.329 antibody, and arthritic mice treated with MTX and rabbit IgG control antibody. These mice were monitored for 70 days and the VAS, ankle and paw thickness were determined throughout the study. At day 70, mice were sacrificed and ankles were harvested for histological scoring and immunofluorescence (IF) staining of the membrane attack complex (C5b-9), as a marker of complement activation.

Results: The MTX-induced remission CIA model was optimized, with a dose of 35 mg/kg MTX IP twice/week showing the lowest arthritis incidence (80%) and clinical severity. This model was used to test the pathological effect of the anti-UH-RA.329 antibody on therapy response. In mice receiving anti-UH-RA.329 antibody alongside MTX, arthritis severity was high with an average VAS of 3.8 out of 4, and average ankle thickness of 3.4 mm. Histological analysis confirmed severe joint pathology, with a mean score of 11.6 out of 12, characterized by pronounced synovial hyperplasia, inflammatory infiltrates and cartilage destruction. In contrast, MTX-only treated mice displayed significantly milder disease when compared to the anti-UH-RA.329 antibody group (VAS: 1.8/4, p < 0.0001; ankle thickness: 2.7 mm, p = 0.005; histology score: 4.1/12, p < 0.0001). Similarly, mice treated with MTX and control IgG antibody also demonstrated reduced disease severity compared to the anti-UH-RA.329 antibody group (VAS: 2.0/4, p: <0.0001; ankle thickness: 2.8 mm, p = 0.002; histology score: 4.4/12, p = 0.001). Moreover, ankle sections from mice treated with MTX and anti-UH-RA.329 antibody showed enhanced complement activation, as evidenced by increased deposition of the membrane attack complex C5b-9, compared with all other groups.

Conclusions: Passive transfer of the anti-UH-RA.329 antibody abolished the effect of first-line MTX treatment in the CIA model, both in comparison to animals treated with MTX only and those treated with MTX and IgG control antibodies. Additionally, The increased C5b-9 activation in anti-UH-RA.329/MTX–treated mice suggests complement activation as a potential effector mechanism contributing to loss of MTX efficacy. These results highlight the functional relevance of the anti-UH-RA.329 antibody in treatment response and/or RA pathology in vivo.

REFERENCES: [1] Vandormael P, Fadlallah S, Ruytinx P, Pues A, Sleurs E, Liesenborgs J, et al. Fibroblast-like synoviocyte targeting antibodies are associated with failure to reach early and sustained remission or low disease activity after first-line therapy in rheumatoid arthritis. RMD Open. 2024 Nov;10(4):e004743.

Acknowledgments: NIL.

Disclosure of Interests: None declared.