¹Institut für Immunologie, Universitätsmedizin Johannes Gutenberg-Universität, Mainz
²Gene Transfer Department, Translationale Onkologie an der Universitätsmedizin der Johannes-Gutenberg-Universität, Mainz
³Johannes Gutenberg-Universität, Universitätsmedizin, Schwerpunkt klinische Immunologie und Rheumatologie, Mainz

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Introduction: ACPAs are prominent autoantibodies associated with poor prognosis in the pathogenesis of Rheumatoid Arthritis (RA) [1]. Selectively eliminating ACPA-producing B cells is a popular area of research. This study presents data on a Universal-CAR T (Uni-CAR) cell approach using targeting modules of citrullinated peptides, which are specific epitopes of ACPAs.

Methods: ACC22 and E43 ACPA-B cell receptor expressing Raji cells were created as target cell models, and their surface expression characteristics and binding properties for citrullinated peptide epitopes (collagen-II359-369, collagen-II483-498, fibrinogen-β60-74, fibrinogen-β36-52, and α-enolase) were validated using PE-labeled streptavidin tetramers and FACS method. To enable the integration of peptides with Uni-CAR, citrullinated peptides containing a myc-tag (targeting module: TM) were synthesized, and co-culture experiments with varying ratios of cells and peptides were conducted. The activation status of Uni-CAR T cells was determined by changes in IFN-γ levels in the medium supernatants. To evaluate efficacy in real patient cells, B cells were isolated from three patients in the active disease stage with Anti-CCP levels >200 U/mL and co-cultured with Uni-CAR T cells. A Luciferase Assay System was used to assess cytotoxic activity.

Results: In co-culture experiments, we observed that the production of IFN-γ by Uni-CAR T cells increased proportionally with TM dosage. In the Luciferase Assay, we noted that the cytotoxic effect observed on ACC2 and E4-BCR Raji cells increased with TM doses. Tetramer binding analysis of B cells isolated from RA patients revealed positive cell rates of 10.1%, 7.05%, and 4.45% for collagen-II359-369, as well as 42.4%, 25.9%, and 30% for fibrinogen-β36-52. In cytotoxicity assays with these cells, we again detected an effect consistent with TM dosage.

Conclusion: This study highlights the effectiveness of targeting strategies using Uni-CAR T cells against ACPA-producing B cells. The increased IFN-γ production and enhanced cytotoxic effect correlated with TM dosage demonstrate the potential of ACPA-targeted therapies in treating rheumatoid arthritis. The specific binding rates observed in B cells isolated from RA patients suggest that these approaches could be developed as next-generation immunotherapy strategies. The findings provide a significant foundation for future clinical research.

References

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