Background: In the R4RA biopsy-driven randomized clinical trial[1] rheumatoid arthritis (RA) patients were randomised to rituximab or tocilizumab based on their synovial tissue B cell rich/poor signature. Response, defined as 50% improvement of clinical disease activity index (CDAI50), was assessed at 16 weeks. Samples from this cohort were utilised to improve understanding of pathogenic mechanisms determining response to rituximab. This is still a major issue with up to 5-20% of RA patients not responding to all current medication. While rituximab was designed to target CD20 on B cells, effects on CD4 T cells[2] and a highly inflammatory CD20dim T cell population[3] has been described. Several mechanisms of non-response have been proposed, including incomplete B cell depletion in synovium after rituximab.

Objectives: Investigate mechanisms of response and non-response to rituximab through deep molecular and cellular phenotyping of blood and synovial biopsies pre and post treatment.

Methods: B cell receptor (BCR) repertoires from synovial biopsies (n=9) and matched peripheral blood (n=7) RNA from RA patients at baseline and 16 weeks post rituximab were amplified and sequenced. Blood T and B cells (total n=39 baseline, n=33 post-rituximab) were phenotyped and quantified with a 27-marker panel on a spectral flow cytometer and matched with serum protein profiling (proteomics platform) and bulk RNA-Seq from blood pre- and post-rituximab.

Results: The baseline BCR repertoire in blood showed no differences in isotype usage between responders and non-responders. In contrast high levels of IgG2 in synovial tissue was associated with non-response at baseline, and IgA2 was higher at week 16 in the response group compared to non-response (p<0.05). Incomplete depletion of synovial B cells was observed in both responders and non-responders. Highly expanded clones in the joint post-rituximab were found to originate from the baseline synovium. However, there was no association with response with any specific individual clones. This suggests that while failure of depletion of specific B cell clones is linked to treatment failure, these individual clonotypes vary between patients, but tend to originate from within synovium.

At baseline, non-responder blood samples showed a more inflammatory serum profile with higher levels of proteins such as IL18RAP, IL1R1, associated with higher levels of inflammatory cells such as Th17 cells with increased rates of activation markers ICOS and PD1 on NKT cells and CD95 on B cells. Furthermore, the ratio of CD20 expressing B cells compared to CD19 only expressing B cells was significantly 3.8 times higher in response patients compared to non-response patients (p=0.02). Additionally, response patients had 2.04 times higher levels of CD3 ⁺ CD20 ^dim T cells compared to non-responders (p=0.02).

In post-rituximab patients higher NK cell levels were associated with greater response to treatment (p=0.03).

Conclusion: Our results show that there may be multiple mechanisms explaining rituximab response including i) failure to deplete B cell clonotypes in the synovium, ii) higher NK-cell levels that may help clear rituximab-coated B cells and higher levels of inflammatory, CD20 targetable T cells, iii) higher activation profile in T cells preventing response and iv) a B cell profile skewed towards CD19 in non-responders and towards CD20 in responders in the blood. These findings enrich our understanding of mechanisms of response and non-response to rituximab, and could be used as biomarkers for patient stratification at baseline. We observed B cell repopulation originating from within synovium in all patients, which may also explain future relapse.

REFERENCES: [1] Humby , et al. Lancet (2021).

[2] Lavielle, et al. Arthritis Research and Therapy (2016).

[3] Palanichamy and Jahn, et al. Journal of Immunology (2014).

Acknowledgements: We thank all patients participating in the trial and the Patient Advisory Group. The R4RA trial was funded by the Efficacy and Mechanism Evaluation (EME) Programme, a partnership between the Medical Research Council (MRC) and the National Institute for Health and Care Research (NIHR) (grant no. 11/100/76), Versus Arthritis (Experimental Arthritis Treatment Centre, grant number 20022) and Barts Charity (grant number 523/819), MRC and Arthritis Research UK (ARUK) by joint funding of Maximizing Therapeutic Utility in Rheumatoid Arthritis (MATURA) (grant numbers MR/K015346/1 and 20670 respectively), NIHR (grant 131575) and MRC TRACT-RA (MR/V012509/1). This work acknowledges the support of the National Institute for Health Research Barts Biomedical Research Centre (NIHR 203330) as well as the Wellcome Trust for providing funding for the stipend (Wellcome Trust GMS stipend: BST00080.H508.01) for Lauren Overend.

Disclosure of Interests: Anna Surace: None declared, Lauren Overend: None declared, Elisabetta Sciacca: None declared, Liliane Fossati-Jimack: None declared, Edyta Jaworska: None declared, Elena Pontarini: None declared, Rachael J.M. Bashford-Rogers Co-founder of Alchemab Therapeutics Ltd, Alchemab Therapeutics Ltd and GSK, Costantino Pitzalis: None declared, Myles Lewis: None declared.