Senolt, L; Vencovský, J; Pavelka, K; Ospelt, C; Gay, S (2009). Prospective new biological therapies for . Autoimmunity Reviews, 9(2):102-107. Postprint available at: http://www.zora.uzh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch Originally published at: Autoimmunity Reviews 2009, 9(2):102-107.

Winterthurerstr. 190 CH-8057 Zurich http://www.zora.uzh.ch

Year: 2009

Prospective new biological therapies for rheumatoid arthritis

Senolt, L; Vencovský, J; Pavelka, K; Ospelt, C; Gay, S

Senolt, L; Vencovský, J; Pavelka, K; Ospelt, C; Gay, S (2009). Prospective new biological therapies for rheumatoid arthritis. Autoimmunity Reviews, 9(2):102-107. Postprint available at: http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Autoimmunity Reviews 2009, 9(2):102-107. Prospective new biological therapies for rheumatoid arthritis

Ladislav Šenolt 1, Jiří Vencovský 1, Karel Pavelka 1, Caroline Ospelt 2, Steffen Gay 2

1 Institute of Rheumatology and Connective Tissue Research Laboratory, Department of Rheumatology of the First Faculty of Medicine, Charles University in Prague, Czech Republic

2 Center of Experimental Rheumatology, University Hospital Zurich, and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.

March 2009

Corresponding author: Ladislav Šenolt, MD, PhD. Institute of Rheumatology Na Slupi 4 Prague 2, 128 50 Czech Republic Email: [email protected] Abstract Advances in the current knowledge of pathogenetic mechanisms of rheumatoid arthritis have contributed to the development of biological therapy, and translated research findings into clinical practice. TNF- (, , ), IL-1 () and IL-6 () inhibitors, a B-cell depleting agent () and a drug blocking T-cell costimulation () have been approved for rheumatoid arthritis. The progress in manufacturing biotechnology has contributed to the development of several other prospective agents that may form the basis for the therapy of rheumatoid arthritis in the near future. New or modified TNF- inhibitors (, ), new monoclonal against other cytokines (e.g. IL-1, IL-6, IL-12, IL-15, IL-17, IL-23), and other agents targeting B-cell depletion (e.g. , ofatumumab) are in various stages of development. Many pharmaceutical companies have focused on developing small molecule inhibitors with possible peroral administration, which are considered promising drugs for rheumatoid arthritis. In most cases, these small molecules inhibit cellular kinases (e.g. p38, JAK or Syk) that mediate the signaling and transcription of proinflammatory genes. In this review, we describe the cytokine inhibitors and modulators of the immune response currently in ongoing clinical trials, the results of which may further expand the spectrum of efficient therapies for chronic autoimmune diseases.

Key words: rheumatoid arthritis, biological therapy, clinical trials, monoclonal antibodies, small molecules

Take-Home messages:  Spectrum of monoclonal antibodies are currently tested in clinical trials including TNF- inhibitors (golimumab, certolizumab pegol), inhibitors of several pro-inflammatory cytokines such as IL-1, IL-6, IL-12, IL-15, IL-17, or IL-23, and agents targeting B-cells (ocrelizumab, ofatumumab, TRU-015)  Small molecule inhibitors, particularly inhibitors of JAK3 and Syk kinases, are currently the most encouraging therapeutics for rheumatoid arthritis; however long-term efficacy and treatment safety have yet to be shown.  Treg cells, innate , several new cytokine-like molecules, RNA interference or epigenetic alterations expand the spectrum of new promising targets for autoimmune disorders

1 Introduction

Biological therapy has become a cornerstone in the treatment of rheumatoid arthritis (RA) with inadequate response to standard disease modifying antirheumatic drugs (DMARDs). Currently, seven approved biological agents are available (Table 1). Biological therapy targeting molecules and cells specific for processes associated with the pathogenesis of RA is very efficient yet still unable to induce remission or even cure in most patients. Expanding spectrum of potential therapies is currently being tested in various stages of clinical trials that have been facilitated by recent understandings regarding the pathogenesis of RA. The aim of this report is to review potential new therapeutic concepts, which may be promising for patients with RA.

Prospective biological agents

Pharmaceutical companies aim to modify existing agents or develop further biological therapeutics and treatment strategies in order to reduce the costs of manufacturing technology, as well as to improve the clinical efficacy and perhaps even induce total remission of the disease.

The most promising agents currently being tested in various stages of clinical trials are summarized in table 2. Biological agents can be divided into monoclonal antibodies and small molecules (1, 2).

Monoclonal antibodies

The use of monoclonal therapy in rheumatology has increased in recent years (1).

Initially, these treatments consisted of chimeric antibodies with human constant regions of light and heavy chain and the variable murine binding site for the target molecule. Progress in manufacturing technology led to a reduction of immunogenicity, and humanized and eventually fully human antibodies were created. These antibodies function by neutralizing a target cytokine or its receptor, blocking costimulation molecules, and inducing cytolysis, apoptosis or depletion of target cell molecules.

2 TNF- inhibitors. Two additional TNF- blocking agents are likely to be approved in 2009 for the treatment of RA. Preliminary data of subcutaneously administered golimumab (CNTO148), a fully human monoclonal anti-TNF- antibody and certolizumab pegol (Cimzia), a humanized monoclonal anti-TNF- antibody exhibit the same efficacy and spectrum of adverse events as currently available TNF- inhibitors (3, 4).

IL-1 inhibitors. To simplify the dosing, decrease the risks of local adverse events, increase the affinity to IL-1, and thus improve the efficacy compared to anakinra, a humanized targeting IL-1, (ACZ885), has been recently developed. The preliminary results of clinical trials in patients with systemic juvenile idiopathic arthritis are promising (5), and a phase II clinical trial in patients with RA began in 2008 (http://clinicaltrials.gov).

IL-6 inhibitors. Tocilizumab is a humanized monoclonal antibody against IL-6 receptor. The results of several phase III clinical trials suggest tocilizumab as an agent with extreme potential for the treatment of active RA (6). As of 2009, the European Medicines Agency (EMEA) described a favorable benefit-to-risk balance for tocilizumab in combination with MTX, and approved this indication for the treatment of moderate to severe active RA in adult patients who have either responded inadequately or were intolerant to previous therapy with one or more

DMARDs or TNF- inhibitors (http://www.emea.europa.eu). In 2008, a phase II clinical trial of a subcutaneously administered monoclonal antibody against IL-6 (CNTO 136) was initiated to establish the suitable dosage, and confirm the efficacy and safety of the drug

(http://clinicaltrials.gov).

IL-15 inhibitor. Blocking IL-15 in an experimental model of arthritis led to a significant reduction of cartilage and bone destruction. The first open, placebo-controlled, double blind study

3 proved the humanized monoclonal antibody against IL-15 (HuMaxIL-15) to be efficient (7), however, a phase II clinical trial of a fully human monoclonal antibody against IL-15 (AMG 714) failed to confirm significant efficacy. Other cytokines, such as IL-2, IL-4, IL-7, IL-9 and IL-21, share an identical receptor subunit of the γ chain and thus some functions of IL-15. So far, no data is available on the use of these cytokines as a therapeutic target in RA patients.

IL-17 inhibitor. Th17 cells, a subset of memory T-cells that play a key role in an autoimmune inflammation, are a major source of IL-17. IL-17 increases production of several proinflammatory cytokines such as IL-1, IL-6, or TNF- and regulates osteoclastogenesis.

Inhibition of IL-17 generated successful results in the treatment of an experimental arthritis model (8). A phase I/II clinical trial of a monoclonal antibody against IL-17 (AIN457) in patients with RA is currently in progress (http://clinicaltrials.gov).

IL-12/IL-23 inhibitor. IL-12 is a key inducer of Th1 polarization and IL-23 is responsible for the proliferation of a Th17 subset of memory T-cells (9). A recent clinical trial evaluating the effect of a specific monoclonal antibody, , against the p40 subunit of cytokines IL-12 and

IL-23 in patients with psoriasis has generated promising results (10). On an experimental level, a positive effect of an IL-23 blocking antibody was described in collagen-induced arthritis (11) and one proof-of-concept study has shown that besides associated psoriatic skin lesions, ustekinumab treatment can also reduce the signs and symptoms of arthritis in patients with psoriatic arthritis

(12). Recently initiated clinical trials could show if ustekinumab is of any practical use in the treatment of RA (http://clinicaltrials.gov).

Inhibitors of the TNF superfamily members. The results of a phase II clinical trials of baminercept (BG9924), an inhibitor of LT (lymphotoxin-), and , a fully human

4 monoclonal anti-BAFF (B-cell activating factor) antibody, have not been encouraging thus far in

RA patients (http://clinicaltrials.gov). To increase the effect on B-cell maturation, atacicept, a recombinant of BAFF and the APRIL (a proliferation-inducing ligand) receptor

(TACI-Ig) was designed. Results of a phase I clinical trial shows a depletion of peripheral B-cells as well as decrease in rheumatoid factors (RFs) and anti-citrullinated peptide antibodies (ACPA).

During the three-month study, a positive clinical effect was observed in a small group of patients; however, no significant decrease of acute phase reactants was observed (13).

Inhibitors of osteoclastogenesis. A monoclonal antibody against receptor activator for nuclear factor B ligand (RANKL) denosumab is an efficient inhibitor of osteoclastogenesis that is in phase III clinical trial in patients with postmenopausal osteoporosis. Subcutaneous administration of denosumab every six months led to significant retardation of radiographic progression in patients with active RA after one year (14). The therapy was well tolerated, yet does not exhibit a larger effect on the disease activity.

Inhibitors of chemokines and angiogenesis. On an experimental level, blocking several specific chemokines or their receptors has been successfully tested; however, early phases of clinical trials in patients with active RA have not proven a sufficient clinical effect of monoclonal antibodies against CCL-2/MCP-1 or its receptor CCR-2 (15, 16). Angiogenesis occurs in the early phase of

RA and enables chemotactic migration of inflammatory cells into the synovial tissue. A monoclonal antibody against vascular endothelial growth factor (VEGF), bevacizumab (Avastin), significantly expanded therapeutic possibilities in oncology (17). A preclinical trial recently demonstrated a prophylactic effect of a VEGF beta monoclonal antibody in a murine model of arthritis; however, there has been no significant impact on an established disease (18).

5 Regulators of apoptosis. In general, cells from RA synovial tissue are resistant to apoptosis, and activated synovial fibroblasts and/or other joint tissue resident cells may presumably escape the effect of the current biological therapies (Fig. 1). Over 10 years ago, a strategy for treatment of

RA that induced apoptosis by binding the FAS receptor had been introduced in an in vivo model of arthritis (19). The first monoclonal anti-FAS IgM antibody (ARG098) was subsequently developed, and is being tested as a single intraarticular application into an active knee in a phase I clinical trial in patients with RA (http://clinicaltrials.gov).

T-cell inhibitors. Clinical trials directly targeting T-cells - (anti-CD52, Campath-

1H), (chimeric monoclonal anti-CD4 antibody) or (the IgG4 version of the previous antibody) were accompanied by serious adverse events linked with severe CD4+ T- lymphocytopenia and a rash, leading to discontinuation of the clinical trials of these agents (20).

The improved knowledge of the co-stimulation regulatory mechanisms and good efficacy and safety profile in clinical trials facilitated recently abatacept approval for the treatment of RA (21).

B-cell inhibitors. Progress in manufacturing technology led to the development of ocrelizumab, a humanized monoclonal antibody, and ofatumumab (HuMax-CD20), a fully human monoclonal antibody against CD20, which are in phase III clinical trials (22, 23). The results of early phases of studies show a good effect of both agents in patients refractory to both and TNF-

 inhibitors. Compared to rituximab, minimum immunogenicity was observed. A phase I clinical trial of a fully human monoclonal anti-CD19 antibody (MDX-1342) is currently in progress in patients with active RA (http://clinicaltrials.gov). Trubion Pharmaceuticals developed SMIP

(small modular immunopharmaceutical), which binds and specifically blocks the CD20 molecule

(TRU-015) and induces a peripheral B-cell depletion (http://www.trubion.com). A phase II clinical trial is currently in progress (http://clinicaltrials.gov), and results should indicate potential advantages of this agent in comparison with other therapeutics targeting B-cells. As this molecule

6 is smaller than a standard monoclonal antibody, it is thought to have better tissue penetration, efficacy and safety profile.

Small molecules

More than half of the new anti-inflammatory therapies in preclinical and clinical trials involve small molecules, the molecular weights of which do not exceed 1kDa (2). These agents are available for peroral administration, should be less expensive; furthermore should have at least the same efficacy as the current biological therapy, and a reduced risk of adverse events. Recently developed small molecules have a targeted impact on various intercellular factors or cell structures, including receptors, intracellular signaling pathways and enzymes participating in the pathogenesis of RA.

Inhibitors of intracellular signaling molecules

Binding of an extracellular factor (e.g. a cytokine or a pathogen) to a cell-surface receptor activates cytoplasmic kinases: mitogen-activated protein kinases (p38, ERK, JNK) or tyrosin kinases (JAK, Syk), which can subsequently modulate the activity of transcription factors, thus regulating the expression of target genes (Fig. 2). Perorally administered drugs that modify the regulation of complex intracellular signaling pathways have been tested with a variable clinical response.

Inhibitors of MAP kinase p38. Based on very promising preclinical data, a number of pharmaceutical companies have developed inhibitors of MAP kinase p38 that predominantly impact its alpha subunit. Most of the therapeutics are in phase II clinical trials; the therapeutic effects have not yet reached expectations. Moreover, the latest data has shown that pamapimod was even less effective than MTX in patients with active RA (24). Based on these data, one can speculate that other signaling pathways are predominantly involved in the pathogenesis of RA.

7 Inhibitors of the JAK/STAT signaling pathway. Tyrosine Janus kinases (JAK) can be activated by - and other cytokines playing a significant role in the pathogenesis of RA (25).

Pfizer developed a JAK3 inhibitor (CP-690,550), which has already been in a phase II clinical trial in patients with moderately- to severely-active RA (26). The results of this study have been very encouraging so far; after 12 months of administration, remission was achieved in approximately one-third of the individuals, and a relatively good tolerance and safety profile of the therapy was observed. A multicentre study is currently in progress comparing the effect of five dosing regimens of JAK-3 inhibitor with TNF inhibitor (adalimumab) and DMARDs in patients with active RA (http://clinicaltrials.gov).

Inhibitors of Syk kinase. Intracellular Spleen tyrosine kinase (Syk) has significant immunomodulatory activity, and is stimulated by activation of Fc receptors and B-cell receptors.

This signaling pathway is upstream MAP-kinases, especially JNK, and its activation plays a key role in the TNF- induced expression of proinflammatory cytokines and proteolytic enzymes by synovial fibroblasts (27). Use of a Syk kinase inhibitor (Fostamatinib disodium) has produced very good results with regard to attenuation of clinical activity of RA (28). After 12 weeks of peroral administration of 150 mg of the agent twice a day, remission was observed in almost half of the individuals. Nevertheless, the prevalence of adverse events (e.g. diarrhea, increased blood pressure, neutropenia and elevation of liver enzymes) increased due to this relatively high dosage.

Inhibitors of transcription factors. The NFB (nuclear factor B), NFAT (nuclear factor for activation of T-cells) and AP-1 (activator protein) transcription factors have been shown to play the most critical roles in the pathogenesis of RA. The first preclinical data from an experimental model of arthritis have shown that treatment with c-Fos/AP-1 inhibitor (T-5224) prevents synovitis, osteoclastogenesis, and the subsequent destruction of a joint (29). I kinases (IKK)-2 has been shown to play a central role in regulating NFB signaling and a highly selective IKK-2

8 inhibitor has been very recently introduced to inhibit the expression of various inflammatory mediators as well as to block joint swelling and bone destruction in an experimental model of arthritis (30). Another approach to inhibit NFB, permeable NBD peptide, has been demonstrated through blocking the regulatory activity of NFB. Its intraarticular administration into a rat joint with adjuvant arthritis led to significant attenuation of synovitis and joint destruction (31).

Inhibitors of cytokines and chemokines

Synta Pharmaceuticals has developed a small molecule IL-12/IL-23 inhibitor (Apilimod mesylate), which, based on clinical results, is presumed to have an impact on immune response showing decrease of several proinflammatory cytokines and a retardation of RA progression. This drug has been successfully tested in patients with Crohn’s disease, and a phase II clinical trial in patients with RA is currently in progress (32). A selective antagonist of a chemokine receptor

CCR5, maraviroc, was well tolerated, without severe adverse events; however, due to lack of efficacy, the phase II clinical trial in patients with RA was terminated at the end of 2008.

Inhibitors of cell surface markers

RhuDex, a small molecule blocking the CD80 marker on presenting cells, is one of the first modulators of T-cell costimulation available in peroral administration. A phase I clinical trial was already in progress, and 80 patients have been enrolled in this study so far; however, it was discontinued in July 2008 due to death of one volunteer from myocardial infarction. The pharmaceutical company was requested to complete in vitro tests to exclude possible drug interactions supporting atherogenesis (http://www.medigene.com).

Conclusions

A growing knowledge of the pathogenesis of an autoimmune inflammatory process has expanded the spectrum of new molecules and some of these may become the therapeutic targets of RA (33).

9 The therapeutic possibilities have been expanded by new monoclonal antibodies against several proinflammatory cytokines, chemokines, growth factors, cytokine and chemokine receptors, or agents targeting cell surface markers. Most encouraging therapeutic strategies of recent years are small molecules; mostly inhibitors of intracellular signaling pathways. These have the advantage of peroral administration. Since intracellular signaling pathways have pleiotropic effects, focusing on the efficacy and especially the long term safety in further studies will be needed.

There are many other targets with potential therapeutic utility approaching for example regulatory

T cells (Treg) (34), Toll like receptors (35), complement pathway mediated by C5a (36) or adipokines – e.g. visfatin/PBEF (37). The future of biological therapy may also develop according to new scientific advancements in genetic engineering, possibilities of affecting gene transcription using RNA interference with small interfering RNA or microRNA as well as possibilities of epigenetic alterations (DNA methylation and histone modifications), which may affect the regulation of inflammation and autoimmune process during the course of rheumatic disorders (38, 39). In this regard it needs to be stressed that no therapy has been targeting so far the epigenetically activated synovial fibroblast operating the cytokine independent pathway of

RA (39, 40).

Acknowledgments: The study was supported by Ministry of Health of Czech Republic, research project no. 00023728

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15 Tables

Table 1. Currently used biological therapies for rheumatoid arthritis. ______TNF- inhibitors Infliximab (Remicade) - chimeric monoclonal antibody Etanercept (Enbrel) - TNF- receptor Adalimumab (Humira) - human monoclonal antibody

IL-1 inhibitor Anakinra (Kineret) - IL-1 receptor antagonist

IL-6 inhibitor Tocilizumab (Actemra) - humanized monoclonal anti- IL-6 receptor antibody

B-lymphocyte depletion Rituximab (MabThera) - monoclonal anti-CD20 antibody

Inhibition of costimulation Abatacept (Orencia) - fusion protein CTLA-4 with immunoglobulin ______TNF, tumor necrosis factor; IL, interleukin; CTLA-4, cytotoxic T-lymphocyte antigen 4.

16 Table 2. Perspective biological therapies for rheumatoid arthritis in clinical trials

Therapeutic aim/drug Phase of clinical trial ______Novel TNF- inhibitors Golimumab - fully human monoclonal antibody, under regulatory review Certolizumab pegol (Cimzia) - humanized monoclonal antibody, under regulatory review

Novel IL-1 inhibitors Canakinumab (ACZ885), humanized monoclonal antibody against IL-1 II

Inhibitors of cytokines from the TNF superfamily Atacicept/TACI-Ig - recombinant fusion protein of BAFF- and APRIL-receptor II

Inhibitors of other cytokines HuMaxIL-15 - humanized monoclonal anti-IL-15 antibody II AIN457 - monoclonal anti-IL-17 antibody I/II

Inhibitors of osteclastogenesis Denosumab - monoclonal antibody against RANKL III

Inhibitors of B-lymphocyte cell-surface markers Ocrelizumab - humanized monoclonal anti-CD20 antibody III Ofatumumab - fully human monoclonal anti-CD20 antibody III TRU-015 - small antibody polypeptide (SMIP) blocking CD20 II

Small molecules Inhibitors of p38 kinase II Inhibitor of JAK3 kinase (CP-690,550) II Inhibitor of Syk kinase (Fostamatinib) II IL-12/IL-23 inhibitor (Apilimod, STA-5326) II Inhibitor of CD80-CD28 costimulation (RhuDex) I ______TNF, tumor necrosis factor; BAFF, B-cell activating factor; APRIL, a proliferation-inducing ligand; IL, interleukin; RANKL, receptor activator for nuclear factor B ligand; SMIP, small modular immunopharmaceutical; MAPK, mitogen-activated protein kinase; JAK, Janus tyrosine kinase; Syk, spleen tyrosine kinase.

17 Legends to figures

Figure 1. Regulation of proinflammatory cytokines and immune cells within a joint by currently available biological therapies for rheumatoid arthritis. Therapeutical agents inhibiting activated synovial fibroblasts and/or other resident tissue cells are still unavailable, which may explain that not all patients are sufficiently responsive to current biological therapy. Thereby other targets, modulators of apoptosis or small molecule inhibitors regulating intracellular signaling pathways could be beneficial (see Fig. 2).

Figure 2. Schematic drawing of signal transduction pathways and transcription factors, and their potential modulation by peroral inhibitors. Upon exposure of a cell to stress, proinflammatory environment, cytokines or pathogens, several regulatory enzymes are phosphorylated and activated. As a result, an intracellular signaling cascade is activated to transmit the signal from a receptor via MAP and tyrosine kinases to transcription factors, which affect the expression of genes for cytokines, matrix metalloproteinases, apoptosis regulating molecules, proliferation, etc. Inhibitors of Syk kinase and Jak3 have been most successful in clinical trials in patients with rheumatoid arthritis so far.

MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; JAK, Janus tyrosine kinase; STAT, signal transducer and activator of transcription; Syk, spleen tyrosine kinase; IKK, inhibitor IB; JNK, c-Jun N-terminal kinase; ERK, extracellular signal- regulated kinases; NF-B, nuclear factor-B; AP1, activator protein 1; ATF2, activating transcription factor 2, TNF, tumor necrosis factor; IL, interleukin; MMPs, matrix metalloproteinases.

18 Inhibitors of cytokines (inhibitors of TNF, IL-1, IL-6)

Inhibitors of co-stimulation Inhibitors of B-cells (abatacept) (rituximab)

M

T-cells B-cells APC Immune cells

Resident tissue cells

Synovial fibroblasts Osteoclasts Chondrocytes

Endothelial cells Adipose tissue Extracellular signals – cytokines, pathogens, stress

Cell membrane

Cytoplasm JAK JAK P

MAPKKK Jak inhibitors Syk inhibitors P STAT IKK P P MAPKK Syk IKK IKK P

MAPK – p38, ERK, JNK IB STAT P P p50 p65 STAT p38 inhibitors P P P NF B ERK P p38 P JNK p65 P IB p50 degradation Transcription factors - ATF2, AP1, … nucleus STAT P P DNA STAT

TNF, IL-1, IL-6, MMPs, etc Inflammation, matrix degradation, apoptosis