Background/Purpose: Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and despite many therapeutic options, an unmet medical need persists for novel therapies in RA. Protein citrullination is a post-translational modification in which arginine is converted to citrulline through a process mediated by the activity of peptidylarginine deiminase enzymes. Citrullination is an important driver of RA pathogenesis and autoantibodies against citrullinated proteins are observed in majority of RA patients. We have developed a novel autologous chimeric antigen receptor (CAR) Treg therapy, SBT777101, that is designed to suppress inflammation in RA patients by utilizing a targeting domain directed against a subset of citrullinated antigens (Van der vurst de vries, ACR 2022). An enzyme-linked immunosorbent assay (ELISA) was developed to characterize the level of CAR-reactive antigens in RA patients.

Methods: An ELISA assay using a capture antibody derived from the binding domain of SBT777101 was used to measure the level of citrullinated proteins in synovial fluid (n = 176 RA, n = 54 non-RA) and serum (n = 130 RA, n = 15 non-RA). Levels of citrullinated proteins as evaluated by the ELISA assay, were compared to the activity produced by the antigen in a CAR-expressing cell-based assay. Additionally, the level of citrullinated antigens were compared to the levels of inflammatory markers in a set of matched serum and synovial fluid samples (n = 50).

Results: In RA patients with active disease, elevated protein citrullination was detected in 84% of synovial fluid samples and in 48% of serum samples (n = 50 matched synovial fluid and serum). Citrullinated antigen in synovial fluid correlated with levels of the antigen in serum (n = 50, Spearman R = 0.41, p < 0.05). The levels were higher by 4.6-fold in synovial fluid compared to serum which was reflected by more activity as detected by the CAR-expressing cell-based assay in synovial fluid. Additionally, levels of CAR-specific citrullinated antigens as measured by ELISA significantly correlated with the level of activity measured in the CAR-expressing cell-based assay (n =50, Spearman R = 0.48, p < 0.05) and with several markers of inflammation measured in synovial fluid (TNFα, IL1β, IL6, IL12p70, IP10 and MIP1α) and in serum (CRP and IL6) of these patient samples.

Conclusion: In summary, we were able to detect CAR-reactive citrullinated antigens for SBT777101 in synovial fluid and serum and these levels correlated with ability to activate the CAR in a cell-based assay, and with several inflammatory markers both in synovial fluid and serum. The presence of these CAR-reactive citrullinated antigens and inflammatory markers will be further evaluated in an upcoming planned phase 1 study.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/detection-of-citrullinated-proteins-recognized-by-a-novel-chimeric-antigen-receptor-tregtherapy-in-both-synovial-fluid-and-serum-from-patients-with-rheumatoid-arthritis/