MICA: Interleukin-21 in rheumatoid arthritis: exploring its therapeutic potential for the development of a novel targeted biologic therapy.

Rheumatoid arthritis (RA) is a chronic, autoimmune disease that affects ~1% of the population and an important cause of disability and premature mortality. The disease is driven by chronic inflammation of the joint synovial tissue in which there is accumulation of activated immune cells (macrophages, dendritic cells, T-cells, B-cells and plasma-cells).

The therapeutic benefit of B-cell depletion with anti-CD20 mAb in RA demonstrated that autoreactive B-cells play a crucial pathogenic role. Likewise, blockade of inflammatory cytokines (TNFa, IL-6) results in significant clinical response. However, ~30-40% of patients remain refractory to current therapies. Thus, the development of better therapies in this area of unmet need remains of critical importance particularly in "resistant" patients where the identification of additional/alternative inflammatory pathways may lead to novel therapeutics.

IL-21 is a potent pleiotropic cytokine involved in the activation/differentiation of many immune cell types including B and T cells but also stromal cells with the induction of metalloproteinases and inflammatory cytokines including TNFa. IL-21 is a member of the common cytokine receptor gamma chain-binding family and is mainly produced by activated T-cells including Th17 cells and TFh. TFh play a fundamental role in B cell activation and antibody production in secondary lymphoid organs and control humoral immune responses.

IL-21 has been associated with RA based on genetic studies and increased expression in RA patients; however its pathogenic function in RA joints and its potential as a therapeutic target have not been fully addressed. Blockade of IL-21 receptor (IL-21R) considerably ameliorates disease progression in animal models of RA indicating that modulation of the IL-21/IL-21R pathway is a promising therapeutic approach.

In support of this application we studied a large cohort of RA patients and demonstrated that i) the IL-21/IL-21R pathway is highly increased in a subset of RA patients presenting focal lesions called ectopic lymphoid structures (ELS) but not in patients with diffuse synovitis; ii) synovial IL-21 protein is expressed in strict association with a subset of TFh-like cells; iii) RA synovial fibroblasts, but not RA dermal fibroblasts, express high levels of IL-21R suggesting that IL-21 acts not only on infiltrating immune cells but also on resident synovial cells; iv) IL-21 and IL-21R expression is maintained for several weeks when RA synovial tissue is engrafted into immunodeficient (SCID) mice and support the production of human autoantibodies.

Thus, we ultimately aim to develop effective IL-21 targeting strategies in RA through the following 4 steps: Step 1) Identification of the cellular sources and targets of IL-21 within the joints of RA patients. Step 2) Analysis of IL-21 function on immune and stromal cells isolated from the RA joints in vitro and in vivo in the RA/SCID model. Step 3) Testing IL21/IL21R blocking reagents for human use (in preclinical development with the industrial partner) in the human RA/SCID model. Step 4) Evaluating IL-21 contribution to disease outcome and response to existing traditional and biological therapies in patients with early arthritis and in those treated with anti-TNFa or anti-IL-6R Biologics.

By addressing key aspects of IL-21 biology in RA this proposal will advance our knowledge of the role of IL-21 in chronic inflammation, B cell autoimmunity and ELS function with important implications for other autoimmune/chronic inflammatory diseases. Proof of concept studies of the effectiveness of human IL-21 blocking reagents on synovial inflammation and humoral autoimmunity in diseased tissue will advance the development of novel IL-21 therapeutics for the treatment of RA. Finally, the patient-based studies may enable better identification of key responders of IL-21 mediated arthritis and have a means of stratifying subjects in early clinical trials.

Our developmental plan follows 4 steps

Step 1 Identification of IL-21 producing/target cells in RA
We will employ confocal microscopy on RA synovial tissues and FACS on isolated RA synovial cells to identify IL-21 and IL-21R+ cells in in association with specific markers for Th1 (IFNg,T-bet, CXCR3), Th2 (IL-4/GATA-3), Th17 (IL-17A/F, Rorgt, CCR6) and TFh (CXCR5, ICOS, PD-1) cells. Additionally, IL-21R expression will be investigated on synovial B cells subsets and CD8+ T cells.

Step 2 Functional role of IL-21 in inflammation, autoimmunity and tissue damage
We will define the functional role of IL-21:
in vitro: i) on human IL-21R+ RA synovial fibroblasts assessing the capacity of IL-21 to induce cytokines and MMPs (mRNA and protein) and ii) on synovial B cell activation, including B cell differentiation (FACS), AID/circular transcripts (rt-PCR) and class-switched antibodies (ELISA).
in vivo: in the RA/SCID chimera by analysing IL-21-induced activation of pro-inflammatory pathways such as ELS function, B cell autoreactivity, production of inflammatory cytokines and stromal cell activation.

Step 3 Pre-clinical development of IL-21 blocking agents
We will directly contribute to the development of pre-clinical IL-21 therapeutics by testing their efficacy in vivo in the HuRA-SCID chimera on i) synovial inflammation and ELS; ii) B/T cell function; iii) autoantibody production and iv) stromal cell activation.

Step 4 IL-21 in disease outcome and response to treatment in RA: a roadmap to patient stratification
We will assess IL-21 and related pathways as biomarkers of disease evolution and response to treatment in prospective clinical studies in the MRC Pathobiology of Early Arthritis Cohort and in RA patients treated with IL6-R and TNFa biologics.

Emerging data will be applied to clinical trial design based on IL-21 target validation, role in synovial immunopathology, development of IL-21 blocking reagents and stratification of patients for treatment

The project will have a significant impact both on basic research and applied translational science.

1. Impact on basic research and knowledge acquisition within and beyond the field of RA and rheumatology.

At the basic science level a better understanding of IL-21 in key inflammatory immune mechanisms in a complex disease such as RA will broadly benefit the scientific community with considerable advance in knowledge not only in the specific field of RA but with significant repercussion on other autoimmune diseases, particularly those characterized by ELS, both within the rheumatology field (i.e. Sjogren's syndrome, lupus) and beyond with applications in numerous other medical conditions including: inflammatory bowel diseases, multiple sclerosis, as well as potentially on hematological malignancies.

2. Impact on translational and clinical research.

- Shifting therapeutic paradigms within the RA field. Demonstration of functionality in ELS at synovial tissue level will provide supporting evidence to target IL-21 therapeutically in RA. Furthermore, the opportunity to dissect the molecular functions in sophisticated in vitro and in vivo models (SCID/RA chimeric model) using human tissues will provide invaluable data for the translational relevance of this important cytokine pathway to the disease and may lead to potential biomarkers that could be used to help identify anti-IL-21 responding patients.
Additionally, this project aims to transform the notion of "blind" clinical trial to that of biopsy-guided, pathology driven patient stratification. In this regard, IL-21 seems an ideal candidate as it is clear that IL-21 expression is linked to the formation of ELS in the synovium, which occurs in ~40-50% of RA patients. Thus, a better comprehension of IL-21 function in diseased tissue subtypes will provide a stronger understanding on how to stratify subjects for IL-21 based therapies. In addition, the evaluation of IL-21 expression in disease evolution in deeply phenotyped early arthritis patients [Pathobiology of Early Arthritis Cohort (PEAC) http://www.peac-mrc.mds.qmul.ac.uk/] and following treatment with current biologics (IL6-R and TNFa) may identify novel biomarkers of disease prognosis and/or response to anti-IL-21 therapy.

- Broader impact on translational and clinical science through the development of IL-21 blocking agents for clinical use. MedImmune is a biopharmaceutical company and world player in antibody therapeutics with significant expertise in the development of novel therapeutics in autoimmune diseases. IL-21 blockade is highly ranked in the translational pipeline. The opportunity to develop unique reagents in partnership with MedImmune could enable the acceleration of the development programme to bring targeted IL-21 therapies for clinical use not only in patients with RA, but also in other autoimmune conditions in which IL-21 is emerging as a critical pathogenic player.

3- Commercial impact on the public and private sector.

A defined development programme described in this application coupled with the formation of a strong Academic Industry partnership will maximise the opportunity of success by translating novel scientific and clinical observation into commercially valuable assets such as diagnostic or therapeutic agents. The equitable Head of Terms agreed between QMUL and MedImmune will also ensure that economic benefits will profit both private and public partners.

4- Impact on patient health.

The ultimate beneficiaries of this research it is hoped will be patients with RA. Although the time-scale for the direct impact on patient health of IL-21 blocking agents in RA is likely to be long-term, the definition of biomarkers of disease evolution and clinical response to current treatments in RA and the role of IL-21 in this context would provide more immediate impact on RA patients through biopsy-based stratification allowing higher response rates to available therapies.