Title: Evaluating treatment response mechanisms of Rituximab and Tocilizumab in Rheumatoid Arthritis in the R4RA randomised clinical trial
Funder: Medical Research Council

Abstract: Rheumatoid arthritis (RA) is one of the most common inflammatory disorders. It is an autoimmune disease in which the immune system mistakenly attacks the joints, causing pain, stiffness and swelling of the joints. The immune system is very complex with lots of different types of cells and molecules involved. Established medicines, which are tried first when treating patients, act by damping down the immune system in a non-specific way. If these are ineffective patients are treated with newer, more targeted medicines but because we don’t know which part of the immune system has gone wrong we don’t know which one to use. Our research is designed to understand more about how these targeted medicines work and so to help identify the medicine that works best for the patient._x000D_ We previously ran a clinical trial, known as the R4RA trial, comparing Rituximab and Tocilizumab, both interfere with the immune system but in different ways- Rituximab attaches to a molecule (CD20) on B-cells and Tocilizumab blocks the action of a molecule Interleukin-6 (IL6). In the R4RA clinical trial, samples from the joints (synovial tissue) of patients were examined to see if the type of cells or molecules present could help guide doctors to choose the best treatment.. One hundred and sixty-four people with RA joined the R4RA trial and had a biopsy to remove tiny pieces of the joint lining (synovium) from one of their swollen joints, they were followed up every month to see if the treatment was working. Because Rituximab works by reducing B-cells we expected that in the patients with low numbers of B- cells Tocilizumab would work better than Rituximab. Overall, the results strongly suggested that this was the case, but the results were not clear cut._x000D_ In this research project, we will investigate the tissue and blood samples already donated by patients in the R4RA trial in order to better understand the reasons why Rituximab and Tocilizumab either work or are ineffective. We will look at a range of genes and molecules associated with B-cells and IL-6 in both the synovial tissue and blood of patients to see whether the activity of genes and quantities of these molecules are useful in predicting the effectiveness of these two medicines. For example, we know that most but not all B-cells have CD20, the target for Rituximab, so we will investigate whether one of the reasons for treatment with Rituximab not working is because B cells lacking the CD20 cause the disease to continue. Similarly, we will explore if there are mechanisms that modify the production or action of IL6 when Tocilizumab is ineffective._x000D_ The work will generate a large amount of data which will be analysed by experienced bioinformaticians who will use sophisticated computer systems and statistical programmes to identify the important biological pathways underling the reasons for medicines working, or not._x000D_ This work may help us to understand the mechanisms underlying why these targeted medicines work or are ineffective. This could lead to more effective prescribing which will be better for patients and will save money for the NHS._x000D_ This work has been shared with, and driven by, the patient group involved in the R4RA trial and the group will continue to be involved throughout this project. They will review our progress, put the patients’ perspective on the results and help to raise awareness of this work in the patient community.

Lay abstract: Background_x000D_ The proposed research capitalises on the NIHR-EME funded R4RA Randomised Clinical Trial (RCT) to evaluate the mechanisms of action of Rituximab (RTX) and Tocilizumab (TOC). R4RA is the first synovial biopsy-driven multicentre RCT aimed at testing the hypothesis that specific cellular/molecular signatures in rheumatoid arthritis (RA) joint tissue (high v low B cells; high v low IL-6 signalling system) influence therapeutic outcome to RTX or TOC. Analysis of the R4RA trial has shown that in RA patients with low B cells in synovial biopsy classified by histopathology (number of CD20+ve B cells), RTX was less effective compared to TOC. This result was enhanced when an RNA-seq based B-cell poor/rich classification was utilised, implicating additional B cell differentiation genes in response/non-response to RTX and TOC._x000D_ _x000D_ Research question_x000D_ We hypothesise that i) low expression of RTX and TOC targets (CD20 and IL-6/IL6-R pathways) and/or critical downstream cells and signalling apparatus (plasmablasts, plasma cells, IL-6R/gp130 complex) in RA synovium is associated with failure to respond to RTX and TOC; ii) failure to deplete synovial B cell clonotypes, plasmablasts, plasma cells (CD20-ve) or repopulation of pathogenic B cell clones from synovial niche or primary/secondary lymphoid organs is associated with persistent disease and treatment failure in RA; iii) blood markers of synovial pathotypes predict response to therapy._x000D_ _x000D_ Aims & objectives_x000D_ 1. Determine whether pathogenic B cell clonotypes and diverse B cell differentiation states drive non-response to RTX or TOC._x000D_ 2. Define whether low expression of IL-6/IL-6R gene module and/or B-cell signatures predict poor response to TOC._x000D_ 3. Test the hypothesis whether previously reported biomarkers in the peripheral blood (PB) act as surrogates of tissue pathology (akin to a ‘liquid biopsy’), and are mechanistically linked to response to RTX and TOC._x000D_ _x000D_ Methods_x000D_ 1. Deep histological phenotyping of synovium by Hyperion mass cytometry using antibody markers for B/T cell, plasma cell, macrophage, FDC & fibroblast populations._x000D_ 2. Plasmablasts/ pre-plasma cell PB repopulation will be measured by high sensitivity flow cytometry (hsFACS)._x000D_ 3. B cell receptor repertoire sequencing and family tree analysis will be used to map B cell clonality in tissue and peripheral blood as a mechanism of non-response to RTX and TOC._x000D_ 4. Bioinformatic synovial RNA-seq expression analysis for drug targets & linked signalling apparatus and response to TOC/RTX._x000D_ 5. i) Luminex analysis of serum markers associated with synovial pathology, therapeutic response and up/downstream factors associated with B cell maturation and IL-6 secretion._x000D_ ii) QT-PCR analysis of IFN response signature and B cell maturation associated blood transcripts pre/post treatment and with response._x000D_ _x000D_ Timelines for delivery_x000D_ The project will be delivered over a 2 year period adhering to milestones & GANTT chart as documented (see main application)._x000D_ _x000D_ Anticipated impact & dissemination_x000D_ The project will improve scientific knowledge of effective treatment mechanisms in RA, underpinning future funding and industry participation. Elucidating the mechanisms driving response/non-response to RTX and TOC could help identify the right drug for the right patient, and thus prevent delay in starting an effective biologic, reduce exposure to adverse drug effects and avoid wasting several £million/year in NHS and welfare costs.

Research activity codes: 5.1 PHARMACEUTICALS;6.1 PHARMACEUTICALS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM