**2. Biological agents for the treatment of autoimmune diseases**

#### **2.1 Biological agents for autoimmune therapy in clinics**

The immune response is a highly coordinated process and involves complex interactions of diverse molecules including cytokines and various cell types such as lymphocytes (Figure 1). Dysregulation in immune response such as overproduction of cytokines and aberrant activation of immune cells is implicated in autoimmune disorders. Therefore, these molecules and/or cells involved in immune response have been targeted to develop therapies in autoimmune disorders (Figure 1).

This figure summarizes the cellular interactions in the pathogenesis of RA and the interaction among antigen presenting cells (APCs), T cells, B cells, macrophages, hematopoietic cells (neutrophil, mast cell) and nonhematopoietic cells (fibroblast, connective tissue cell, and bone). These interactions are facilitated by the actions of cytokines released from the activated cells then induce the production of other pro-inflammatory and inflammatory cytokines, which contribute to the pathogenesis of RA. Also, this figure shows therapeutic biological agents proved as a RA treatment (Brennan and McInnes, 2008; McInnes and Schett, 2007).

Fig. 1. An overview of the pathogenesis of rheumatoid arthritis (RA) and the cytokine targets.

Table 1. Summary of biological agents

224 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

current understanding of biological agents for the treatment with autoimmune diseases and then describe our attempt to identify new biomarkers in autoimmune diseases by taking

The immune response is a highly coordinated process and involves complex interactions of diverse molecules including cytokines and various cell types such as lymphocytes (Figure 1). Dysregulation in immune response such as overproduction of cytokines and aberrant activation of immune cells is implicated in autoimmune disorders. Therefore, these molecules and/or cells involved in immune response have been targeted to develop

This figure summarizes the cellular interactions in the pathogenesis of RA and the interaction among antigen presenting cells (APCs), T cells, B cells, macrophages, hematopoietic cells (neutrophil, mast cell) and nonhematopoietic cells (fibroblast, connective tissue cell, and bone). These interactions are facilitated by the actions of cytokines released from the activated cells then induce the production of other pro-inflammatory and inflammatory cytokines, which contribute to the pathogenesis of RA. Also, this figure shows therapeutic biological agents proved as a RA treatment (Brennan and McInnes, 2008;

Fig. 1. An overview of the pathogenesis of rheumatoid arthritis (RA) and the cytokine targets.

**2. Biological agents for the treatment of autoimmune diseases** 

**2.1 Biological agents for autoimmune therapy in clinics** 

advantage of a new proteomic approach.

therapies in autoimmune disorders (Figure 1).

McInnes and Schett, 2007).

Every biological agent used in clinics today has its own specific targets and can be grouped as follows according to its aims: 1) tolerance induction, 2) inhibition of MHC, antigen, and T cell receptor interaction, 3) Inhibition of cellular function and cell-cell interaction, 4) Interference with cytokines, 5) apoptosis (Table 1). Among them, the anticytokine biological

arthritis, RA: Rheumatoid arthritis, sJIA: Systemic-typed JIA, SLE: Systemic lupus erythematosus. (Textbook of Pediatric

Rheumatology, 6th ed. 2011, Saunders)

Application of Novel Quantitative Proteomic Technologies to

mechanism of action and show different effects on patients.

Identify New Serological Biomarkers in Autoimmune Diseases 227

50.4% in the abatacept group versus 19.5% in the placebo group and sustained improvements in ACR responses were achieved after 2 years of threatment with abatacept (Genovese et al., 2005; Rubbert-Roth and Finckh, 2009). In Randomized Evaluation of Long-Term Efficacy of Rituximab in RA (REFLEX) trial, B cell-depleting anti-CD20 antibody, rituximab, was administered to active RA patients with an inadequate response to TNF inhibitor. Among 208 patients treated with rituximab, 51 % of patients achieved an ACR20 response compared to 18 % of patients treated with placebo (Cohen et al., 2006). In addition, the patients treated with rituximab, by themselves, reported clinically meaningful and statistically improvements of

Recently, the Research on Actemra Determining efficacy after Anti-TNF failures (RADIATE) study examined the efficacy and safety of anti-IL-6 receptor (IL-6R) antibody, tocilizumab, in patients with active RA who had failed TNF inhibitor. Especially, 50.0 % of patients treated with tocilizumab at the 8 mg/kg of dose achieved ACR20 as well as rapid and sustained improvement of RA symptoms compared to 10.1 % of patients treated with placebo achieved ACR20 (Emery et al., 2008) (Figure 2). These findings are in accordance with the supposition that biological agents targeting different molecules have distinctive

The second biological agents with other mechanisms with TNF inhibitors such as abatacept, rituximab and tocilizumab were used for patients who failed to initial TNF inhibitors. Bars show percentages of patients achieving a response according to the American College of Rheumatology 20% improvement criteria (ACR20), 50% improvement criteria (ACR50), and 70% improvement criteria (ACR70). The ACR20, ACR50, ACR70 responses in patients treated with abatacept, rituximab and tocilizumab were

\*

Fig. 2. Responsiveness of treatment with the second biological agents in the patients with

significantly higher than patients treated with placebo (p<.001, \*p=.003).

RA refractory to initial TNF inhibitors.

pain, functional disability, and health-related quality of life (Keystone et al., 2008).

agents that suppress the action of proinflammatory cytokines such as TNF, IL-1 and IL-6 are well-known and widely used in clinics. These agents were developed as therapies in RA and recommended for the treatment of patients whose disease does not respond to conventional therapies (Gomez-Reino and Carmona, 2006). RA patients treated with the anticytokine biological agents show dramatic improvement of their clinical symptoms and the levels of inflammatory biomarkers such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Subsequently, these agents have been applied to the treatment of other inflammatory autoimmune diseases and have had a significant impact on patients' prognosis and survival (Andreakos et al., 2002; Efthimiou and Markenson, 2005; Maini et al., 2006; Nishimoto et al., 2009; Yokota et al., 2008).

However, it has been reported that substantial numbers of patients with autoimmune diseases still do not respond to one or more anticytokine biological agents. Among biological agents, TNF inhibitors have been extensively investigated with regard to the frequency of inadequate responders (Launois et al., 2011; Lovell et al., 2008; Maini et al., 2006; Yokota et al., 2008), because anti-TNF antibodies were the first agents approved as the therapy of RA. For example, 20~40 % of patients treated with a TNF inhibitor failed to achieve an improvement of 20 % in American College of Rheumatology criteria (Emery et al., 2008; Rubbert-Roth and Finckh, 2009). More patients lose efficacy during therapy, as shown by a report that 21 % of RA patients initially treated with etanercept no longer receive this therapy after 24 months (Feltelius et al., 2005).

Recently, patients who had an inadequate response or adverse events with one anticytokine agent are often treated with another biologic agent (Gomez-Reino and Carmona, 2006; Hyrich et al., 2007; Karlsson et al., 2008; Rubbert-Roth and Finckh, 2009). In the case of the treatment failure with the first TNF inhibitor, one survey reported that over 94 % of practicing rheumatologists in the United States of America have switched from one TNF inhibitor to another (Yazici et al., 2009). Interestingly, while some surveys reported that the efficiency of second TNF inhibitors is less than that of the first TNF inhibitor (Gomez-Reino and Carmona, 2006; Hyrich et al., 2007; Karlsson et al., 2008; Rubbert-Roth and Finckh, 2009), a large cohort study from the UK revealed that patients who switched their therapy from an initial TNF inhibitor continued to receive the second TNF inhibitor for mean length of 6 months and only 16 % of patients stopped it again due to poor response (Hyrich et al., 2007). This observation indicates that biological agents that share the common target do not always show the same effect on patients. One reason for the inefficacy of the first TNF inhibitor but not of the second one is the development of neutralizing antibody against the first agent, which may not interfere with the action of the second TNF inhibitor. Nevertheless, this observation also raises the possibility that these agents may have their own mode of action. Supporting the latter notion, there are differences in the efficacy between TNF inhibitors depending on diseases (Ackermann and Kavanaugh, 2007; Nash and Florin, 2005; Ramos-Casals et al., 2008; Sfikakis et al., 2007; Triolo et al., 2002; Veres et al., 2007). For example, while anti-TNF antibodies are effective for both RA and Crohn's disease, TNF receptor-Fc fusion protein (TNFR-Fc) is effective for RA but not for Crohn's disease.

The other treatment option after the failure with TNF inhibitors is to switch from TNF inhibitors to other biological agents with different targets. The Abatacept Trial in Treatment of Anti-TNF INadequate responders (ATTAIN) study investigated the effect of abatacept (CTLA4-Ig), an inhibitor of T cell co-stimulatory signal, on patients with active RA and an inadequate response to previous anti-TNF therapy. At 6 months, the ACR20 response rate was

agents that suppress the action of proinflammatory cytokines such as TNF, IL-1 and IL-6 are well-known and widely used in clinics. These agents were developed as therapies in RA and recommended for the treatment of patients whose disease does not respond to conventional therapies (Gomez-Reino and Carmona, 2006). RA patients treated with the anticytokine biological agents show dramatic improvement of their clinical symptoms and the levels of inflammatory biomarkers such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Subsequently, these agents have been applied to the treatment of other inflammatory autoimmune diseases and have had a significant impact on patients' prognosis and survival (Andreakos et al., 2002; Efthimiou and Markenson, 2005; Maini et al.,

However, it has been reported that substantial numbers of patients with autoimmune diseases still do not respond to one or more anticytokine biological agents. Among biological agents, TNF inhibitors have been extensively investigated with regard to the frequency of inadequate responders (Launois et al., 2011; Lovell et al., 2008; Maini et al., 2006; Yokota et al., 2008), because anti-TNF antibodies were the first agents approved as the therapy of RA. For example, 20~40 % of patients treated with a TNF inhibitor failed to achieve an improvement of 20 % in American College of Rheumatology criteria (Emery et al., 2008; Rubbert-Roth and Finckh, 2009). More patients lose efficacy during therapy, as shown by a report that 21 % of RA patients initially treated with etanercept no longer

Recently, patients who had an inadequate response or adverse events with one anticytokine agent are often treated with another biologic agent (Gomez-Reino and Carmona, 2006; Hyrich et al., 2007; Karlsson et al., 2008; Rubbert-Roth and Finckh, 2009). In the case of the treatment failure with the first TNF inhibitor, one survey reported that over 94 % of practicing rheumatologists in the United States of America have switched from one TNF inhibitor to another (Yazici et al., 2009). Interestingly, while some surveys reported that the efficiency of second TNF inhibitors is less than that of the first TNF inhibitor (Gomez-Reino and Carmona, 2006; Hyrich et al., 2007; Karlsson et al., 2008; Rubbert-Roth and Finckh, 2009), a large cohort study from the UK revealed that patients who switched their therapy from an initial TNF inhibitor continued to receive the second TNF inhibitor for mean length of 6 months and only 16 % of patients stopped it again due to poor response (Hyrich et al., 2007). This observation indicates that biological agents that share the common target do not always show the same effect on patients. One reason for the inefficacy of the first TNF inhibitor but not of the second one is the development of neutralizing antibody against the first agent, which may not interfere with the action of the second TNF inhibitor. Nevertheless, this observation also raises the possibility that these agents may have their own mode of action. Supporting the latter notion, there are differences in the efficacy between TNF inhibitors depending on diseases (Ackermann and Kavanaugh, 2007; Nash and Florin, 2005; Ramos-Casals et al., 2008; Sfikakis et al., 2007; Triolo et al., 2002; Veres et al., 2007). For example, while anti-TNF antibodies are effective for both RA and Crohn's disease, TNF receptor-Fc fusion protein (TNFR-Fc) is

The other treatment option after the failure with TNF inhibitors is to switch from TNF inhibitors to other biological agents with different targets. The Abatacept Trial in Treatment of Anti-TNF INadequate responders (ATTAIN) study investigated the effect of abatacept (CTLA4-Ig), an inhibitor of T cell co-stimulatory signal, on patients with active RA and an inadequate response to previous anti-TNF therapy. At 6 months, the ACR20 response rate was

2006; Nishimoto et al., 2009; Yokota et al., 2008).

receive this therapy after 24 months (Feltelius et al., 2005).

effective for RA but not for Crohn's disease.

50.4% in the abatacept group versus 19.5% in the placebo group and sustained improvements in ACR responses were achieved after 2 years of threatment with abatacept (Genovese et al., 2005; Rubbert-Roth and Finckh, 2009). In Randomized Evaluation of Long-Term Efficacy of Rituximab in RA (REFLEX) trial, B cell-depleting anti-CD20 antibody, rituximab, was administered to active RA patients with an inadequate response to TNF inhibitor. Among 208 patients treated with rituximab, 51 % of patients achieved an ACR20 response compared to 18 % of patients treated with placebo (Cohen et al., 2006). In addition, the patients treated with rituximab, by themselves, reported clinically meaningful and statistically improvements of pain, functional disability, and health-related quality of life (Keystone et al., 2008).

Recently, the Research on Actemra Determining efficacy after Anti-TNF failures (RADIATE) study examined the efficacy and safety of anti-IL-6 receptor (IL-6R) antibody, tocilizumab, in patients with active RA who had failed TNF inhibitor. Especially, 50.0 % of patients treated with tocilizumab at the 8 mg/kg of dose achieved ACR20 as well as rapid and sustained improvement of RA symptoms compared to 10.1 % of patients treated with placebo achieved ACR20 (Emery et al., 2008) (Figure 2). These findings are in accordance with the supposition that biological agents targeting different molecules have distinctive mechanism of action and show different effects on patients.

The second biological agents with other mechanisms with TNF inhibitors such as abatacept, rituximab and tocilizumab were used for patients who failed to initial TNF inhibitors. Bars show percentages of patients achieving a response according to the American College of Rheumatology 20% improvement criteria (ACR20), 50% improvement criteria (ACR50), and 70% improvement criteria (ACR70). The ACR20, ACR50, ACR70 responses in patients treated with abatacept, rituximab and tocilizumab were significantly higher than patients treated with placebo (p<.001, \*p=.003).

Fig. 2. Responsiveness of treatment with the second biological agents in the patients with RA refractory to initial TNF inhibitors.

Application of Novel Quantitative Proteomic Technologies to

**3.1 A need for new biomarkers in the era of biological agents** 

biomarkers that predict individual treatment response before therapy.

**3. Biomarkers** 

disease flares.

Identify New Serological Biomarkers in Autoimmune Diseases 229

Given the difference in mechanism and therapeutic effect of each biologic agent, it is desirable to select an effective biological agent on each patient before initiating therapy or after failure of the initial therapy. However, no reliable guidance is available at present for the selection of biological therapies. There is a growing need for the development of

In addition, in patients treated with biological agents in whom immune response is substantially suppressed, conventional laboratory biomarkers such as CRP and ESR do not always reflect disease activity. In particular, since serum CRP is primarily dependent on liver by circulating IL-6, CRP is unable to reflect disease activity in patients treated with IL-6 inhibitors. Moreover, conventional markers may also be inadequate for the detection of inflammation unrelated to original diseases. In RA patients after joint surgery, anti-IL-6R mAb tocilizumab completely suppressed the increase in CRP and partially suppressed the rise in body temperature (Hirao et al., 2009). More importantly, biological agents may mask typical symptoms of bacterial infection and inhibit the elevation of serum biomarkers. Indeed, RA patients treated with tocilizumab did not present characteristic clinical symptoms and typical elevation of serum CRP after bacterial pneumonia and septic shock (Fujiwara et al., 2009). Even without biologic treatment, current inflammatory biomarkers are not useful to distinguish infection from flares of autoimmune diseases. This is an important issue in clinical settings, because therapeutic strategies for infection and disease flares are completely opposite. Infection must be treated primarily with antibiotics and discontinuation of biological agents should be considered. In contrast, disease flares should be treated intensively with the same or alternative biological agents. Thus, new biomarkers are needed for the detection and discrimination of inflammation by either infection or

Even after the successful repression of disease with biologic therapies, it remains unknown yet whether biological agents can be terminated safely without disease recurrence. Therefore, a biomarker that indicates clinical remission or cure of autoimmune diseases is

Collectively, the development of a number of novel biomarkers, such as those that can help to select biological agents before therapy, can precisely evaluate disease activity and therapeutic effect during the therapy or can instruct the timing of therapy completion after achievement of remission, are warranted for the appropriate clinical management of

The pathogenesis of autoimmune diseases involves alterations in the expression of genes that control pathways regulating self tolerance. However, gene transcripts may not faithfully reflect their protein levels. In addition, post-translational modifications are not amenable to the study of transcriptional profiling (Hueber and Robinson, 2006). Recently, there has been the remarkable improvement of the proteomic approaches as represented by the development of sophisticated methods of protein sample preparation and the improvement of the sensitivity, accuracy and resolution in mass spectrometer. Therefore, direct proteomic measurement may provide greater utility for the discovery of new biomarkers monitoring autoimmune diseases in the post genomic era. Current efforts to

helpful to determine the timing to stop biological agents.

**3.2 Serum proteome analysis using the new technology iTRAQ** 

patients receiving biological therapies.

#### **2.2 Biological therapeutic agents tested in animal models of autoimmune disorders**

The analyses on murine disease models have contributed greatly to gain insight into pathogenesis and therapeutic strategy of autoimmune disorders. These models are also useful to clarify the detailed mechanisms of action of biological agents. We have recently investigated several disease models and reveled that anticytokine biological agents have different mechanism of action and show different effects on clinical manifestations of disease models (Fujimoto et al., 2008; Terabe et al., 2011).

We analyzed the effect of two anticytokine agents, anti-IL-6R monoclonal antibody (mAb) and TNFR-Fc, on collagen-induced arthritis (CIA), a murine model of human RA (Fujimoto et al., 2008). In accordance with the pivotal proinflammatory role of IL-6 and TNF in this arthritis model, both agents could inhibit the development of arthritis. However, while anti-IL-6R mAb potently inhibited the differentiation of Th17 cells, a highly inflammatory subset of T helper cells, TNFR-Fc exhibited no effect on Th17 cells. This observation suggests that these two agents have different action points: IL-6 blockade acts on initial phase of adoptive immune response and regulates T helper cell differentiation, whereas TNF inhibitors act much later, presumably at inflamed sites. Our study also suggests that IL-6 inhibitors may be applicable to other Th17-related autoimmune diseases. Indeed, anti-IL-6R mAb suppressed disease in a murine model of multiple sclerosis via the inhibition of Th17 cell differentiation (Serada et al., 2008). The different modes of action in anti-IL-6R mAb and TNF inhibitors may explain the difference in their efficiency in a murine model of uveoretinitis. Anti-IL-6R mAb treatment had a significant protective effect in experimental autoimmune uveoretinitis (EAU) mice, but either TNFR-Fc or anti-TNF mAb treatment did not (Hohki et al., 2010). Interestingly, in the EAU model, anti-IL-6R mAb not only suppressed Th17 cell differentiation but also suppressed autoantigen-specific Th1 cells via the generation of induced regulatory T cells, supporting the notion that IL-6 inhibitors act on initial phase of adoptive immune response (Haruta et al., 2011).

Confusingly, biological agents may act differently on different autoimmune diseases. Indeed, anti-IL-6R mAb and anti-TNF mAb, but not TNFR-Fc exerted similar effect on a murine inflammatory bowel disease (IBD) model (Terabe et al., 2011). This model is a T cell dependent colitis and is induced by the transfer of purified naïve CD4 T cells into lymphopenic mice. Both anti-IL-6R mAb and anti-TNF mAb successfully inhibited colitis, whereas TNFR-Fc did not show any protective effect on colitis. In addition, anti-IL-6R mAb and anti-TNF mAb could comparably inhibit the expansion of colitogenic T cells in this model, although like in other models, anti-IL-6R mAb additionally could modulate the profile of T helper cell differentiation (Terabe et al., 2011). Thus, anti-IL-6R mAb and anti-TNF mAb may share a similar mode of action in the inhibition of IBD. It is also notable that TNFR-Fc failed to inhibit inflammation in this colitis model (Terabe et al., 2011). Similar discrepancy in the effect of anti-TNF mAb and TNFR-Fc has been observed in human IBD. Many mechanisms have been proposed so far to explain the difference of action between these two agents. For example, anti-TNF mAb binds not only to soluble TNF-α, but also to membrane-bound TNF-α, leading to the induction of antibody-dependent and complement dependent cytotoxicity (Maini, 2004). The anti-TNF mAb may also have more capacity than TNFR-Fc to induce apoptosis via reverse signaling with cross-linking by binding firmly to transmembrane TNF(Terabe et al., 2011). Nevertheless, these hypotheses are still controversial and it remains to be explained why anti-TNF mAb and TNFR-Fc have differential effectiveness in some autoimmune diseases such as Crohn's disease. We believe that further study on this murine IBD model is useful for elucidation of this issue.
