**2. The blockade of cytokines inducing inflammatory responses**

#### *TNF-blocking agents (Etanercept, Adalimumab, Infliximab)*

Currently, there are 3 major TNF-blockers available for patients who do not react well to standard therapies like methotrexate or other disease modifying anti-rheumatic drugs (DMARDs), these are: etanercept, infliximab and adalimumab. Most common side effects of anti-TNF therapy are a higher susceptibility for infections and possible flares of TB.

Etanercept, a fusion protein consisting of two extracellular binding domains of the TNF receptor 2 and the Fc-part of a human IgG1 molecule is acting like a soluble decoy receptor by inhibiting ligand-binding to TNF-receptors, only with an extended in vivo half-life due to the presence of the Fc-part. It is licensed by the FDA for the treatment of RA, polyarticular juvenile idiopathic arthritis (JIA), psoritic arthritis, ankylosing spondylitis and plaque psoriasis (1).

Infliximab (Remicade) is a chimeric monoclonal antibody specific for TNF that was approved by the FDA in 1998 for the treatment of Crohn's disease (2). Its use has been extended since then to the treatment of psoriasis, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis and ulcerative colitis.

Adalimumab (Humira), another monoclonal antibody of fully human origin was derived by a phage display library and used to treat RA patients first. Since then, clinical trials proved

Application of Monoclonal Antibody Therapies in Autoimmune Diseases 241

and systemic juvenile idiopathic arthritis. Tocilizumab is a potential candidate drug for the therapy of several other disorders including SLE, Crohn's disease or multiple sclerosis (9).

Like TNF or IL-6, IL-1 and also induce a wide spectrum of biological responses that contribute to fight infections: these include the production of acute phase proteins, raising body temperature (hence the term endogenous pyrogens) or mobilization of neutrophils, thus promoting microbe clearance by phagocytosis. The main source for IL-1 and are macrophages and epithelial cells, whereas IL-1Ra, a naturally occurring IL-1R antagonist is released also by monocytes and hepatocytes (10). The IL-1 receptors CD121a and b are

Recombinant IL-1a (anakinra) is an approved therapeutic drug for RA that mimics the effects of endogenous IL-1Ra, thus blocking the IL-1 binding site on the receptor without inducing any further signaling events. The treatment with anakinra is well tolerated, with less occurring opportunistic infections than in case of TNFblockage, and it was shown to improve joint swelling, pain and inflammation, although with less efficacy (11, 12, 13).

IL-21 is a type I cytokine expressed by activated CD4+ T cells and NKT cells (14), and induces the differentiation and activation of NK cells, promotes NKT proliferation, enhances the differentiation of Th17 cells and was found to regulate mature B cell responses depending on the type of co-stimulation (within a wide range of inducing proliferation to cell death) (15). IL21R-/-mice showed no defects in B cell development, but had severe problems with class-switch to IgG1 and IgG2b, and the down-regulation of the germinal center reaction. As a consequence they experienced a decrease in the number of plasma cells

Although IL-21 fulfills a complex role in the immune regulation, experimental animal models indicate that its targeting could be of therapeutic benefits. In MRL/lpr mice inhibition of IL-21 improved symptoms of the disease, the mice showing a reduction in proteinuria, skin lesions, circulating dsDNA autoantibodies and lymphadenopathy (17). In collagen-induced arthritis and in adjuvant-induced arthritis, the blockade of IL-21R with an IL-21R-Fc fusion protein reversed clinical disease activity, most probably via the downregulation of TNF and production of IL-17 (18). In RA patients, IL-21R is expressed in the synovial macrophages and fibroblasts. In addition, a significantly higher percentage of IL-21R is to be found in the blood and synovial fluid of these individuals, where it might contribute to an increase in TNFand IFN secretion upon T cell activation, thus, in up-

While initially systemic autoimmunity was considered as a T cell-driven condition, multiple functions of B cells have been described in orchestrating autoimmune disorders, including self-reactive antibody production, auto-antigen presentation and co-stimulation of T lymphocytes, formation of ectopic lymphoid structures (neo-organogenesis) in the end-

Due to their central role in the immune pathogenesis of systemic autoimmunity and the observation that patients treated with non-Hodgkin's lymphoma and coexisting RA showed

*Inhibition of the IL-1 mediated responses with a recombinant IL-1R antagonist (anakinra)* 

expressed on different subsets of lymphocytes, monocytes and macrophages.

*Induction of alterations in IL-21 mediated cellular responses* 

and an increase in memory B cells (16).

regulating the pro-inflammatory response (19, 20).

target organs and pro-inflammatory cytokine production.

*CD20-mediated B cell depletion* 

**3. B cell depletion therapies mediated via CD20 and CD22** 

its effectiveness in psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and juvenile idiopathic arthritis (3).

Infliximab and adalimumab were shown to neutralize biological activity of TNF by binding to its soluble, membrane- or receptor-bound forms, while etanercept is unable to neutralize the receptor-bound form of TNF due to its structural features. Additionally, the anti-TNF monoclonal antibodies can induce Fc-receptor-mediated cell lysis and infliximab has been also shown to induce apoptosis of lamina propria T cells in Crohn's patients in a TNF-dependent manner.

In a follow-up comparative study Bacquet-Deschryver et al. evaluated the effects of the 3 different anti-TNF biologics on the re-emerginig of anti-nuclear antibodies (ANA), antidsDNA antibodies, RF and anti-CCP in rheumatoid arthritis and spondyloarthropathy patients (3). They found that the response to treatment is independent of the induction of ANA production and anti-dsDNA autoantibody variations regardless of the rheumatism and the anti-TNF treatment prescribed.

Another study conducted in human TNF transgenic mice showed that in a strictly TNF driven model of RA the number of CD3+CD25+FoxP3+ Treg cells is initially lower than in wild-type counterparts, but gets elevated during the course of the disease. This population of regulatory T cells is attenuated in its suppressor activity, which can be restored with either passive (infliximab treatment) or active (TNF-K immunization) TNF-blocking approaches. Moreover, the differentiation of a CD62L regulatory T cell population is induced (4).

#### *Blockade of IL-6 (tocilizumab)*

IL-6 is a widely expressed pleiotropic cytokine, best known as main mediator of fever and acute phase reactions alongside IL-1 and TNF. In hepatocytes it strongly induces production of acute phase proteins e.g. C-reactive protein, mannan-binding lectin, or serum amyloid protein A, and it also causes immobilization of neutrophil granulocytes from the bone marrow. Besides supporting B cell differentiation into antibody plasma cells, it has been shown to be essential in Th17 cell differentiation as well. The IL-6R consists of two chains, the 80-kDa IL-6-binding subunit and the 130-kDa membrane glycoprotein gp130 that is responsible for signal transduction (5). The expression of membrane bound IL6R is restricted to only few cell types including macrophages, neutrophils, some T-cell subpopulations and hepatocytes. On the other hand, gp130 is ubiquitously expressed. IL-6R is either shed from the cell surface by matrix metalloproteases or in human, expressed as a result of alternative splicing. Association of the IL-6/sIL-6R complex to gp130 mediates agonistic signaling events (trans-signaling) (6).

Excessive levels of the IL-6/IL-6R complex can be detected in the synovial fluid of many RA patients, which could highly contribute to osteoclast-like cell formation and therefore, joint destruction (7). Also, IL-6 production of synovial fibroblasts induces excess production of vascular endothelial growth factor (VEGF) resulting in enhanced angiogenesis and increased vascular permeability of synovial tissue. Serum IL-6 levels were found elevated in other autoimmune conditions e.g. in SLE as well (8).

Tocilizumab is a humanized IL6R-specific monoclonal antibody that blocks IL-6 mediated signal transduction via the inhibition of ligand-binding to the IL-6Rs. Phase III clinical studies showed a remarkable inhibition of radiological damage of joints. It has been approved as a therapeutic drug for the treatment of RA and in Japan for Castelman's disease

its effectiveness in psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and

Infliximab and adalimumab were shown to neutralize biological activity of TNF by binding to its soluble, membrane- or receptor-bound forms, while etanercept is unable to neutralize the receptor-bound form of TNF due to its structural features. Additionally, the anti-TNF monoclonal antibodies can induce Fc-receptor-mediated cell lysis and infliximab has been also shown to induce apoptosis of lamina propria T cells in Crohn's patients in a

In a follow-up comparative study Bacquet-Deschryver et al. evaluated the effects of the 3 different anti-TNF biologics on the re-emerginig of anti-nuclear antibodies (ANA), antidsDNA antibodies, RF and anti-CCP in rheumatoid arthritis and spondyloarthropathy patients (3). They found that the response to treatment is independent of the induction of ANA production and anti-dsDNA autoantibody variations regardless of the rheumatism

Another study conducted in human TNF transgenic mice showed that in a strictly TNF driven model of RA the number of CD3+CD25+FoxP3+ Treg cells is initially lower than in wild-type counterparts, but gets elevated during the course of the disease. This population of regulatory T cells is attenuated in its suppressor activity, which can be restored with either passive (infliximab treatment) or active (TNF-K immunization) TNF-blocking

IL-6 is a widely expressed pleiotropic cytokine, best known as main mediator of fever and acute phase reactions alongside IL-1 and TNF. In hepatocytes it strongly induces production of acute phase proteins e.g. C-reactive protein, mannan-binding lectin, or serum amyloid protein A, and it also causes immobilization of neutrophil granulocytes from the bone marrow. Besides supporting B cell differentiation into antibody plasma cells, it has been shown to be essential in Th17 cell differentiation as well. The IL-6R consists of two chains, the 80-kDa IL-6-binding subunit and the 130-kDa membrane glycoprotein gp130 that is responsible for signal transduction (5). The expression of membrane bound IL6R is restricted to only few cell types including macrophages, neutrophils, some T-cell subpopulations and hepatocytes. On the other hand, gp130 is ubiquitously expressed. IL-6R is either shed from the cell surface by matrix metalloproteases or in human, expressed as a result of alternative splicing. Association of the IL-6/sIL-6R complex to gp130 mediates

Excessive levels of the IL-6/IL-6R complex can be detected in the synovial fluid of many RA patients, which could highly contribute to osteoclast-like cell formation and therefore, joint destruction (7). Also, IL-6 production of synovial fibroblasts induces excess production of vascular endothelial growth factor (VEGF) resulting in enhanced angiogenesis and increased vascular permeability of synovial tissue. Serum IL-6 levels were found elevated in

Tocilizumab is a humanized IL6R-specific monoclonal antibody that blocks IL-6 mediated signal transduction via the inhibition of ligand-binding to the IL-6Rs. Phase III clinical studies showed a remarkable inhibition of radiological damage of joints. It has been approved as a therapeutic drug for the treatment of RA and in Japan for Castelman's disease

regulatory T cell population is

juvenile idiopathic arthritis (3).

TNF-dependent manner.

induced (4).

*Blockade of IL-6 (tocilizumab)* 

and the anti-TNF treatment prescribed.

approaches. Moreover, the differentiation of a CD62L-

agonistic signaling events (trans-signaling) (6).

other autoimmune conditions e.g. in SLE as well (8).

and systemic juvenile idiopathic arthritis. Tocilizumab is a potential candidate drug for the therapy of several other disorders including SLE, Crohn's disease or multiple sclerosis (9).

*Inhibition of the IL-1 mediated responses with a recombinant IL-1R antagonist (anakinra)* 

Like TNF or IL-6, IL-1 and also induce a wide spectrum of biological responses that contribute to fight infections: these include the production of acute phase proteins, raising body temperature (hence the term endogenous pyrogens) or mobilization of neutrophils, thus promoting microbe clearance by phagocytosis. The main source for IL-1 and are macrophages and epithelial cells, whereas IL-1Ra, a naturally occurring IL-1R antagonist is released also by monocytes and hepatocytes (10). The IL-1 receptors CD121a and b are expressed on different subsets of lymphocytes, monocytes and macrophages.

Recombinant IL-1a (anakinra) is an approved therapeutic drug for RA that mimics the effects of endogenous IL-1Ra, thus blocking the IL-1 binding site on the receptor without inducing any further signaling events. The treatment with anakinra is well tolerated, with less occurring opportunistic infections than in case of TNFblockage, and it was shown to improve joint swelling, pain and inflammation, although with less efficacy (11, 12, 13).

#### *Induction of alterations in IL-21 mediated cellular responses*

IL-21 is a type I cytokine expressed by activated CD4+ T cells and NKT cells (14), and induces the differentiation and activation of NK cells, promotes NKT proliferation, enhances the differentiation of Th17 cells and was found to regulate mature B cell responses depending on the type of co-stimulation (within a wide range of inducing proliferation to cell death) (15). IL21R-/-mice showed no defects in B cell development, but had severe problems with class-switch to IgG1 and IgG2b, and the down-regulation of the germinal center reaction. As a consequence they experienced a decrease in the number of plasma cells and an increase in memory B cells (16).

Although IL-21 fulfills a complex role in the immune regulation, experimental animal models indicate that its targeting could be of therapeutic benefits. In MRL/lpr mice inhibition of IL-21 improved symptoms of the disease, the mice showing a reduction in proteinuria, skin lesions, circulating dsDNA autoantibodies and lymphadenopathy (17). In collagen-induced arthritis and in adjuvant-induced arthritis, the blockade of IL-21R with an IL-21R-Fc fusion protein reversed clinical disease activity, most probably via the downregulation of TNF and production of IL-17 (18). In RA patients, IL-21R is expressed in the synovial macrophages and fibroblasts. In addition, a significantly higher percentage of IL-21R is to be found in the blood and synovial fluid of these individuals, where it might contribute to an increase in TNFand IFN secretion upon T cell activation, thus, in upregulating the pro-inflammatory response (19, 20).
