Background: Despite therapeutic advancements in rheumatoid arthritis (RA), a significant subset of patients (20-40%) remains unresponsive to current treatments, including biologics and targeted-synthetic DMARDs. A deeper understanding of the molecular impacts of these drugs could pave the way for tailored therapeutic strategies.

Objectives: 1. To delineate the molecular shifts triggered by TNFi, IL6Ri, and JAKinibs in RA immune cells via ex-vivo assays. 2. To identify RA patient subgroups with active disease exhibiting molecular patterns parallel to those influenced by these medications.

Methods: Peripheral blood mononuclear cells (PBMC) and neutrophils from 24 treatment-naïve RA patients were cultured for 24h and 12h, respectively, with autologous serum and one of three agents: etanercept (TNFi), sarilumab (IL6Ri), or baricitinib (JAKinib) (all at 10 micromolar). Changes in PBMC and neutrophil proliferation, adhesion, and NETosis-derived products were quantified using specialized kits. The impact of each drug on protein expression was gauged via the proximity extension assay (PEA) technology (Olink), analyzing a 92-protein inflammation panel. This proteomic signature’s presence was validated in the serum of a new cohort of 183 RA patients, demonstrating a similar duration of disease evolution, moderate activity, and clinical response to DMARDs as observed in the patients subjected to the in vitro studies.

Results: TNFi, IL6Ri, and JAKinibs downregulated PBMC and neutrophil proliferation and adhesion in 94% of RA patients. Notably, TNFi and IL6Ri outperformed JAKinibs. All inhibitors effectively countered NETosis triggered by autologous serum. The autologous serum enhanced inflammatory protein secretion by both cell types, with each drug uniquely influencing various cytokines, chemokines, and growth factors. Specifically, Etanercept and Baricitinib exhibited broad-spectrum modulation of inflammatory proteins.

Lastly, examination of the circulating inflammatory proteome revealed that approximately 50% of active RA patients displayed at least one altered proteomic signature associated with the one modulated ex vivo by any of the drugs. Specifically, 23,5% of patients exhibited an altered proteomic signature modulated ex vivo by the 3 drugs, 11,5% by two of them and 15% by only one precise drug.

Conclusion: The specific cellular and molecular changes elicited by TNFi, IL6Ri and JAKinibs in ex vivo assays of RA immune cells, were consistent with singular altered serum profiles observed in subgroups of active RA patients.

These findings provide compelling evidence for the therapeutic potential of each treatment, underscoring their alignment with individual patients’ baseline molecular profile alterations.

REFERENCES NIL.Acknowledgements: Supported by the EU/EFPIA Innovative Medicines Initiative Joint Undertaking 3TR, Projects no. PI21/0591 & CD21/00187 funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union. Project no. RD21/0002/0033 funded by ISCIII and funded by the European Union-NextGeneration EU, via Plan de Recuperación, Transformación y Resiliencia (PRTR) and MINECO (RYC2021-033828-I, and PID2022-141500OA-I00).

Disclosure of Interests: None declared.