WO2009147362A1

WO2009147362A1 - Method of rheumatoid arthritis treatment

Info

Publication number: WO2009147362A1
Application number: PCT/GB2008/002420
Authority: WO
Inventors: Richard Owen Williams; Clare Angela Notley
Original assignee: Imperial Innovations Limited
Priority date: 2008-06-03
Filing date: 2008-07-17
Publication date: 2009-12-10

Abstract

A method of treating rheumatoid arthritis (RA) in a patient, the method comprising administering to the patient an anti-TNF agent and an anti-IL17 agent.

Description

METHOD OF RHEUMATOID ARTHRITIS TREATMENT

The present invention relates to a method of treating rheumatoid arthritis (RA).

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Rheumatoid arthritis (RA) is a chronic autoimmune disease in which pro-inflammatory cytokines, such as TNFa₁ iL-6 and IL-1 play dominant pathological roles. More recently,

IL-17 has been suggested to play an important additional role in the induction and maintenance of RA (1 , 2). Thus, IL-17 is present in the synovium of RA patients and contributes to the production of IL-6 and MMP-1 in the joint (2, 3), whereas treatment of human macrophages with IL-17 in vitro stimulates the production of TNFα and IL-1 β (4). IL-17 can also synergise with TNFα to induce cytokine and chemokine production by synovial fibroblasts and cartilage destruction in vitro and can promote osteoclastogenesis

(1 , 5, 6).

IL-17 is produced predominantly by T helper cells (Th 17 cells) and although there is controversy over the signals required for the differentiation of murine and human Th17 cells, both murine and human CD4⁺ Th17 T cells require IL-23 for their proliferation and maintenance (7). IL-23 is a heterodimeric protein composed of a p19 subunit and a p40 subunit whereas IL-12, an important cytokine for Th1 cell differentiation, is formed when the p40 subunit dimerises with p35 (8).

The role of TNFα in RA is well documented, with TNFα blocking biologies causing amelioration of clinical symptoms (eg pain, joint swelling and stiffness), laboratory parameters of inflammation (eg CRP, ESR) and radiological progression of disease (9, 10). Although TNFα plays a direct pathological role in disease pathogenesis in RA, its contribution to disease pathogenesis is amplified by its ability to promote the expression of other pro-inflammatory cytokines, including IL-1 , IL-8 and GM-CSF (11-13). More recently, TNFα has been shown in vitro to drive the production of IL-17 by equipping! DC with the ability to differentiate T cells towards a Th 17 phenotype (14). On this basis it would be predicted that TNFα blockade would result in reduced IL-17 expression aild in order to test this hypothesis in vivo we investigated the dependence of IL-17 expression on TNFα in ClA. Surprisingly, our data show that TNFα is an important negative regulator of IL-17 and IFNγ production by T cells and we propose that this forms part of a negative feedback loop that limits the intensity and/or duration of Th 17 and Th1 responses. Thus, blockade of TNFα, whilst relatively effective in RA, leads to upregulation of IL-17 which is deleterious. Hence, we propose combined therapy with anti-TNFα and anti-IL17.

A first aspect of the invention provides a method of treating rheumatoid arthritis (RA) in a patient, the method comprising administering to the patient an anti-TNF agent and an anti-IL17 agent.

Hereafter TNF is tumour necrosis factor α. IL17 is interleukin 17.

The patient is typically a human, but may be an animal, particularly a mammal such as a horse, dog or cat, or other companion or farm mammal.

It is preferred that the anti-TNF agent is an agent that is directed to the TNF of the species of the patient to be treated. For example, when the patient is a human, it is preferred that the agent is an anti-human TNF agent.

Similarly, it is preferred that the anti-IL17 agent is an agent that is directed to the IL17 of the species of the patient to be treated. For example, when the patient is a human, it is preferred that the agent is an anti-human IL17 agent.

TNF from many species is known. For example, the amino acid sequence of human TNF is disclosed in Pennica et al (1984) Nature 312, 724-729.

IL17 from many species is known. For example, the amino acid sequence of human IL17 is disclosed in Strausberg et al (2002) Proc. Natl. Acad. ScI. USA 99, 16899-16903.

The combination treatment of the invention is believed to be particularly suited to patients with severe RA.

The combination treatment of the invention may be used at any stage of the disease, but is believed to be particularly applicable to patients who have shown a poor response to conventional therapies, including patients who have shown a poor response to anti-TNF alone.

The anti-TNF and the anti-IL17 may be administered by any suitable route. Typically, administration of the anti-TNF agent or the anti-IL17 agent or both agents is intravenously or subcutaneously, for example by injection, or orally.

In one particular embodiment, the anti-TNF agent and the anti-IL17 agent are administered simultaneously. In another particular embodiment, the anti-TNF agent and the anti-IL17 agent are administered sequentially. If sequential, they may be administered in any order.

The anti-TNF agent and the anti-IL7 agent are administered either together or separately in a dose which is effective for the treatment of RA. Efficacy of the treatment may be determined by standard methods, such as improvements in one or more of the following: number of swollen joints, number of tender joints, erythrocyte sedimentation rate and C-reactive protein levels, or by patient assessment, physician assessment, disability/functional questionnaire or pain scale.

Doses of the anti-TNF agent and the anti-IL17 agent may be determined empirically.

A second aspect of the invention provides the combination of an anti-TNF agent and an anti-IL17 agent for use as a medicament.

A third aspect of the invention provides the combination of an anti-TNF agent and an anti-IL17 agent for use in treating RA in a patient.

A fourth aspect of the invention provides an anti-TNF agent for use in treating RA in a patient, wherein said patient is administered an anti-IL17 agent. The patient may be administered the anti-IL17 agent prior to or at the same time as or after administration of the anti-TNF agent.

A fifth aspect of the invention provides an anti-IL17 agent for use in treating RA in a patient, wherein said patient is administered an anti-TNF agent. The patient may be administered the anti-TNF agent prior to or at the same time as or after administration of the anti-IL17 agent.

A sixth aspect of the invention provides the use of the combination of an anti-TNF agent and an anti-IL17 agent in the manufacture of a medicament for the treatment of RA in a patient.

A seventh aspect of the invention provides the use of an anti-TNF agent in the manufacture of a medicament for the treatment of RA in a patient, wherein the patient is administered an anti-il_17 agent. The patient may be administered the anti-TNF agent prior to or at the same time as or after administration of the anti-IL.17 agent.

An eighth aspect of the invention provides the use of an anti-!l_17 agent in the manufacture of a medicament for the treatment of RA in a patient, wherein the patient is administered an anti-TNF agent. The patient may be administered the anti-TNF agent prior to or at the same time as or after administration of the anti-IL17 agent.

A ninth aspect of the invention provides a system for treating RA in a patient, the system comprising an anti-TNF agent and an anti-!L17 agent. The system suitably contains means for delivering the agents to the patient, for example a hypodermic needle and a syringe.

A tenth aspect of the invention provides a kit of parts for treating RA in a patient, the system comprising an anti-TNF agent and an anti-!L17 agent.

In all aspects of the invention, the anti-TNF agent may be any agent that neutralises TNF or blocks the TNF receptor or inhibits the production of TNF or inhibits signalling via the TNF receptor. Typically, the anti-TNF agent neutralises TNF by binding to TNF.

In all aspects of the invention, the anti-IL17 agent may be any agent that neutralises IL17 or blocks the IL17 receptor or inhibits the production of IL17 or inhibits signalling via the IL17 receptor. Typically, the anti-IL17 agent neutralises IL17 by binding to IL17.

It is preferred that the anti-TNF agent is an anti-TNF antibody or a soluble TNF receptor (TNFr). It is preferred that the anti-IL17 agent is an anti-IL17 antibody or a soluble anti-IL17 receptor (IL17r).

By antibody, we include any antibody-like molecule which is able to bind TNF (or, as the case may be, IL17) in a similar way to an intact antibody. Thus, we include intact antibodies, antibody fragments such as Fab, F(ab')₂ and Fv fragments, genetically engineered antibodies such as humanised antibodies, chimaeric antibodies, single-chain Fv molecules (scFv), domain antibodies (dAbs) and the like. Also included are monoclonal antibodies and polyclonal antibodies.

Antibodies to TNF and IL17 are known and can in any event be made using well known techniques, such as monoclonal antibody production using hybridoma cells or transfected cells or by using antibody phage display, and by using the known amino acid sequences.

Antibodies to TNF are commercially available for use as anti-TNF agents, including HUMIRA (adalimumab; Abbott Laboratories) and REMICADE (infliximab; Centocor, Inc). Soluble TNF receptor is commercially available as an anti-TNF agent, including ENBREL (etanercept; Immunex Corporation).

Soluble receptors may be readily engineered from the intact membrane bound receptor using methods well known in the art. For example, the TNF (or !L17)-binding portion of the respective receptor (as the case may be) may be joined to a soluble protein molecules to create a molecule which retains the ability to bind TNF (or IL17 as the case may be) and is soluble. Typically, a portion of the receptor is joined to an Fc portion of an antibody.

It will be appreciated that the anti-TNF agent and the antilL7 agent are prepared in a form suitable for therapeutic administration. The agents are typically present in a pharmaceutical composition in combination with a pharmaceutically acceptable carrier. Typically, the pharmaceutical composition is sterile and pyrogen free.

The invention will now be described by reference to the following non-limiting Examples and Figures wherein: Figure Legends

Figure 1. Increased IL-17 and IFNγ production in CIA after blockade of TNFα. DBA/1 mice with CIA were treated with TNFR-Fc or isotype control mAb (100 μg/mouse on alternate days) from the time of disease onset. LN cells were taken 10 days post disease onset and levels of IL-17 (A) and IFNγ (B) were determined by ELISA in the supematants without further stimulation (Nil) or after stimulation with type Il collagen (CII) or anti-CD3 mAb (CD3). Data show individual mice (n=8; ^*p<0.05). Clinical scores were assessed over the 10 day period in TNFR-Fc treated and control mice (C).

Figure 2. Amplification of Th17 and Th1 cell activity in p55 TNFR^''^' mice. LN cells from WT, p55 TNFR^"'^" and p75 TNFR^"'^" mice were taken 14 days post immunisation with type Il collagen in CFA. LN cells were either unstimulated (Nil) or stimulated with collagen (CIl) or with anti-CD3 mAb (CD3) and the level of proliferation was determined by ³H thymidine incorporation (A). The percentage of CD4⁺ T cells in the LN was determined by flow cytometry on day 14 after immunisation (A). Levels of IL-17 and IFNγ were determined by ELISA (B). The proportion of CD4⁺ cells in the LN producing IL-17 and lFNγ were determined by flow cytometry (C). Histograms show mean ±SEM (n=8). *p<0.05 ^**p<0.01.

Figure 3. TNFα inhibits expression of IL-12/IL-23 p40. Thioglycolate elicited macrophages were cultured in the presence or absence of TNFα (30 or 100 ng/ml) for 8 h, then stimulated for a further 18 h with LPS (1 ng/ml). Levels of p40 protein were determined in the culture supematants by ELISA in pg/ml (A). Relative levels of p40 mRNA from WT, p55 TNFR^"'^" and p75 TNFR^"'^" LN cells 14 days post immunisation were determined by real time PCR (B). Histograms show mean ± SEM (n=4). **p<0.01 ***p<0.001.

Figure 4. Blockade of IL-12/IL-23 abrogates the increased production of IL-17 and IFNγ in p55 TNFR^"'^' mice. LN cells from immunised WT (white bars) or p55 TNFR^"'^" (grey bars) mice treated with control Ig (Ig) or rat anti-mouse p40 Ab (p40) were either unstimulated (Nil) or stimulated with collagen (CII) or anti-CD3 mAb (anti-CD3). Levels of IL-17 (A) and IFNγ (B) were determined by ELISA and the proportion of CD4⁺ cells in the LN producing IL-17 and lFNγ were determined by flow cytometry (C). Histograms show mean ± SEM (n=5). ^*p<0.05 ^**p<0.01 ***p<0.001.

Figure 5. Synergistic effect of anti-TNF and anti-IL-17 in collagen-induced arthritis (CIA). CIA is a well-established animal model of rheumatoid arthritis. Arthritis was induced in DBA/1 mice by immunisation with bovine type Il collagen in adjuvant. After onset of clinical arthritis, mice were treated with anti-TNF alone (50 μg), anti-IL-17 alone

(50 μg), anti-TNF (50 μg) plus anti-IL-17 (50 μg) or vehicle alone (PBS). All injections were given intraperitoneally at the times indicated by the arrows. Clinical severity of arthritis was assessed using an established clinical scoring system. There were six mice per group.

Example 1: Blockade of TNFα in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cell differentiation

IL-17 has been implicated in the pathogenesis of a number of autoimmune diseases, including rheumatoid arthritis. TNFα has previously been shown to promote IL-17 expression in vitro and the aim of this study was to assess the impact of anti-TNFα therapy on IL-17 production in collagen-induced arthritis, a model of rheumatoid arthritis. TNFα blockade using TNFR-Fc fusion protein or anti-TNFα antibody reduced arthritis severity but, unexpectedly, increased the production of IL-17, as well as IFNy, by draining lymph node cells, an effect that was mimicked in p55 TNFR^"'^", but not p75 TNFR^"'^" mice. The increases in IL-17/IFNγ production were accompanied by expansion of Th17 and Th1 subsets without any significant changes in Th2 or FoxP3⁺ regulatory T cell populations.

The expression of IL-12/IL-23 p40 was upregulated in p55 TNFR^"y" mice and pre- treatment of thioglycolate-elicited macrophages with rTNFα suppressed LPS-stimulated p40 production. Furthermore, the increased production of IL-17 and IFNy in p55 TNFR^"'^" mice could be completely abrogated by treatment with anti-iL-12/IL-23 p40 mAb. Taken together, these findings indicate that TNFα regulates IL-17/IFNγ production in collagen- induced arthritis by downregulating the expression of IL-12/IL-23 p40. MATERIALS AND METHODS

Mice

Male DBA/1 and C57BL/6 mice were purchased from Harlan Olac (Bicester, UK). TNFRI^"7" and TNFRiI^"'^" mice were bred in-house on a C57BL/6 background. All experimental procedures were approved by the UK Home Office.

Immunisation of mice and assessment of arthritis

DBA/1 and C57BL/6 mice were immunised at 10-16 weeks of age with an emuision of CFA (BD Biosciences, Oxford, UK) plus bovine or chicken type Il collagen, respectively (30). Paw-swelling was measured with calipers and arthritis severity was assessed as follows; 0 = normal, 1 = slight swelling and/or erythema, 2 = pronounced edematous swelling, and 3 = joint rigidity. Each limb was graded giving a maximum score of 12 per mouse.

Anti-cytokine therapy

Blockade of TNFα was achieved using murine p75 TNFR-Fc (kindly donated by GSK, Harlow, UK) or rabbit anti-mouse TNFα IgG (purified using protein G from a rabbit immunised with rTNFα). Blockade of IL-12/IL-23 was achieved using rat anti-mouse p40 !gG2a mAb (clone c17.8; kindly donated by Dr G Trinchieri, then at Wistar Institute, Philadelphia, PA).

Lymph node cell culture

LN cells were cultured in RPMl containing 10% FCS, L-glutamine, penicillin/streptomycin, sodium pyruvate and 2-ME at 2x10⁶/ml and stimulated with type Il collagen (50 μg/ml) or anti-CD3 mAb (0.1 μg/ml). Supernatants were collected for cytokine analysis by ELISA after 48h. Cells were then incubated for a further 24h in the presence of 1 μCi per well of [³H] thymidine to quantify cell proliferation. Thioglycolate elicited macrophages

Mice were injected intraperitonealiy with 1 ml 3% thiogiycoiate. After 3d, mice were killed and peritoneal macrophages collected by PBS lavage. Following overnight adherence, cells were incubated for 8 h in the presence or absence of 30 ng/ml rTNFα (Peprotech, London, UK) followed by 18h in the presence of 1 ng/ml LPS (Sigma-Aldrich, Poole, UK). Supernatants collected for analysis of p40 protein by ELISA.

Cytokine measurement

Cytokines were measured following the manufacturers' instructions using commercially available kits as follows: IFNγ, BD Biosciences; IL-17A, R&D Systems (Abingdon, UK); p40, eBiosciences (Middlesex, UK).

Flow cytometry

For intracellular cytokine staining, cells were cultured for 12h in complete RPMI containing PMA and ionomycin. Brefeldin A was added for the last 4h. For surface staining, cells were incubated with anti-CD4 or anti-CDδ (BD Biosciences) for 20 min at 4⁰C, washed and then fixed in CellFix (BD Biosciences). Cells were permeabilised using PBS containing 1 % FCS₁ 0.01 % sodium azide and 0.05% saponin and stained with anti- mouse IFNγ (BD Biosciences), anti-mouse IL-4 (BD Biosciences) and anti-mouse IL-17 (Cambridge Bioscience, Cambridge, UK) and analysed on FACS Canto Il using FACSDIVA software.

Real-time quantitative RT-PCR

RNA was isolated using the RNeasy protect mini kit (QlAGEN, Crawley, UK) and cDNA transcribed using the reverse transcription system (Promega, Southampton, UK). p40 gene expression was determined by real-time PCR using pre-designed TaqMan® primers and probe (Applied Biosystems, Warrington, UK) by the comparative method of relative quantitation. HPRT mRNA was used as an endogenous control to check for RNA and cDNA differences within samples. Differences in the mean threshold cycle (C_t) for the target gene p40 and the C_t for HPRT RNA, indicated by ΔC_t, were calculated to normalise differences in the mRNA extractions and the efficiency of the reverse transcription. The relative mRNA amount for each target gene was calculated as ΔΔC_t and expressed as fold change compared to a control sample.

Statistical Analysis

The unpaired t test or one way ANOVA with Dunnett's multiple comparison test was used to test statistical significance. Results were considered significant when the P value was less than 0.05.

RESULTS AND DISCUSSION

Blockade of TNFα enhances production of IL-17 and IFNγ

To investigate the effect of blockade of TNFα on the production of IL-17, arthritic DBA/1 mice were treated from the time of arthritis onset with soluble TNFR-Fc for 10 days and the production of IL-17 and IFNγ by LN cells determined by ELISA (Fig 1A and 1 B). Without further stimulation in vitro, a trend towards enhanced IL-17 production was observed in TNFR-Fc treated animals and significantly increased IL-17 production was observed after stimulation of LN cells from TNFR-Fc treated mice in vitro with collagen or anti-CD3 mAb (Fig 1A). A similar finding was observed when the production of I FNy was investigated. lFNγ production was enhanced when LN cells were left unstimulated, stimulated with collagen or stimulated with anti-CD3 mAb (Fig 1 B). This same effect was also observed when TNFα was blocked in CIA using a polyclonal anti-TNFα Ab (data not shown).

Although blockade of TNFα enhanced IL-17 production, arthritis severity was significantly reduced in TNFR-Fc treated mice (Fig 1 C) or anti-TNFα Ab treated mice (data not shown). The data presented in this study therefore confirm previous findings on the therapeutic efficacy of TNFα blockade in CIA (15). The fact that disease activity remained suppressed in anti-TNFα treated mice despite the increases in IL-17 and IFNγ production may be explained in a number of different ways. First, it is possible that IL-17 and IFNγ mediate their pathogenic effects through induction of TNFα, so that their pathogenicity is blunted in the absence of TNFα. A second possibility is that although TNFα inhibitors may result in increased IL-17 and IFNγ production in the periphery, they may prevent the accumulation of pathogenic T cell subsets within the joint, for example by reducing expression of chemokines and adhesion molecules by cells (particularly endothelial cells) in the joint, as has been proposed for human RA (16, 17). A third possibility is that the impact on pathology of increased IL-17 production following anti- TNFα therapy is counterbalanced by the anti-arthritic activity of IFNγ, which has been shown to play a protective role in CIA (18, 19).

TNFα inhibits IL-17 and IFNγ production via p55 TNFR but not p75 TNFR

To confirm that blockade of TNFα signalling through its receptors was responsible for enhancing the production of IL-17 and IFNγ by LN cells from arthritic mice, IL-17 and

IFNγ levels were determined in immunised p55 TNFR^"7", p75 TNFR^"'^" and WT mice. The proliferative responses of cells from the LN of immunised p55 TNFR^''^", p75 TNFR^"'^" and

WT mice did not vary significantiy in response to collagen or anti-CD3 mAb stimulation

(Fig 2A), and the proportions of CD4⁺ T cells in the LN of WT and p55 TNFR^"'^" mice were comparable, although slightly reduced in p75 TNFR^"'^" mice (Fig 2A). The proportions of regulatory CD4⁺Foxp3⁺ T cells also remained unchanged in the p55 TNFR^"'^" and p75

TNFR^"'^" mice when compared to WT mice (data not shown). However, the production of

IL-17 and IFNγ was dramatically higher in the collagen and anti-CD3 mAb stimulated LN cultures from p55 TNFR^"'^" animals compared to both WT and p75 TNFR^"'^" animals (Fig 2B).

Further analysis by flow cytometry confirmed that the proportion of CD4⁺ T cells producing IL-17 and IFNγ in the LN of p55 TNFR^"'^" mice was significantly greater than in the LN of WT and p75 TNFR^"'^" mice (Fig 2C). Th2 cytokines IL-4 and IL-5 were undetectable in immunised WT, p55 TNFR^"'^" and p75 TNFR^"'^" animals, even after stimulation with collagen or anti-CD3 mAb, regardless of genotype (data not shown). This was attributed to the strongly Th1/Th17 skewing properties of the CFA used for immunisation.

It was concluded that immunisation in the absence of signalling via p55 TNFR results in greatly increased production of IL-17 and IFNγ as well as expanded populations of Th17 and Th1 cells in draining LN. The coordinate expansion of both Th17 and Th1 cells in vivo in the absence of TNFα is of particular interest because it challenges the assumption that the differentiation of these two subsets is mutually antagonistic (20, 21 ). TNFα suppresses IL-12/IL-23 p40 expression in vitro and in vivo

We next sought to identify the mechanism by which TNFα inhibits Th17 and Th1 cell generation. IL-12 and IL-23 share the common p40 subunit. Dimerisation of p40 with p35 forms IL-12 which is involved in the differentiation of Th1 cells, whereas dimerisation of p40 with p19 forms IL-23 which has an important role in the generation and/or survival of Th17 cells. Two recent studies in human and mouse myeloid cells have shown that

TNFα selectively inhibits p40 expression at the mRNA and protein level by a mechanism that is not dependent on IL-10 and does not involve inhibition of NFKB, IRF- 1 or ets-2, transcription factors known to regulate p40 gene expression (22, 23).

We therefore set out to confirm that TNFα could suppress p40 production using thioglycolate-elicited macrophages stimulated with LPS in vitro. Pre-treatment of macrophages with 30 or 100 ng/ml of TNFα produced caused a dose-dependent reduction of p40 after stimulation with LPS compared to macrophages stimulated with LPS alone (Fig 3A). The maximum inhibition of p40 production by LPS-stimulated macrophages was around 50%, and the failure to obtain greater suppression was attributed to the fact that LPS alone would inevitably produce significant quantities of TNFα.

Next, we addressed the question of whether p40 expression was elevated in vivo in the absence of signalling via TNFRI. In order to quantify p40 expression without culturing cells in vitro, mRNA was extracted from LN cells immediately post-mortem and analysed by real-time PCR. LN cells from immunised p55 TNFR^"'^" mice were found to express 6-fold greater levels of p40 mRNA compared to immunised WT and p75 TN FR^''^" mice (Fig 3B). This confirms the ability of TNFα to suppress p40 expression in vivo. Thus, our data confirm and extend previous findings (22, 23) concerning the inhibitory effect of TNFα on IL-12/IL-23 p40 expression.

Blockade of IL-12/IL-23 p40 in vivo abrogates the increase in IL-17 and !FNγ production in p55 TNFR^"'^' mice

Finally, we investigated whether inhibition of IL-12/1L-23 p40 activity in p55 TNFR^"'^' mice would result in a reduction of IFNγ/IL-17 production and reduced numbers of Th1/Th17 cells. Treatment with a blocking anti-mouse p40 mAb in p55 TNFR^"'^" mice from the day of immunisation to day 14 post immunisation completely abrogated the increase of both IL-17 (Fig 4A) and IFNγ (Fig 4B) production by LN cells stimulated in vitro with collagen or anti-CD3 mAb. In addition, the percentage of CD4⁺ T cells producing either IL-17 or IFNy in the anti-p40 treated p55 TNFR^"'^' group was also significantly lower than the p55 TNFR^"'^" group treated with control mAb (Fig 4C). These findings confirm that TNFσ influences the development and/or survival of Th1 and Th17 cells in vivo by inhibition of IL-12/IL-23 p40 expression.

Relevance of the findings for human RA

As discussed above, TNFα has been reported to decrease p40 expression in human macrophages (22) therefore the findings presented here may have implications for human immunology. In RA (as in CIA)₁ TNFα blockade is clearly beneficial but it is possible that the rare occurrence of side effects, such as anti-DNA antibodies and demyelination (24), could be explained by an amplification of Th 17 and/or Th1 responses.

Relevance of the findings for other autoimmune diseases

TNFα blockade was shown to increase both the rate and frequency of relapse in patients with existing multiple sclerosis (25). In experimental autoimmune encephalomyelitis (EAE), TNFα was associated with spontaneous regression from disease (26), suggesting that TNFα plays an important anti-inflammatory role in later stages of disease. TNFα^"A mice immunised with myelin oligodendrocyte glycoprotein developed severe inflammation and demyelination, whereas treatment of susceptible mice with TNFα reduced the severity of EAE, further suggesting an important disease-limiting role for TNFα in EAE (27). Similarly, in murine lupus, administration of rTNFα was found to be protective (28) whereas TNFα deficiency was associated with accelerated onset of disease (29). Hence, an important question to be addressed is whether the protective effects of TNFα in multiple sclerosis, EAE and lupus are mediated via inhibition of IL-12/IL-23 p40. Conclusions

The results of this study show that TNFα can reduce both Th1 and Th17 responses in CIA by inhibiting the expression of the p40 subunit of IL-12 and IL-23. We suggest that this represents a negative feedback mechanism that normally serves to limit the extent of immune-mediated pathology during inflammation. The implications for human therapy need to be investigated further, given the increasing use of TNF inhibitors for the treatment of autoimmune disease.

Example 2: Synergistic effect of anti-TNF and anti-iL17 in collagen-induced arthritis (CIA)

In order to demonstrate synergy between anti-TNF and anti-IL-17 in arthritis, arthritis was induced in DBA/1 mice by immunisation with bovine type Il collagen in adjuvant. After onset of clinical arthritis, mice were treated with anti-TNF alone (50 μg), anti-IL-17 alone (50 μg), anti-TNF (50 μg) plus anti-IL-17 (50 μg) or vehicle alone (PBS). Clinical severity of arthritis was assessed using an established clinical scoring system. The results showed that combined treatment with anti-TNFα plus anti-IL-17 gave a greatly enhanced therapeutic effect than either agent alone.

The results are shown in Figure 5. There is a synergistic effect of anti-TNF and anti-IL17 in CIA.

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Claims

1. A method of treating rheumatoid arthritis (RA) in a patient, the method comprising administering to the patient an anti-TNF agent and an anti-IL17 agent.

2. The combination of an anti-TNF agent and an anti-IL17 agent for use as a medicament.

3. The combination of an anti-TNF agent and an anti-IL17 agent for use in treating RA in a patient.

4. An anti-TNF agent for use in treating RA in a patient, wherein said patient is administered an anti-IL17 agent.

5. An anti-IL17 agent for use in treating RA in a patient, wherein said patient is administered an anti-TNF agent.

6. Use of the combination of an anti-TNF agent and an anti-IL17 agent in the manufacture of a medicament for the treatment of RA in a patient.

7. Use of an anti-TNF agent in the manufacture of a medicament for the treatment of RA in a patient, wherein the patient is administered an anti-IL17 agent.

8. Use of an anti-il_17 agent in the manufacture of a medicament for the treatment of RA in a patient, wherein the patient is administered an anti-TNF agent.

9. A system for treating RA in a patient, the system comprising an anti-TNF agent and an anti-IL17 agent.

10. A kit of parts for treating RA in a patient, the system comprising an anti-TNF agent and an anti-IL17 agent.

1 1. A method according to Claim 1 , or the combination according to Claims 2 or 3, or the agent of Claims 4 or 5, or the use of Claims 6, 7 or 8, or the system of Claim 9, or the kit of parts of Claim 10, wherein the anti-TNF agent is an anti- TNF antibody or a soluble TNF receptor (TNFr).

12. A method, combination, agent, use, system or kit of parts according to Claim 9 wherein the anti-IL17 agent is an anti-IL17 antibody or a soluble anti-IL17 receptor (IL17r).

13. A method according to Claim 1 wherein the anti-TNF agent and the anti-IL17 agent are administered simultaneously.

14. A method according the Claim 1 wherein the anti-TNF agent and the anti-IL17 agent are administered sequentially.

15. A method, combination, agent, use, system of kit of parts according to any of the previous claims wherein the patient is human.

16. Any novel method of treating RA in a patient as herein described.