WO2011070339A1 - Method of treating disease - Google Patents

Abstract

A method of treating a disease selected from the group consisting of rheumatoid arthritis (RA), Crohn's disease, ankylosing spondolytis, psoriasis and psoriatic arthritis in a patient, the method comprising administering to the patient an anti-TNF agent and an anti-IL23 agent.

Description

METHOD OF TREATING DISEASE

The present invention relates to a method of treating a disease selected from the group consisting of rheumatoid arthritis (RA), Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis.

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 TNFa, IL-6 and IL-1 play dominant pathological roles. Crohn's disease is a inflammatory disease of the intestines that may affect any part of the gastrointestinal tract. Ankylosing spondylitis is a form of chronic inflammatory arthritis that mainly affects joints in the spine and the sacroilium in the pelvis, causing eventual fusion of the spine. Psoriasis is a chronic autoimmune inflammatory disease that affects the skin and joints. Psoriatic arthritis is a form of arthritis that is frequently associated with psoriasis.

RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis are all diseases that have been shown to be treated relatively effectively by drugs that neutralise TNF.

WO 2004/042009 relates to the inhibition of IL17 using an antagonist of I L23. US 2007/0178099 A1 relates to the treatment of TNF-mediated diseases using the combination of a TNF antagonist and an IL12 antagonist. US 7,247,71 1 B2 relates to anti-IL23p40 specific human Ig derived proteins which do not bind to the p40 subunit of IL12 and thus do not neutralise IL12-related activity. US 2007/0218064 A1 relates to human anti-IL23p19 antibodies. US 2008/0038831 A1 relates to anti-IL23 specific human Ig derived proteins. US2008/0095775 A1 relates to molecules that block the activity of anti-IL23 via its p19 subunit. We have now found that, surprisingly, the use of a combination of anti-TNF agent and anti-IL23 agent has a synergistic effect when used therapeutically.

A first aspect of the invention provides a method of treating a disease selected from the group consisting of rheumatoid arthritis (RA), Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, the method comprising administering to the patient an anti-TNF agent and an anti-IL23 agent.

Hereafter TNF is tumour necrosis factor a. IL23 is interleukin 23.

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.

The patient may be suffering from one or more of the aforementioned diseases.

It is preferred that the anti-TNF agent is an agent that is directed to the TNF or TNF receptor 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-IL23 agent is an agent that is directed to the IL23 or IL23 receptor 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 IL23 agent.

It is preferred that the anti-TNF agent is an anti-human TNF agent and that the anti-IL23 agent is an anti-human IL23 agent and that the patient to be treated is human.

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. IL23 from many species is known. In humans, IL23 occurs as a heterodimer composed of two subunits termed p40 and p19, named according to their approximate molecular mass in kDa. The subunit p40 is also found in the heterodimer IL12 where it is present in combination with the subunit p35. See the Background of the Invention on paragraphs [0003] to [0007] in US 2008/0038831 A1 , incorporated herein by reference.

As is also discussed in US 2008/0038831 A1 , the IL23 receptor complex contains a receptor chain, termed IL23R, and the beta-1 subunit of the IL12 receptor. IL23 does not bind the IL12 receptor. The amino acid sequence of the p40 subunit of human IL23 is disclosed in Wolf et al (1991 ) J. Immunol. 146(9), 3074-3081 , Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells, and the amino acid sequence of the p19 subunit of human IL23 is disclosed in Oppmann et al (2000) Immunity 13(5), 715-725, Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. These papers are incorporated herein by reference.

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 to be treated, 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-IL23 may be administered by any suitable route. Typically, administration of the anti-TNF agent or the anti-IL23 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-IL23 agent are administered simultaneously. In another particular embodiment, the anti-TNF agent and the anti-IL23 agent are administered sequentially. If sequential, they may be administered in any order. The anti-TNF agent and the anti-IL23 agent are administered either together or separately in a dose which is effective for the treatment of the disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis.

Efficacy of the treatment of RA or psoriatic arthritis 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. For ankylosing spondylitis, assessment will include total back pain, patient assessment of disease activity, inflammation and physical function. For Crohn's disease, assessment will include general well-being, abdominal pain, number of liquid stools per day, abdominal mass and presence of complications. For psoriasis, assessment will be by the Psoriasis Area Severity Index (PASI) which combines the assessment of the severity of lesions and the total area of skin affected.

Doses of the anti-TNF agent and the anti-IL23 agent may be determined empirically. The patient is administered an amount of each agent, whether together or separately, that is effective for the treatment of the disease, particularly, the amount is effective for the amelioration of the disease.

A second aspect of the invention provides the combination of an anti-TNF agent and an anti- IL23 agent for use as a medicament.

A third aspect of the invention provides the combination of an anti-TNF agent and an anti-IL23 agent for use in treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient.

A fourth aspect of the invention provides an anti-TNF agent for use in treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, wherein said patient is administered an anti-IL23 agent. The patient may be administered the anti-IL23 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-IL23 agent for use in treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis 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-IL23 agent. A sixth aspect of the invention provides the use of the combination of an anti-TNF agent and an anti-IL23 agent in the manufacture of a medicament for the treatment of a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis 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 a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, wherein the patient is administered an anti-IL23 agent. The patient may be administered the anti-TNF agent prior to or at the same time as or after administration of the anti-IL23 agent.

An eighth aspect of the invention provides the use of an anti-IL23 agent in the manufacture of a medicament for the treatment of a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis 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-IL23 agent.

A ninth aspect of the invention provides a system for treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, the system comprising an anti-TNF agent and an anti-IL23 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 a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, the system comprising an anti-TNF agent and an anti-IL23 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.

It is preferred that the anti-TNF agent is an anti-TNF antibody or a soluble TNF receptor (TNFr) or an anti-TNF receptor antibody. In all aspects of the invention, the anti-IL23 agent may be any agent that neutralises IL23 or blocks the IL23 receptor or inhibits the production of IL23 or inhibits signalling via the IL23 receptor. Typically, the anti-IL23 agent neutralises IL23 by binding to IL23. The anti-l L23 agent is selective for IL23 and has substantially no effect on IL12 or the IL12 receptor. The anti-IL23 agent may be considered to be anti-ll_23 specific. It is preferred that the anti-IL23 agent is an anti-IL23 antibody or a soluble anti-IL23 receptor (IL23r) or an anti-IL23 receptor antibody. It is preferred that the combined therapy comprises the administration of an anti-IL23 antibody and an anti-TNF antibody. In this embodiment it is preferred that the anti-IL23 antibody is one which binds to the p19 subunit of IL23 as discussed below. In this embodiment it is preferred that the anti-TNF antibody is adalimumab. By antibody, we include any antibody-like molecule which is able to bind TNF or the TNF receptor (or, as the case may be, IL23 or the IL23 receptor) 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 IL23 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). Anti-TNF receptor antibodies may also be used.

Antibodies that bind IL23 , but do not bind IL12, and antibodies that bind the IL23 receptor, but do not bind the IL12 receptor, are disclosed in, for example, US 7,247,71 1 B2, US 2007/0218064 A1 , US 2008/0038831 A1 and US 2008/0095775 A1 , all of which are incorporated herein in their entirety.

It is preferred if the anti-IL23 antibody is one which binds to the p19 subunit of IL23. Such antibodies are disclosed, for example in US 2007/0218064 A1 , US 2008/0038831 A1 and US 2008/0095775 A1. Preferably, the antibody should not prevent or inhibit the binding of IL12 to the IL12 receptor.

The antibody may conveniently be an antibody that binds to the IL23 receptor chain (but not the IL12 receptor) as disclosed in US 2008/0038831 A1 , incorporated herein by reference.

Soluble receptors may be readily engineered from the intact membrane bound receptor using methods well known in the art. For example, the TNF (or IL23)-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 IL23 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 antilL23 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. Synergy between anti-TNF and anti-IL23 in collagen-induced arthritis. DBA/1 mice were immunized type II collagen in complete Freund's adjuvant. On the first day arthritis was observed, mice were treated with anti-TNF alone (50 pg/mouse), anti-p 9 alone (50 pg/mouse), anti-TNF (50 pg/mouse) plus anti-p19 (50 μg/mouse) or isotype controls. Treatment was on days 1 and 4 of arthritis. (A) Clinical scores. (B) Paw-swelling. Data were analysed using the Dunnett multiple comparisons test (n=5). ^*P<0.05; ^**P<0.01 (compared to controls).

Figure 2. DBA/1 mice were immunised with type II collagen in CFA. After the onset of arthritis mice were treated with anti-TNF alone (300 pg/mouse), anti-p19 alone (200 pg/mouse), anti- TNF (300 pg/mouse) plus anti-p19 (200 pg/mouse) or isotype controls on days 1 , 4 and 7 of arthritis. Arthritis severity was assessed using a clinical scoring system. Figure 3. C57BL/6 mice were immunised with chicken type II collagen in CFA. After the onset of arthritis mice were treated with anti-TNF alone (250 pg/mouse), anti-p19 alone (100 pg/mouse), anti-TNF (250 pg/mouse) plus anti-p19 (100 pg/mouse) or isotype controls on days 1 , 3, 5, 7 and 9 of arthritis. Arthritis severity was assessed using a clinical scoring system and paw-swelling was measured using micocalipers.

Figure 4 Th17 cells are increased in RA. PB Cs were purified from peripheral blood by Ficoll gradient centrifugation and stimulated with PMA and ionomycin. A. Representative FACS profiles following intracellular cytokine staining of one healthy volunteer (top panels) and one RA patient (lower panels) showing on the Y axis percentages of IL-17+, IFN-y+ and IFN-y+ cells and on populations using a lymphocyte gate. B. Pooled data from A. Statistical analysis was performed using Student T test. *^**P<0.001. C. PBMCs were stimulated with anti-CD3 mAb for 24 hours, and supernatants were analysed for the levels of IL-17 and IFN-γ by ELISA. Bars represent means ±SE.

Figure 5. Anti-TNFa therapy increases Th17 cells in RA. Blood was collected prior to the initiation of anti-TNFa (pre), and at 4 and 8 weeks after. A. Representative FACS profiles following intracellular staining of cell populations from one patient treated with adalimumab. B. Dot plots of intracellular cytokine staining of PBLs from all patients in trial. Bar denotes the mean. C. IL-17 and IFN-γ levels in culture supernatants 24-hour after stimulation of PBMCs with anti-CD3. *P<0.05, ^**P<0.01. Statistical analyses were performed using repeated measures one way ANOVA, followed by Dunnet's multiple comparison test. D. Purified PBMCs from RA patients before and after anti-TNFa therapy were lysed and RNA levels of RORy were measured by RT-PCR. The relative concentration of each gene is expressed as relative units using a healthy control as a calibrator after normalizing against HuPO

Figure 6. Increase in Th17 cells is more potent after monoclonal antibody therapy (adalimumab). Intracellular cytokine staining of PBMCs from patients treated with adalimumab or etanercept. A. Dot plots depicting numbers of Th17 or Th1 cells per ml of blood. B. Dot plots depicting percentage of Th17 or Th1 cells as in Figure 4. Bar denotes the mean. C. ELISA of IL-17 and IFN-γ levels in culture supernatants 24h after stimulation of PBMCs with anti-CD3. D. Sera from patients before and after therapy were tested for the ability to neutralise exogenously-added TNFa (10ng/mL) by measurement of TNFa induced Firefly luciferase (FL) expression normalized with respect Renilla luciferase (RL) expression using a cell line transfected with a FL reported gene under the control of a TNFa-sensitive promoter and RL reporter gene under the control of a constitutive promoter as described in the Materials & Methods. Residual bioactive TNFa is plotted as percentage activity. Figure 7. IL-12/23p40 is reduced in RA and induced after anti-TNFa therapy. Whole blood was stimulated with LPS (500 ng/mL) prior to Ficoll purification. After 24 hours, the plasma layer was collected and levels of total p40 and IL-23 were measured by ELISA.

Figure 8. Plasma levels of IL-12/IL-23 p40 following LPS stimulation of whole blood from RA patients before and after anti-TNFa therapy.

Figure 9. TNF regulates IL-12/IL-23 p40 expression. MoDCs from healthy donors were pre- treated with anti-TNF mAb or rTNF and stimulated with LPS after 18h. IL- 2/IL-23 p40 levels in the culture supernatant were measured by ELISA after a further 24h. ^*P<0.05, ^**P<0.01.

Example 1 : Synergistic effect of anti-TNF and anti-IL23 in collagen-induced arthritis

Aim of the study The aim of this study was to establish whether anti-TNF and a neutralising antibody against the p19 subunit of IL23 act in a synergistic manner in collagen-induced arthritis (CIA), a well established mouse model of RA. In this study, suboptimal doses of anti-TNF and anti-p19 were used which, alone, would be predicted to have no significant effect on the outcome of arthritis. Reagents

Type II collagen was purified from bovine cartilage, as described and solubilised by stirring overnight at 4° C in acetic acid (0.1 M) or Tris buffer (0.05 M Tris, containing 0.2 M NaCI, pH 7.4). Anti-TNF mAb (clone TN3-19.12) was purchased from Dr Robert Shreiber (Washington University, St Louis, USA). Anti-p19 mAb (clone G23-8) was purchased from Ebiosciences (Hatfield, UK). Complete Freund's adjuvant (CFA) was purchased from BD (Oxford, UK). Methods

Male DBA/1 mice (8-12 weeks old) were immunized intradermal^ at the base of the tail with bovine type II collagen (200 μg) emulsified in complete Freund's adjuvant (CFA; Difco, West Molesley, UK). Mice were inspected on a daily basis for signs of arthritis (redness, swelling and/or limping). On the first day arthritis was observed, mice were assigned to randomly to one of four treatment controls: anti-TNF alone (50 pg/mouse), anti-p19 alone (50 pg/mouse), anti- TNF (50 pg/mouse) plus anti-p 9 (50 pg/mouse) or isotype controls. Treatment was on days 1 and 4 of arthritis. Arthritis was monitored clinically using the following scoring system: 0 = normal, 1 = slight swelling and/or erythema, and 2 = pronounced oedematous swelling. Each limb was graded, giving a maximum score of 8 per mouse. In addition, paw-swelling was measured using microcalipers. This research was approved by the local ethical review process committee and by the Home Office of Great Britain. Results

Anti-TNF alone, administered at the sub-optimal dose of 50 pg/mouse, failed to affect the clinical scores or the degree of paw-swelling compared to control mice. Similarly, anti-p19 alone had no effect on severity of arthritis compared to controls. However, when administered in combination, anti-TNF plus anti-p19 produced significant reductions in both clinical score (P<0.05) and paw-swelling (P<0.01 ).

Conclusions This study has shown, as expected, that sub-optimal either anti-TNF or anti-p19 failed to influence disease severity when administered alone. Unexpectedly, however, the combination of anti-TNF and anti-p19 produced a significant reduction in arthritis compared to controls. Hence, the results demonstrate that anti-TNF and anti-p19 act in a synergistic fashion in reducing the severity of established arthritis. Example 2: Combined therapeutic effect of anti-TNF and anti-IL23 in collagen-induced arthritis in C57BL/6 mice

Introduction

We demonstrated a synergistic therapeutic effect between anti-TNF antibody and a neutralising antibody against the p19 subunit of IL-23 in collagen-induced arthritis (CIA) in DBA 1 mice (see Example 1 ). Whilst CIA in DBA/1 mice is widely regarded as being predictive for human rheumatoid arthritis (RA), we have recently reported that CIA in C57BL/6 mice more closely resembles human RA in terms of its disease course and response to therapy (Inglis et al (2007) Arthritis Res Then 9:R113). Hence, we set out to study the effect of anti-TNF/anti-IL-23 combination therapy in CIA in C57BL/6 mice.

Reagents

Type II collagen was purified from chicken cartilage, as described (Inglis et al (2008) Nature Protocols 3: 612-8) and solubilised by stirring overnight at 4°C in acetic acid (0.1 M) or Tris buffer (0.05 M Tris, containing 0.2 M NaCI, pH 7.4). Anti-TNF mAb (clone TN3-19.12) was purchased from Dr Robert Shreiber (Washington University, St Louis, USA). Anti-p19 mAb (clone G23-8) was purchased from Ebiosciences (Hatfield, UK). Complete Freund's adjuvant (CFA) was purchased from BD (Oxford, UK).

Mice Male C57BIJ6 mice were purchased from Harlan UK. Methods

Male mice (12-14 weeks old) were immunized at the base of the tail with chicken type II collagen (200 g) emulsified in complete Freund's adjuvant (CFA; Difco, West Molesley, UK). Mice were inspected on a daily basis for signs of arthritis (redness, swelling and/or limping). On the first day arthritis was observed, mice were assigned to randomly to one of four treatment controls: anti-TNF alone (250 pg/mouse), anti-p19 alone (100 pg/mouse), anti-TNF (250 pg/mouse) plus anti-p19 (100 pg/mouse) or isotype controls. There were 8-10 mice per treatment group. Treatment was on days 1 , 3, 5, 7 and 9 of arthritis. Arthritis was monitored clinically for 10 days using an established clinical scoring system. Paw-swelling was measured using microcalipers. This research was approved by the local ethical review process committee and by the Home Office of Great Britain.

Results

Treatment with anti-TNF alone or anti-p19 alone did not have a significant effect on clinical score. However, when administered in combination, anti-TNF plus anti-p19 completely eliminated disease, such that the clinical score was zero on day 10 after disease onset (Figure 7). Similarly, a synergistic therapeutic effect was observed between anti-TNF and anti- pi 9 in the suppression of paw-swelling (Figure 7).

Conclusions

This study has shown that anti-TNF and anti-p19 act synergistically in the amelioration of CIA in C57BIJ6 mice.

Example 3: Combined therapeutic effect of anti-TNF and anti-IL23 in collagen-induced arthritis

Introduction

In Example 1 a synergistic therapeutic effect is demonstrated between sub-optimal doses of anti-TNF antibody and a neutralising antibody against the p19 subunit of IL-23 in collagen- induced arthritis (CIA), an animal model of rheumatoid arthritis. In this example we set out to repeat this experiment using optimal doses of each antibody. In addition, using a system of adoptive transfer, we addressed the question of whether this form of combination therapy has a sustained therapeutic effect.

Reagents

Type II collagen was purified from bovine cartilage, as described and solubilised by stirring overnight at 4°C in acetic acid (0.1 M) or Tris buffer (0.05 M Tris, containing 0.2 M NaCI, pH 7.4). Anti-TNF mAb (clone TN3-19.12) was purchased from Dr Robert Shreiber (Washington University, St Louis, USA). Anti-p19 mAb (clone G23-8) was purchased from Ebiosciences (Hatfield, UK). Complete Freund's adjuvant (CFA) was purchased from BD (Oxford, UK). Mice

Male DBA/1 mice and female CB-17 SCID mice were purchased from Harlan UK. Methods

Male DBA/1 mice (8-12 weeks old) were immunized intradermal^ at the base of the tail with bovine type II collagen (200 pg) emulsified in complete Freund's adjuvant (CFA; Difco, West Molesley, UK). Mice were inspected on a daily basis for signs of arthritis (redness, swelling and/or limping). On the first day arthritis was observed, mice were assigned randomly to one of four treatment controls: anti-TNF alone (300 pg/mouse), anti-p 9 alone (200 pg/mouse), anti- TNF (300 pg/mouse) plus anti-p19 (200 pg/mouse) or isotype controls. There were 8-10 mice per treatment group. Treatment was on days 1 , 4 and 7 of arthritis. Arthritis was monitored clinically for 10 days using the following scoring system: 0 = normal, 1 = slight swelling and/or erythema, and 2 = pronounced oedematous swelling. Each limb was graded, giving a maximum score of 8 per mouse. This research was approved by the local ethical review process committee and by the Home Office of Great Britain.

For the adoptive transfer of arthritis, arthritis was induced in DBA 1 mice as above and treated with anti-TNF alone (300 pg/mouse), anti-p19 alone (200 pg/mouse), anti-TNF (300 pg/mouse) plus anti-p19 (200 pg/mouse) or isotype controls. Treatment was on days 1 , 4 and 7 of arthritis. On day 10 the mice were killed and spleen and inguinal lymph nodes removed and made into a cell suspension. The cells were then injected intraperitoneally into SCID mice together with 100 pg of type II collagen. Onset of arthritis and clinical scores were monitored as above. Results

Anti-TNF treatment alone caused a significant reduction in arthritis severity compared to control mice whereas anti-p19 alone did not have a significant effect on arthritis. However, when administered in combination, anti-TNF plus anti-p19 produced marked reduction in clinical score that exceeded the effect of anti-TNF alone (Figure 2).

CIA in DBA/1 mice is regarded as the "gold standard" model for testing drugs for RA. However, whilst this model is predictive of human FA, it suffers the drawback of being a self-remitting disease therefore is less useful for looking at the long-term therapeutic effects of novel therapies. Hence, we evaluated the tolerogenic capacity of combined anti-TNF/anti-p19 therapy using an adoptive transfer model in which donor cells from treated arthritic DBA/1 mice are injected into immunodeficient SCID recipients. The SCID recipients are also boosted with 100 pg of type II collagen intraperitoneal^. The incidence of arthritis in SCID mice was reduced when the cells were derived from donors treated with anti-p19 alone or anti-p19 plus anti-TNF (Table 1 ), indicating that treatment with p19 mAb had a long-lasting therapeutic effect.

Table 1. Adoptive transfer of arthritis from treated arthritic DBA 1 mice to SCID recipients. 10 million pooled lymph node and spleen cells were injected intraperitoneal^ into SCID mice together with 100 pg of type II collagen.

Donor treatment Incidence of arthritis in SCID recipients Control mAbs 2/2

Anti-TNF alone 4/5

Anti-p 9 alone 0/2

Anti-TNF plus anti-p19 2/7

Conclusions

This study has shown that optimal doses of anti-p19 plus anti-TNF treatment had an additive therapeutic effect in established CIA. Anti-p19 alone did not significantly affect disease severity. Unexpectedly, however, the anti-p19 treatment (alone or in combination with anti-TNF) of arthritic donor mice prevented the adoptive transfer of arthritis to SCID mice which may be regarded as evidence of a long-lasting therapeutic effect. Hence, the combination of anti-TNF plus anti-p19 provides extremely good control of arthritis as well as a sustained therapeutic effect when the antibodies are no longer present. Example 4: TNF blockade increases Th17 cells in rheumatoid arthritis

Summary This study shows that treatment of RA patients with TNF blocking drugs increases the percentage of Th17 cells.

Objective

The aim of this study is to establish whether anti-TNFa therapy reduces disease severity but increases numbers of Th17 and Th1 cells in patients with rheumatoid arthritis.

Methods

PBMCs from 17 patients with rheumatoid arthritis were analysed before and at two time points after initiation of anti-TNFa therapy. Patients were on adalimumab (n=9) or etanercept (n=8).

Results

Compared to healthy controls, the percentage of Th17 cells was higher in rheumatoid arthritis patients and increased significantly at four and eight weeks after anti-TNFa therapy. Adalimumab had a more pronounced effect on Th17 cells than etanercept and this difference was explained by the fact that sera from adalimumab treated patients exhibited greater TNFa neutralising capacity than etanercept. It was also observed that anti-TNFa naive patients failed to upregulate IL-12/IL-23 p40 in response to LPS. However, this inhibition was abrogated after adalimumab treatment, providing compelling evidence that TNFa inhibits p40 expression in vivo. Compared to healthy controls, IL-23 was elevated in rheumatoid arthritis and increased markedly after adalimumab treatment.

Conclusion

In summary, we have shown that there is an increase in numbers of Th17 cells in RA patients treated with TNF blocking drugs. This increase is accompanied by increased expression of IL- 23, which is known to be responsible for promoting the differentiation and survival and Th17 cells. In addition, in Examples 1 and 2 we have shown that anti-TNF and anti-IL-23 act synergistically in the collagen-induced arthritis model. On this basis we conclude that the combination of anti-TNF and anti-IL-23 is suitable as a treatment for RA and other related diseases in which anti-TNF is effective. Introduction to Example 4

The role of Th17 cellls in the pathogenesis of rheumatoid arthritis (RA) is a matter of ongoing debate. In mice, genetic deletion of IL-23, which promotes survival and functional maturation of Th17 cells, confers complete resistance to collagen-induced arthritis (CIA), an animal model of RA [1]. In contrast, deletion of IL-12, which is the chief cytokine responsible for the differentiation of Th1 cells, exacerbates disease [1].

Blockade of TNFa is clearly beneficial in both CIA and RA [2] yet, surprisingly, we have recently discovered that anti-TNFa treatment of CIA leads to an expansion of antigen-specific Th17 and Th1 cells in draining lymph nodes [3]. These findings were then reproduced in TNFaRI^"'^", but not TNFaR2^"A, mice [3]. We also found, by adoptive transfer, that the expanded population of Th17/Th1 cells was highly pathogenic when the anti-TNFa "brake" was removed. On this basis we concluded that, in addition to its well-established pro-inflammatory role, TNFa was also a negative regulator of Th17/Th1 cells. This conclusion was supported by the important discovery that TNFa, acting via TNFaRI , inhibits the expression of p40, the common subunit of IL-12 and IL-23 [3-5], Many patients with RA and other immune-mediated inflammatory diseases, including inflammatory bowel disease, psoriasis and ankylosing spondylitis, are now being successfully treated with TNFa blocking drugs and it is important to establish whether IL-12/IL- 23 p40 expression and the frequency of Th17/Th1 cells are affected by therapy.

Materials and Methods

Study population

17 patients with active RA were evaluated before and after anti-TNFa therapy. This included etanercept given weekly at a dose of 50mg or adalimumab given at a dose of 40 mg fortnightly. Patients who were on stable doses of disease modifying anti-rheumatic drugs (DMARDS) for at least 6 weeks prior to the study but still had active disease were eligible for inclusion in the study. Peripheral blood was sampled prior to then 4 and 8 weeks after anti-TNFa therapy.

Patients were excluded if they had been previously treated with anti-TNFa therapy for whatever reason. Additionally patients were excluded if they had received oral, intra-articular or intramuscular injection of steroid in the preceding 4 weeks prior to commencing anti-TNFa therapy, or had intercurrent, active infections, as these can independently affect T cell subpopulations (Lamas M 1993 cell immune 151 ). Seven age and sex matched healthy volunteers were recruited and underwent a one off procedure of peripheral blood sampling.

Ethical approval was given by an independent ethics committee before recruitment of patients (RREC 07/8070681 ).

Peripheral blood mononuclear cell purification

Blood was collected with tubes with or without EDTA for the collection of PBMCs and plasma, or serum, respectively. PBMCs were purified using Ficoll gradient centrifugation (Cedarlane), followed by wash in 1X PBS twice. Cells were counted and seeded at 1.5x10⁶ or 0.75x10^s per well and stimulated for intracellular FACS analysis or ELISA, respectively.

Flow cytometry

For extracellular staining, stimulated or non-stimulated cells were stained with anti-human anti- CD4 ( BD Biosciences), CD8 (BD Biosciences ), CD11 c (eBioscience), CD11 b (eBioscience) and B220 (eBioscience) for 30 min at 4°C prior to fixation with Cytofix (BD Biosciences). For intracellular cytokine staining, cells were stimulated for up to 5h with PMA and ionomycin in the presence of Brefeldin A. After extracellular staining, cells were permeabilised with PBS containing 1 % BSA and and 0.05% saponin and stained with anti-human IFNy and anti-human IL-17 (BD Biosciences) for 30 minutes at room temperature. Cells were acquired and analyzed on FACS Canto II using FACSDIVA software (BD Biosciences).

ELISA

Whole blood was stimulated with 100ng/mL of LPS (Sigma-Aldrich) for 24 hours. Blood was spun down and plasma was collected for analysis of IL12/23p40, IL12p35 and IL23p19 levels by ELISA. Plates were coated with purified anti-human p40 (BD bioscience) or purified anti-human p19 (Biolegened). Bound cytokine was detected using biotinylated anti-human p40 (BD bioscience). Cytokine standards were obtained from Peprotech, UK. Real-Time PCR

Comparative mRNA levels of cytokines or transcription factors were assessed in patients before and anti-TNFa therapy. PBMCs were lysed in RLT lysis buffer with DTT and RNA was extracted from the cells using the RNAeasy mini kit (QIAGEN) according to the manufacturer's instructions. Reverse transcription for the production of cDNA was carried out on the RNA samples using the ABI High capacity reverse transcription system with random hexamer primers according to the manufacturer's protocol. Amplification of cDNA was performed on an ABI AB7900HT real-time PCR machine in a 384 well plate using TaqMan® Gene Expression Assays sets from the ABI inventoried library for all genes and Human acidic ribosomal protein (HuPO) control. Amplification was by a three-step PCR. The relative concentration of each gene of interest was calculated using the AACt method and expressed as relative units using a healthy control as a calibrator after normalizing against HuPO.

TNFa bioactivity

Jurkat cells were transfected with the Firefly luciferase (FL) reporter gene regulated by a TNFa-sensitive chimeric promoter and Renilla luciferase (RL) under the control of a constitutive promoter. TNFa activity was measured after the addition of 10ng/mL TNFa to sera from patients prior to and after therapy. FL and RL activity was determined sequentially for each sample in the same assay well using the Dual-Glo luciferase assay system (Promega, Madison, Wl). Results are expressed as residual TNFa activity determined from FL expression normalized with respect to RL expression.

Multiplex bead array

PBMCs were seeded at 0.75x10⁶ cells per well in a 96 well u bottom plate. Cells were stimulated with either 1 ug/mL anti-human anti-CD3 (functional grade OKT3, eBioscience) or 500ng/mL LPS (Sigma) for 24 hours before supernatant collection. Supernatants were assessed for levels of IL-12, IL-23, IL- 7, IFN-y, IL-Ι β, IL-6, IL-10, IL-8, IL-2, IL-5 using multiplex bead array (Bender Med Systems) and analysed using BMS software. Results

Th17 cells are increased in RA patients

We have recently reported that TNFa blockade or genetic deletion of TNFaRI in mice results in the expansion of Th17 and Th1 cells in the draining lymph nodes [3]. We have now investigated whether anti-TNFa treatment in patients with RA leads to an increase in peripheral Th17 Th1 cells. However, prior to embarking on the human study it was necessary to establish that the increase in numbers of Th17/Th1 cells found in lymph nodes of anti-a treated mice was paralleled by an increase in peripheral blood. Hence, the frequency of Th17 Th1 cells in peripheral blood of arthritic DBA/1 mice that have been treated for 7 days with anti-TNFa mAb (TN3-19.12) were compared with control arthritic mice. There was a significant increase in both Th17 and Th1 cells in the peripheral blood of anti-TNFa treated mice, thereby providing the rationale for monitoring Th17/Th1 cells in the blood of patients treated with TNFa blocking drugs.

Blood was collected from 17 patients with active RA before and after anti-TNFa therapy (Table 2) in order to monitor changes in Th17 and Th1 populations by intracellular cytokine staining following stimulation with PMA/ionomycin. In addition, cytokine secretion was measured by ELISA following stimulation with anti-CD3 mAb. The first finding to emerge was that Th17 cells were markedly increased in RA compared to healthy controls (0.68% versus 0.2% of total PBMCs), whilst percentages of Th1 cells did not significantly differ between the two groups (Fig. 4A and B). This finding is important to set a baseline for Th1 responses in anti-TNFa naive patients, and is in agreement with published reports [6] to provide clear evidence of a heightened Th17 response in RA. To confirm this, we measured the levels of IL-17 and IFN-γ in the supernatants of PBMCs stimulated with anti-CD3 mAb for 24 hours. In agreement with results from intracellular staining, IL-17 was elevated in RA compared to healthy controls, while IFN-γ levels were insignificantly increased (Fig. 4C).

TNFa blockade increases circulating Th17 cells in RA

Next, we compared numbers of Th17 and Th1 before and at 4 and 8 weeks after anti-TNFa therapy. There was a 50% increase in the percentage of CD4+IL17+ cells after 4 weeks of therapy, and this increase was further enhanced at 8 weeks. No such changes were observed for Th1 cells (Fig. 5A and B). Disease activity score 28-eryrthrocyte sedimentation rate (DAS28-ESR) scores indicate that all patients responded to treatment at 4 weeks and this response was sustained at 8 weeks in 15 out of 17 patients (Table 2). The levels of IL-17 were significantly upregulated after 4 weeks of therapy, with no significant change in IFNy levels (Figure 5C). We also lysed PBMCs from healthy controls and RA patients before and after therapy, and measured RNA levels of the transcription factor for Th17 cells, RORy. Consistent with the increase in Th17 cells, PBMCs from RA patients had a marked up-regulation of RORy, and TNFa blockade induced a further increase in its expression (Figure 5D).

RA (Adalimumb) RA (Etanercept) Healthy

Age (mean ± SD) 50.55 + 12.1 52.75 ± 17.2 48.37 ± 17.1

Sex M=1 F=8 =1 F=7 M=1 F=8

Disease duration in months (mean ± SD) 133.1 1 1 ± 106.794 162.25 ± 179.091 N.A. DMA Ds (n) N.A.

None 1 0

MTX 3 3

SSZ 2 0

HCQ 1 0

MTX+HCQ 1 4

SSZ+HCQ 1 1

DAS28-ESR at baseline (mean ± SD) 5.278 + 1.630 5.433 + 0.864 N.A.

DAS28-ESR 5.1 (n) 5 5 N.A.

3.2 < DAS28-ESR > 5.1 (n) 3 3 N.A.

DAS28-ESR < 3.2 (n) 1 0 N.A.

DAS28-ESR at 4 wk after anti-TNF (mean + SD) 4.225 + 1.352 3.28 ± 0.574 N.A.

DAS28-ESR at 8 wk after anti-TNF (mean ± SD) 4.069 ± 1.371 3.441 ± 1.285 N.A.

Good-responders at 4 wk 9 8 N.A.

Non-responders at 4 wk 0 0 N.A.

Good-responders at 8 wk 7 8 N.A.

Non-responders at 8 wk 2 0 N.A.

RF+ (%) (pre anti-TNF) 47 0

CCP+ (%) (pre anti-TNF) 64 0

Table 2. Clinical parameters of RA study. 17 RA patients and 8 healthy controls were recruited into the study. RA patients, grouped by the type of anti-TNFa drug (adalimumab or etanercept), were assessed for the duration of disease, disease severity and response to treatment on the basis of disease activity score 28-erythrocyte sedimentation rate (DAS28- ESR), and whether patients are also on concurrent disease modifying anti-rheumatic drugs (DMARDs). Abbreviations for DMARDS are as follows: Methotrexate (MTX), sulfasalazine (SSZ), Hydroxychloroquine (HCQ). Of the two non-responders to adalimumab, one patient developed pneumonia and renal failure after 8 weeks of therapy. The other patient was losing response by 8 weeks and finally stopped treatment 12 weeks after the initiation of therapy.

Anti-TNF therapy increases Th17 cells in RA

When anti-TNFa treated patients were divided according to type of TNFa blocking agent, a much more pronounced increase in the number and percentage of Th17 cells was observed in patients receiving adalimumab (a fully humanized monoclonal antibody) compared to those receiving etanercept (a p75 TNFaR-Fc fusion protein) (Fig. 6A and B), although a trend towards increased percentage of Th17 cells was observed in both groups (Figure 6B). Consistent with results from the intracellular staining, the levels of IL-17 in the supernatants of anti-CD3 stimulated PBMCs were significantly higher from patients treated with adalimumab but not etanercept after 8 weeks (Mean IL-17 levels (pg/mL)= 2021 ±1022 versus 563±530, respectively) (Fig. 6C top panels). However, a significant increase in the levels of IFN-γ was also observed with both treatments (Fig. 5C lower panels). The reasons for this are not clear but it is possible that un-physiological stimulation by mitogens followed by intracellular staining fails to detect physiological differences in the production of certain cytokines.

It is possible that the difference in Th17 cell induction between adalimumab and etanercept is a reflection of different pharmacokinetics of the two biologies. It is known for example that etanercept forms relatively unstable complexes with soluble TNFa, resulting in the potential release of biologically active TNFa [7]. To address this question, we compared the ability of sera from adalimumab and etanercept treated patients to neutralize "spiked" TNFa. This experiment clearly showed that after eight weeks of treatment, sera from adalimumab treated patients had greater TNFa neutralizing capacity than sera from etanercept treated patients (Fig. 6D). Circulating levels of IL12/23p40 are restored by adalimumab treatment

We and others have reported that pre-treatment of APCs with TNFa inhibits p40 induction by LPS in vitro [3, 4]. Furthermore, our findings attributed the increase in murine Th17 and Th1 cells, at least in part, to the upregulation of the p40 subunit that is common to IL-23 and IL-12, necessary to support the differentiation and/or survival of Th17 and Th1 cells, respectively. We therefore measured the levels of total p40, as well as IL-23 and IL-12 in RA patients before and after anti-TNFa therapy. Un-purified whole blood from RA patients or healthy controls was stimulated with 500 ng/mL of LPS for 24 hours. The lack of purification ensured that in vivo levels of circulating cytokines or drugs were maintained at the time of stimulation. The levels of p40 following LPS stimulation were below the detectable limit in the plasma of most RA patients prior to anti-TNFa therapy, while all healthy controls secreted modest levels of p40 in response to LPS (Mean=85.88±18.1 pg/mL). Adalimumab treatment was able to restore p40 to the levels to those of healthy controls after 4 weeks, and dramatically increase p40 after 8 weeks of therapy (Fig. 7 top panels). In contrast, etanercept failed to upregulate the levels of p40. IL-23 levels were elevated in RA compared to healthy controls (Fig. 7 bottom panels), and adalimumab, but not etanercept, induced a further and significant increase in IL-23 after 8 weeks of treatment (Fig. 7 bottom panels). The fact that levels of IL-23 as well as numbers of Th17 cells were both increased in RA and that both IL-23 and Th17 cells increased after TNFa blockade are consistent with the role of IL-23 in the maintenance of pathogenic Th17 cells [12, 13]. IL-12 levels were below the limit of detection in the blood of most patients and controls, and LPS addition did not induce IL-12 levels significantly (data not shown). Furthermore, PCR analysis IL-12p35 levels in PBMCs from patients and healthy controls show no difference between the two groups or in patients before or after treatment (data not shown). TNFa blockade may affect other cytokines produced by both T cells and APCs. To address this and confirm the above findings, purified PBMCs were stimulated with LPS for 24 hours and supernatants were assessed for a panel of cytokines (Table 3). In agreement with plasma levels, IL-23 was markedly upregulated after adalimumab treatment. Additionally, IL-1 β and IL- 10 levels appear to be elevated after both therapies, while TNFa blockade did not appreciably vary most of the other cytokines measured.

IL-12^* IL-23-† IL-1b'J IL-6^*† IL-10*+. IL-8^*† IL-2^**† IL-10^**t IL-8^**† IL-5"†

Adalimumab

Pre (n=9) N.D. 70.84+16.64 0.803±4.566 106.598±409 3.260±1.508 17.059±262 308.995+732 1.19210.900 136.366+276 88.358 +157

4wks post (n=8) N.D. 780.7+372.8 3.370±3.583 80.498 ±100 6.075±5.432 95.716+170 43.3811745 0.79010.853 172.04+405 N.D.

8 ks post (n=6) N.D. 544.8±321.5 4.338+4.031 172.882±214 3.40711.756 78 922+232 271.8551349 1.137+2.574 154.1411261 40.88H62

Etanercept

Pre (n=8) N.D. 376.1+331.3 1.776+2.190 325.111+453 1.977±3.738 84.242±53 149.828+106 0.691±0.715 211.333±388 27.926+76

4wks post (n=7) N.D. 87.98±20.30 3.151 +4.308 101.679±73 3.566+6.566 125.413±168 279.522+201 0.76810.374 252.5551310 N.D.

8 ks post (n=6) N.D. 69.7±29.23 2.852±3.160 301.502±97 5.990±6.106 36.545 ±90 327.8291527 1.06710.765 151.4121319 80.559+266

Table 3. Cytokine levels of PBMCs from RA patients before and after anti-TNF therapy. Purified PBMCs were stimulated with 1 ug/mL anti-CD3 mAb or 500ng/mL LPS for 24 hours. Supernatants were collected and cytokine levels were measure using Th1/Th2 cytokine bead multiplex array. Symbols as shown: ^* LPS stimulated, ^** anti-CD3 stimulated,† values in pg/mL, $ values in ng/mL.

The percentage of T cells and myeloid cells in the blood by FACS analysis, as well as the total number of PBMCs in the blood remained fairly consistent before and after treatment, and between the two therapies (Table 4), indicating that the changes observed are not due to a general change in cellular distribution, but may be due to a more specific change in the distribution of pathogenic cells.

PBMC numbers (cells

per rtiL of blood) %CD4+ %CD8+ %CD11 c+ %CD11 b+

Adalimumab

Pre 1.4+0.158 (n=9) 53.5+3.8 (n=8) 16.5±2.7 (n=8) 12.8+3.6 (n=6) 9.0+2.0 (n=4)

4 weeks 1.3+0.104 (n=9) 39.7+3.0 (n=9) 13.911.3 (n=9) 9.5+1.8 (n=8) 4.911.6 (n=4)

8 weeks 1.8+0.358 (n=7) 42.3+2.9 (n=7) 15.011.4 (n=7) 8.9+3.2 (n=7) 12.4+8.4 (n=3)

Etanercept

Pre 1.4+0.222 (n=7) 44.2+5.2 (n=7) 20.413.4 (n=7) 7.9+2.6 (n=7) 8.0+1.0 (n=3)

4 weeks 1.5+0.185 (n=7) 54.1 +5.0 (n=7) 14.813.7 (n=7) 13.2+4.7 (n=7) 16.2+8.1 (n=4)

8 weeks 1.8±0.269 (n=7) 49.6±3.3 (n=7) 16.813.2 (n=7) 9.4+3.5 (n=6) 7.0+1.2 (n=4)

Healthy

1.6±0.346 (n=6) 45.6±4.4 (n=6) 18.011.8 (n=6) 10.5+2.6 (n=6) 5.9+1.2 (n=3)

Table 4. Number of PBMCs and percentage of T cell and APC populations in the blood of RA patients before and after anti-TNF therapy. Numbers represent the mean number or percentage ± SEM. PBMCs were counted after Ficol separation. Purified PBMCs were stained for the various surface receptors for 30 minutes at 4°C and florescence was read using FACS Canto II machine.

We have previously, with others, described that 10% of RA patients treated with infliximab develop anti-dsDNA antibodies [14-16]. We therefore investigated a panel of IgG and IgM autoantibodies associated with rheumatic diseases to examine whether the increase in Th 7 cells was correlated with an increase in autoantibodies. No significant increase was observed in the frequency or level of autoantibodies commonly found in RA (IgG antibodies to CCP, CEP-1 , CEP-11 , IgG or IgM rheumatoid factor) or in autoantibodies found in other autoimmune diseases (IgG antibodies to U1-RNP, Sm, Ro, La, Jo-1 , ssDNA, IgG or IgM dsDNA) (data not shown). Discussion

In summary, this study has shown that Th17 cells are increased in RA compared to healthy controls and that TNFa blockade increases numbers of Th17 cells despite reducing disease activity. This may argue against an important pathological role for Th17 cells in RA but it is also possible that their pathogenicity only reaches full expression in the presence of TNFa. This is supported by the observation that IL-17 induces TNFa production by myeloid cells [17] and has been shown to synergise with TNFa in a number of cellular systems [18-20], Our previous study sheds light on the possibility that TNFa-blockade results in the accumulation of pathogenic Th17 cells in the lymph nodes by redirecting them away from the joints, thus relieving inflammation. The induction of p40 expression and increase in Th17 cells following anti-TNFa therapy is consistent with the findings in CIA but the chief difference between the mouse and human data is that Th1 cells are induced by anti-TNFa in mice with CIA but not patients with RA, despite the increase in IFN-γ levels. Therefore, it was of interest to observe that the expression of IL-23, but not IL-12, was increased by anti-TNFa therapy in the blood of RA patients, a finding consistent with an expansion of Th17 but not Th1 cells.

It is clear that the two TNFa blocking agents result in different functional outcomes. Additional differences between the two drugs include the fact that adalimumab is more effective than etanercept in the elimination of cells bearing membrane-bound TNFa [8] and, unlike etanercept, adalimumab is effective in the treatment of Crohn's disease [9]. Interestingly, we have also observed that serum from anti-TNFa naive patients also displayed TNFa-neutralising properties, albeit modestly (Fig. 6D). This is not surprising as it has been documented that RA patients develop autoantibodies to TNFa [10] . Additionally, TNFa may be neutralised by biologically active levels of soluble TNFaRs, which are upregulated in RA [1 1].

Example 5: Plasma levels of IL-12/IL-23 p40 following LPS stimulation of whole blood from RA patients

We measured plasma levels of IL-12/IL-23 p40 following LPS stimulation of whole blood from RA patients before and after anti-TNFa therapy. The levels of p40 following LPS stimulation were below the limit of detection prior to treatment with adalimumab but increased progressively after 4 and 8 weeks of therapy (Figure 8). We tested the effect of TNF blockade on p40 production following LPS stimulation in vitro. In addition, we studied the effect of increasing TNFR signalling by addition of rTNF. Monocyte-derived DCs were generated from healthy donors and pre-treated with adalimumab or rTNF and stimulated with LPS. Blockade of TNF caused a significant increase in p40 expression whereas rTNF caused a significant decrease in p40 expression (Figure 9).

References to Example 4

1. Murphy, C.A., et al., Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med, (2003) 198(12): p. 1951-7.

2. Feldmann, M. and R.N. Maini, Lasker Clinical Medical Research Award. TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases. Nat Med, (2003) 9(10): p. 1245-50.

3. Notley, C.A., et al., Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and T 17 cells. J Exp Med, (2008) 205(1 1 ): p. 2491 -7.

4. Ma, X., et al., Inhibition of IL-12 production in human monocyte-derived macrophages by TNF. J Immunol, (2000) 164(4): p. 1722-9.

5. Zakharova, M. and H.K. Ziegler, Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells. J Immunol, (2005) 175(8): p. 5024-33.

6. Shen, H., J.C. Goodall, and J.S. Hill Gaston, Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis. Arthritis Rheum, (2009) 60(6): p. 1647-56.

7. Scallon, B., et al., Binding and functional comparisons of two types of tumor necrosis factor antagonists. J Pharmacol Exp Ther, (2002) 301 (2): p. 418-26.

8. Mitoma, H., ef al., Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor alpha-expressing cells: comparison among infliximab, etanercept, and adalimumab. Arthritis Rheum, (2008) 58(5): p. 1248-57.

9. Targan, S.R., et al., A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med, (1997) 337(15): p. 1029-35.

10. Wildbaum, G., M A. Nahir, and N. Karin, Beneficial autoimmunity to proinflammatory mediators restrains the consequences of self-destructive immunity. Immunity, (2003) 19(5): p. 679-88. Cope, A. P. , et al. , Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases. Arthhtis Rheum, (1992) 35(10): p. 1160-9.

Langrish, C.L., et al. , IL-23 dnves a pathogenic T cell population that induces autoimmune inflammation. J Exp Med, (2005) 201 (2): p. 233-40.

McGeachy, M.J., et al., TGF-beta and IL-6 drive the production of IL-17 and 11-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol, (2007) 8(12): p. 1390- 7.

Charles, P. J., et al., Assessment of antibodies to double-stranded DNA induced in rheumatoid arthritis patients following treatment with infliximab, a monoclonal antibody to tumor necrosis factor alpha: findings in open-label and randomized placebo-controlled trials. Arthritis Rheum, (2000) 43(11): p. 2383-90.

Maini, R.N., et al., Immunological intervention reveals reciprocal roles for tumor necrosis factor-alpha and interleukin-10 in rheumatoid arthhtis and systemic lupus erythematosus. Springer Semin Immunopathol, (1994) 16(2-3): p. 327-36.

De Rycke, L, et al., Infliximab, but not etanercept, induces IgM anti-double-stranded DNA autoantibodies as main antinuclear reactivity: biologic and clinical implications in autoimmune arthritis. Arthhtis Rheum, (2005) 52(7): p. 2192-201.

Jovanovic, D.V., et al., IL-17 stimulates the production and expression of proinflammatory cytokines, IL-beta and TNF-alpha, by human macrophages. J Immunol,

(1998) 160(7): p. 3513-21 .

Chabaud, M., G. Page, and P. Miossec, Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines. J Immunol, (2001) 167(10): p. 6015- 20.

Van Bezooijen, R.L., et al., Interleukin 17 synergises with tumour necrosis factor alpha to induce cartilage destruction in vitro. Ann Rheum Dis, (2002) 61 (10): p. 870-6.

Katz, Y., O. Nadiv, and Y. Beer, lnterleukin-17 enhances tumor necrosis factor alpha- induced synthesis of interleukins 1,6, and 8 in skin and synovial fibroblasts: a possible role as a "fine-tuning cytokine" in inflammation processes. Arthhtis Rheum, (2001 ) 44(9): p. 2176-84.

Claims

A method of treating a disease selected from the group consisting of rheumatoid arthritis (RA), Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, the method comprising administering to the patient an anti- TNF agent and an anti-IL23 agent.

The combination of an anti-TNF agent and an anti-IL23 agent for use as a medicament.

The combination of an anti-TNF agent and an anti-IL23 agent for use in treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient.

An anti-TNF agent for use in treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, wherein said patient is administered an anti-ll_23 agent.

An anti-IL23 agent for use in treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, wherein said patient is administered an anti-TNF agent.

Use of the combination of an anti-TNF agent and an anti-IL23 agent in the manufacture of a medicament for the treatment of a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient.

Use of an anti-TNF agent in the manufacture of a medicament for the treatment of a disease consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, wherein the patient is administered an anti-IL23 agent.

8. Use of an anti-IL23 agent in the manufacture of a medicament for the treatment of a diseases selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, wherein the patient is administered an anti-TNF agent.

9. A system for treating a diseases selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, the system comprising an anti-TNF agent and an anti-IL23 agent.

10. A kit of parts for treating a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient, the system comprising an anti-TNF agent and an anti-IL23 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 an anti-TNF receptor 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-IL23 agent is an anti-IL23 antibody or an anti-IL23 receptor antibody or a soluble form of a component of thelL23 receptor.

13. A method according to Claim 1 wherein the anti-TNF agent and the anti-IL23 agent are administered simultaneously.

14. A method according the Claim 1 wherein the anti-TNF agent and the anti-IL23 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 a disease selected from the group consisting of RA, Crohn's disease, ankylosing spondylitis, psoriasis and psoriatic arthritis in a patient as herein described.