Evaluating treatment response mechanisms of Rituximab and Tocilizumab in Rheumatoid Arthritis in the R4RA randomised clinical trial

Background

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.

Research question

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.

Aims & objectives

1. Determine whether pathogenic B cell clonotypes and diverse B cell differentiation states drive non-response to RTX or TOC. 2. Define whether low expression of IL-6/IL-6R gene module and/or B-cell signatures predict poor response to TOC.

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. Methods

1. Deep histological phenotyping of synovium by Hyperion mass cytometry using antibody markers for B/T cell, plasma cell, macrophage, FDC & fibroblast populations. 2. Plasmablasts/ pre-plasma cell PB repopulation will be measured by high sensitivity flow cytometry (hsFACS).

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.

4. Bioinformatic synovial RNA-seq expression analysis for drug targets & linked signalling apparatus and response to TOC/RTX.

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.

ii) QT-PCR analysis of IFN response signature and B cell maturation associated blood transcripts pre/post treatment and with response. Timelines for delivery

The project will be delivered over a 2-year period adhering to milestones & GANTT chart as documented (see main application). Anticipated impact & dissemination

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.