Discovery of TNF-α as a therapeutic target in rheumatoid arthritis: preclinical and clinical studies

Joint Bone Spine 2002 ; 69 : 12-8 © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1297319X01003359/REV

REVIEW

Discovery of TNF-α as a therapeutic target in rheumatoid arthritis: preclinical and clinical studies Marc Feldmann*, Ravinder N. Maini Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, 1 Aspenlea Road, London W6 8LH, UK

Summary – The development of effective new treatments is greatly facilitated by the understanding of the mechanisms of disease. In rheumatoid arthritis, there has been progress in understanding its immunology, the HLA class II predisposition including the ‘shared epitope’ and more recently in understanding the importance of proinflammatory cytokines. Here we review our work in defining TNFα as a therapeutic target in rheumatoid arthritis, from an understanding of molecular pathogenesis in vitro, to formal proof in the clinic in vivo. There is now extensive clinical use of anti-TNFα biologicals for severe rheumatoid arthritis in the US and Europe. Joint Bone Spine 2002 ; 69 : 12-8. © 2002 Éditions scientifiques et médicales Elsevier SAS antibody / cytokines / infliximab / rheumatoid arthritis / therapy At the first EULAR meeting in June 2000, the coveted European Courtins-Clarins award was shared between two groups, one headed by Professor Marc Feldmann and Professor Ravinder N. Maini and the other by Professor Jean-Michel Dayer. Presented at EULAR, June 2000, Nice, France, in receipt of Prix Courtin-Clarins

PRECLINICAL STUDIES The progress in defining the nature of the soluble protein mediators of inflammation, immunity, tissue growth, repair, fibrosis, which are now collectively termed cytokines (reviewed in [1-3]), prompted several groups in the mid-80s to investigate their possible role in various diseases (e.g., [4-7]). Our interest in this field was stimulated by awareness that a number of disease features of human autoimmune sites were possibly dependent on cytokines. Thus human thyroid Grave’s disease tissue expresses abundant HLA class II (the molecules involved in antigen presentation of peptides to CD4 T cells), even on cells which are not normally

* Correspondence and reprints. E-mail address: [email protected] (M. Feldmann).

considered capable of such function, such as thyroid epithelial cells [8]. On this basis we considered that cytokines might be of importance in the pathogenesis of other human autoimmune disease [9]. Rheumatoid arthritis, representing one of the most frequent autoimmune diseases, with the disease tissue being relatively accessible and expressing similar histological features [10, 11], was an obvious choice for in-depth studies to define the role of cytokines. With the cloning of the cDNAs encoding cytokines, first the interferons [12], then IL-2 [13], IL-1 [14] etc., the molecular tools became available in the 1980s to evaluate cytokine expression in the tissue samples made available from biopsies and surgical procedures upon inflamed joints. We, and others, rapidly documented that many of the known proinflammatory cytokines are expressed in RA synovium ([4-7, 15], reviewed [2, 16]). Of interest was that this was the case in essentially all

TNFα is an effective therapeutic target

Figure 1. Cytokine disequilibrium in rheumatoid arthritis. (Reprinted with permission from Feldmann et al., Cell 1996 ; 85 : 307-10.)

such tissues, irrespective of the stage of disease. The simplest interpretation of this universal expression of cytokines in diseased tissue was that, unlike their transient expression during a self-limiting normal inflammatory or immune response, in disease there is continuous cytokine expression. Perhaps, surprisingly, it was also found that the antiinflammatory cytokines (e.g., IL-10 [17, 18], TGFβ [19, 20]) were also upregulated in RA synovium. This led us to formulate the concept that it is the disequilibrium between pro-inflammatory and anti-inflammatory cytokines that leads to a chronic inflammatory state (figure 1). With a plethora of proinflammatory cytokines being expressed, it was a problem to decide which one might be a therapeutic target. Many groups in the field came to the conclusion that blocking multiple cytokines was unrealistic, and hence cytokines were not good therapeutic targets. Our own view, based on the rate-limiting nature of cytokines, with their transient expression of minute quantities, was that cytokines must be therapeutic targets, the dilemma being which one. To address that issue, our approach was to investigate the regulation of IL-1. IL-1 was chosen as it is not only a powerful

Figure 2. Cytokine cascade in rheumatoid arthritis. (Reprinted with permission from Feldmann et al., Cell 1996 ; 85 : 307-10.)

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proinflammatory cytokine, but especially because it had recently been reported by Saklatvala and colleagues that IL-1 could provide a powerful signal for inducing destruction of cartilage and bone in joints [21]. To study cytokine dysregulation in RA joints the experimental system that we chose was to culture cells from enzymatically disaggregated synovium, and not the ‘synoviocytes’ obtained by multiple passages of cells, which leaves just synovial fibroblast-like cells. Our cultures contained the entire complex cell mixture represented in the joint, comprising chiefly macrophages (30–40%), T lymphocytes (30%) and fibroblasts [15, 22]. Using this system, our colleague, Fionula Brennan, found that the dissociated rheumatoid cell mixture spontaneously produced cytokines in a continuous manner over the entire culture period of 6 days [22]. This continuous expression, unlike the normal brief response to a stimulus, was an indication of disordered regulation and pathology. Most importantly, we had found an in vitro model to study the signals driving chronic IL-1 production in RA synovium. The next step was to use a specific antagonist to block signals occurring within the synovial cell mixture; driving IL-1 we used antibodies; the surprising result that was obtained was that neutralizing anti-TNFα antibodies inhibited IL-1 production virtually completely within 3 days in these cultures [23]. This provided the first clue in 1998 that TNFα might be a useful therapeutic target, as blocking TNFα would not only neutralize its own proinflammatory effects but also downregulate IL-1 production. This observation led to studies of what effects anti-TNFα antibodies had on the synthesis of other cytokines, and diminution of GM-CSF, IL-6, IL-8 was rapidly documented (e.g., [24, 25]), thus establishing TNFα at the apex of a proinflammatory ‘cascade’ (figure 2). These in vitro studies have subsequently been confirmed in vivo ([26, 27], vide infra).

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The results of the work thus focused our attention on further evaluation of TNFα as a therapeutic target. Immunohistological studies by our fellows, Field, Chu, etc., verified the upregulation of TNFα and TNF receptors in synovial tissues and at the invasive front of the pannus-cartilage interface [28]. Most relevant was the evaluation of anti-TNFα antibody effects in an animal model of RA. With a generous gift from Bob Schrieber of abundant amounts of monoclonal anti mouse TNF antibody (TN3), we were able to verify that even after the onset of arthritis repeated, anti-TNFα antibody treatment ameliorated joint inflammation in collageninduced arthritis in DBA/1 mice, and also protected joints from destruction, as evaluated by histology. This work performed by Richard Williams in our group at the Kennedy Institute of Rheumatology in 1991 [29], and was also simultaneously published by two other groups, those of Jeanette Thorbecke [30] and Pierre Piguet [31]. Together these studies indicated that anti-TNFα was a rational therapeutic target for RA, and that antiTNFα antibody was an appropriate tool to test the hypothesis in patients with RA. CLINICAL STUDIES Our work in defining TNFα as a therapeutic target from 1988 to 1991 led us to seek an industrial partner who could provide us with an anti-TNFα antibody for clinical use to help us verify our ideas. Eventually we found a willing company, Centocor, Inc., who had produced a chimaeric (mouse Fv, human IgG1) neutralizing antibody with high affinity for TNFα for clinical trials in sepsis from a murine anti-TNF hybridoma produced in Jan Vilcek’s laboratory [32]. It was termed cA2, subsequently known as infliximab, and now Remicade™ as its marketed trade name. With the assistance of James N. Woody, Head of Research at Centocor, an open-label clinical trial was initiated in our hospital, infusing a total dose of 20 mg/ kg administered in divided doses by intravenous injections over a 2-week period. This was the equivalent dose that we had shown to be effective in mice. The results were striking, considering that for ethical reasons, the patients were all multidrug failures, doing badly on existing treatment, and for which no suitable alternative therapy existed. Within a few hours there was evidence of increased well-being, less tiredness, etc. Within a day or two considerably less pain and stiffness was recorded, serum C-reactive protein concentration

was dramatically reduced, and by 2 weeks there was a statistically significant reduction in the number of swollen joints. All 20 patients in the first trial responded, although to various degrees, and for various durations, from 8–26 weeks. All relapsed, so transient TNFα blockade in these patients did not lead to a cure [33]. This first trial led to a multicentre European clinical trial to generate formal proof of efficacy, a randomized double-blind placebo-controlled trial of a single infusion of either one or two doses of anti-TNFα antibody (1 or 10 mg/Kg) for the assessment was at 4 weeks, with the composite criteria devised by Harold Paulus [34] to judge success. At the low dose, 44% of patients met the predetermined criteria of response, at the high dose 79%, compared to 8% with a placebo infusion [35]. While this success was meaningful, and has ushered in a new era, further important clinical trials were necessary to establish the optimum dose and whether TNFα blockade with antibodies with or without methotrexate could be effective in the long term. This was accomplished in a retreatment study [36] and a further multicentre phase II trials with five infusions given over 14 weeks (with or without methotrexate coadministration) and an observation period of 6 months [37]. However, one of the biggest successes had to wait for the phase III trials, which were planned to last for a primary end-point at 1 year, i.e., long enough to permit evaluation of whether joint damage assessed by radiographs of hands and feet is retarded by anti-TNFα therapy. In the phase III study, termed ‘ATTRACT’(AntiTNF Therapy of Rheumatoid Arthritis with Concomitant Therapy), it was found that joint damage to both cartilage and bone was virtually arrested in 50% of patients, at all doses of infliximab (Remicade™) given in combination with methotrexate [38]. This was in contrast to the progression of joint damage in the control group of patients with active disease despite ongoing methotrexate (and placebo infusion) therapy (figure 3). The combination of MTX plus Remicade™ has proved effective in arresting damage to joints up to the end of 2 years in the ATTACT trial [39, 40]. Our studies were rapidly disclosed in 1992 and have prompted competition to the benefit of the patient community. A number of other anti-TNFα therapeutics have been successful in trials. These include: – CDP571, a human complementing determining region (CDR) grafted antibody from Celltech [41]; – Lenercept, a human Fc, e.g., [42, 43], dimeric human p55 TNF receptor chimeric fusion protein from Roche;

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The success of these diverse products, with the anticipated variation in efficacy, testifies to the robustness of TNFα as a therapeutic target in RA, and hence its central role in pathogenesis. However, many questions remain to be answered. MECHANISM OF ACTION STUDIES

Figure 3. Changes in (a) American College of Rheumatology 20% response, (b) tender joint score and (c) serum C-reactive protein in patients treated with infliximab with 8-weekly (left) and 4-weekly (right) intervals in the phase III ATTRACT study of infliximab and methotrexate in rheumatoid arthritis. (Adapted with permission from Maini et al., Lancet 1999 ; 354 : 1932-9.)

– Etanercept, a human Fc, dimeric human p75 TNF receptor fusion protein from Immunex, whose clinical development was strikingly successful. Starting 2 years after Centocor, they reached the market for RA one year earlier, testifying to the value of a focused wellresourced clinical development strategy. This drug is now widely used under the trade name Enbrel™ [4345]; – A human antibody, D2E7 made by Cambridge antibody technology for BASF, highly successful in clinical trials, now in phase III [46]; – Polyethylene glycol TNF-R, from Amgen [47]; and – Polyethylene glycol-treated Fab [48] domains (of anti-TNF antibody), from Celltech.

The efficacy of infliximab prompted us to try to ascertain the important pathways controlled by TNFα in RA, and thus learn more about the pathogenesis of RA. For this purpose we used blood and sometimes synovial samples from patients in the clinical trials. We were able to establish the following: – TNFα regulates a cytokine cascade in vivo. Blockade of TNFα leads to a rapid diminution in the serum levels of IL-6, IL-1, IL-8, MCP-1, VEGF, etc. This confirmation in vivo is important, as in vitro studies are not always representative of physiological systems (e.g., [4951]). Synovial IL-1 and IL-6 are also reduced, as judged by immunohistology, and interestingly, TNFα synthesis also [27], confirming the role of TNFα in regulating its own synthesis; – There is a marked reduction in leucocyte trafficking to the joints. Serological and synovial studies indicated that components of the leucocyte recruitment pathways were reduced, such as soluble E-selectin, ICAM-1 and chemokines [52, 53] (figure 4). This led to a study, performed by Peter Taylor, in our group, who demonstrated a reduction in the recruitment of indium-labeled granulocytes into joints before and after infliximab therapy [54], which established the importance of this mode of action; and – Anti-TNF therapy is associated with a reduction in vascular endothelial growth factor (VEGF) and angiogenesis. Serum VEGF levels were measured after infliximab therapy [50]. Angiogenesis was assessed by serial biopsy and immunohistology [55]. CONCLUSION Clinical science moves slowly. It does so because of the paramount concern over safety, from the Hippocratic tradition, of doing no harm to patients. As TNFα is an important component of host defence, we and others had major worries about the safety of anti-TNFα therapy, especially in the long term. Thankfully, these concerns are not major, and the overall safety of antiTNFα therapies, be it infliximab (Remicade™) or etanercept (Enbrel™), is at least as good as that of any other anti-rheumatic drugs. With hindsight this result

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Figure 4a. Gamma camera images of the hands and of the knees of a rheumatoid arthritis patient. Images were taken 22 hours after a bolus injection of autologous radiolabelled (111indium) granulocytes before (left) and after (right) a single 10 mg/kg intravenous bolus of anti-tumour necrosis factor α antibody (infliximab). There was a reduction in signal after treatment. Images kindly provided by P.C. Taylor. Figure 4b. Mean percentage change from baseline of results from ten patients treated as described above. Histogram drawn from data published in Taylor PC, Peter AM, Paleolog E, et al. Arthritis Rheum 2000 ; 43 : 38-47.

could be anticipated, as immunoglobulins and soluble TNF receptors are intrinsically nontoxic, and what these drugs chiefly do is remove the excess TNFα. Nevertheless, there are some reports of infection, and caution is warranted in the long term. The omens for the future are good. Anti-TNF therapy has provided relief for over 150,000 patients, and the testament to its efficacy and the effect it has had on their lives can be seen on US television, where advertising of medicines is permitted, but not in Europe. Our demonstration that TNFα was an effective therapeutic target for RA, presented first in Arad, Israel, in October 1992, has led to work to define other diseases for which TNFα blockade is also effective. These include Crohn’s disease, including fistulae, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriasis and psoriatic arthropathy. Nevertheless, there is ample scope for improvement. More research is needed since not all treated patients improve and there is a need to find ways of delivering the same degree of benefit in a cheaper, more convenient and potentially safer way. We eagerly look forward to the day when rheumatology research will provide a cure to this dreadful disease. ACKNOWLEDGMENTS The work described here would not have been possible without the financial support of the Arthritis Research Campaign, nor the dedication of many colleagues and

collaborators in an increasing network. Involved in preclinical studies include F. Brennan, G. Buchan, R. Williams, C. Haworth, E. Paleolog, C. Chu, M. Field, P. Gray, M. Shepard, T. Taniguchi, T. Kishimoto. Of importance in clinical studies include F. Breedveld, J. Kalden, J. Smolen, P. Lipsky, M. Elliott, P. Taylor, and from Centocor, J.N. Woody, H. Weisman, G. Harriman, T. Scheible and others. REFERENCES 1 Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987 ; 30 : 1205-13. 2 Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996 ; 14 : 397-440. 3 Oppenheim JJ, Feldmann M, Eds. The cytokine reference. A comprehensive guide to the role of cytokines in health and disease. London: Academic Press; 2000. 4 Symons JA, Wood NC, Di Giovine FS, Duff GW. Soluble IL-2 receptor in rheumatoid arthritis. Correlation with disease activity, IL-1 and IL-2 inhibition. J Immunol 1988 ; 14 : 2612-8. 5 Hopkins SJ, Humphreys M, Jayson MI. Cytokines in synovial fluid. I. The presence of biologically active and immunoreactive IL-1. Clin Exp Immunol 1988 ; 7 : 422-7. 6 Xu WD, Firestein GS, Taetle R, Kaushansky K, Zvaifler NJ. Cytokines in chronic inflammatory arthritis. II granulocytemacrophage colony-stimulating factor in rheumatoid synovial effusions. J Clin Invest 1989 ; 83 : 876-82. 7 Malyak M, Swaney RE, Arend WP. Levels of synovial fluid interleukin-1 receptor antagonist in rheumatoid arthritis and other arthropathies. Arthritis Rheum 1993 ; 36 : 781-9. 8 Hanafusa T, Pujol-Borrell R, Chiovato L, Russell RCG, Doniach D, Bottazzo GF. Aberrant expression of HLA-DR antigen

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