**3. Tumor necrosis factor inhibitors**

#### **3.1. Classification of tumor necrosis factor inhibitors**

There are presently five TNF-α inhibitors available. They are classified based on their mech‐ anism of inhibition of tumor necrosis factor into TNF neutralizing monoclonal antibodies that are capable of neutralizing the soluble as well as the tissue bound TNF and TNF receptor fusion protein that is capable of neutralizing the effects of the soluble TNF in circulation.

#### **3.2. Tumor necrosis factor monoclonal antibodies**

Infliximab: A chimeric human/mouse monoclonal anti-TNF antibody composed of the constant regions of human (Hu) IgG1κ, coupled to the Fv region of a high-affinity neutralizing murine anti-Hu TNF-α antibody. The antibody exhibits high affinity for recombinant and natural hu TNF-α, and neutralizes TNF-mediated cytotoxicity and other functions in vitro. The drug is given at a dose of 3-10mg/ Kg intravenous infusion at 0-2-6 and 8 weeks then every 8 weeks. Because of the potential for an immune reaction to the mouse protein components of a chimeric antibody, an alternate strategy has been to develop a fully human anti-TNF monoclonal antibody. [4]

Adalimumab: The first fully human monoclonal anti-tumor necrosis factor (TNF)-α antibody approved in the year 2002 as a second line anti-tumor necrosis factor for the treatment of refractory rheumatoid arthritis. Such antibody, known as D2E7, also known as adalimumab, was generated by phage display technology. A high affinity murine anti-TNF monoclonal antibody was used as a template for guided selection, which involves complete replacement of the murine heavy and light chains with human counterparts and subsequent optimization of the antigen-binding affinity. Adalimumab is considered a highly specific TNF-a inhibitor. It binds to both soluble and membrane-bound TNF-a. The drug is believed to exert its pharmacological effect by binding to soluble TNF-a preventing its interaction with TNFR1 and TNFR2 cell receptors. The drug is given at a dose of 40mg subcutaneously every 1-2 weeks (initially 80mg loading dose then 40 mg as maintenance therapy). [5]

Golimumab: is a human immunoglobulin (Ig) G1-kappa monoclonal antibody (mAb) that is specific for TNF-alpha. Golimumab binds to both the soluble and transmembrane forms of human TNF-alpha. The drug is given in a dose of 50 mg subcutaneously every 4 weeks. Golimumab is approved for the treatment of rheumatoid arthritis, and seronegative arthropathies. [6]

Certolizumab: Certolizumab is a PEGylated recombinant, humanized antibody Fab' fragment specific for human tumor necrosis factor alpha (TNFα) that is indicated for treatment of moderately to severely active Rheumatoid Arthritis (RA), treatment and maintenance of remission of moderate to severe active Crohn's disease (CD) in adult patients who have an inadequate response to conventional therapy. [7]

The latest two new monoclonal antibody TNF inhibitors, certolizumab and golimumab have been genetically engineered recently aiming to improve affinity and specificity to TNF with better tolerability and less autoimmunity. Neither golimumab nor certolizumab have been tried in patients with vasculitis.

#### **3.3. Tumor necrosis factor receptor block**

The primary immunopathogenic events that initiate the process of vascular inflammation and blood vessel damage remain largely unknown. Granulomatous inflammation involving the vessel itself, the adjacent tissue, or distant sites is a feature of several systemic vasculitic syndromes and tumor necrosis factor is being identified as a major contributor to granulom‐ atous tissue inflammation via its' stimulatory effects on tissue macrophages. The unopposed actions of TNF via its' receptors highly potentiates and sustains inflammatory granuloma formation and integrity. The recognition of the role of this pro-inflammatory cytokine sets it as a potential therapeutic target. Tumor necrosis factor inhibitors have been approved for the

There are presently five TNF-α inhibitors available. They are classified based on their mech‐ anism of inhibition of tumor necrosis factor into TNF neutralizing monoclonal antibodies that are capable of neutralizing the soluble as well as the tissue bound TNF and TNF receptor fusion

Infliximab: A chimeric human/mouse monoclonal anti-TNF antibody composed of the constant regions of human (Hu) IgG1κ, coupled to the Fv region of a high-affinity neutralizing murine anti-Hu TNF-α antibody. The antibody exhibits high affinity for recombinant and natural hu TNF-α, and neutralizes TNF-mediated cytotoxicity and other functions in vitro. The drug is given at a dose of 3-10mg/ Kg intravenous infusion at 0-2-6 and 8 weeks then every 8 weeks. Because of the potential for an immune reaction to the mouse protein components of a chimeric antibody, an alternate strategy has been to develop a fully human anti-TNF

Adalimumab: The first fully human monoclonal anti-tumor necrosis factor (TNF)-α antibody approved in the year 2002 as a second line anti-tumor necrosis factor for the treatment of refractory rheumatoid arthritis. Such antibody, known as D2E7, also known as adalimumab, was generated by phage display technology. A high affinity murine anti-TNF monoclonal antibody was used as a template for guided selection, which involves complete replacement of the murine heavy and light chains with human counterparts and subsequent optimization of the antigen-binding affinity. Adalimumab is considered a highly specific TNF-a inhibitor. It binds to both soluble and membrane-bound TNF-a. The drug is believed to exert its pharmacological effect by binding to soluble TNF-a preventing its interaction with TNFR1 and TNFR2 cell receptors. The drug is given at a dose of 40mg subcutaneously every 1-2 weeks

Golimumab: is a human immunoglobulin (Ig) G1-kappa monoclonal antibody (mAb) that is specific for TNF-alpha. Golimumab binds to both the soluble and transmembrane forms

(initially 80mg loading dose then 40 mg as maintenance therapy). [5]

treatment of rheumatoid arthritis as well as sero-negative spondylo-arthropathies.

protein that is capable of neutralizing the effects of the soluble TNF in circulation.

**3. Tumor necrosis factor inhibitors**

242 Updates in the Diagnosis and Treatment of Vasculitis

**3.1. Classification of tumor necrosis factor inhibitors**

**3.2. Tumor necrosis factor monoclonal antibodies**

monoclonal antibody. [4]

Etanercept: Etanercept: is a dimeric fusion protein composed of two extracellular TNF-receptor domains bound to the Fc portion of human IgG and is injected once or twice weekly at a dose of 50mg. It effectively binds soluble TNF, thereby blocking TNF-receptor activation. [5]

#### **3.4. Tumor necrosis factor inhibitors in vasculitis**

TNF-α is increasingly being implicated in the etio-pathogenesis of several autoimmune diseases, including systemic vasculitis, featuring an interesting therapeutic target. Several case series studies and case reports addressing the role of suppressing tumor necrosis factor in vasculitis have been issued. Such studies have shown that anti TNF therapy might provide a promising therapeutic alternative in the management of refractory systemic vasculitides [8, 9, 10] especially of granulomatous inflammatory nature including; Takayasu's arteritis, probably in giant cell arteritis (GCA) and granulomatous polyangiitis (GPA) amongst other forms of vasculitis.

#### *3.4.1. Takayasu vasculitis — TAK*

Takayasu's arteritis is a rare, chronic, systemic panarteritis of unknown etiology characterized by granulomatous inflammation of the aorta and its major branches (occasionally including the pulmonary arteries as well) with progressive fibrosis and stenosis of the affected vessel wall and, less commonly, aneurysm formation. [11, 12, 13] The disease typically presents in women before the age of 40 years old. Glucocorticoids and methotrexate are the mainstays of treatment.[] Amongst the identified pathogenic targets in Takayasu arteritis displayed in (Figure 2), TNF alpha is the only cytokine under evaluation. There have been several case series [14, 15, 16] and case reports [17, 18, 19] that have shown clinical benefit of TNF inhibition for refractory cases with TAK. The use of TNF blockade therapy was associated with significant improvement in BVAS, successful reduction in the dose of oral corticosteroids and longer glucocorticoid drug free remission in cases with refractory Takayasu arteritis. In a case series study by Hoffman et al. including 15 patients with treatment-resistant TAK, patients were

**Figure 4.** Mode of action of Tumor necrosis factor inhibitors.

treated with either infliximab or etanercept for disease relapse with 93% of patients showing marked improvement with significant reduction in the dose of oral corticosteroids from a median dose of 20mg/day to 0 mg/day and 67% of patients sustaining up to 3 years glucocor‐ ticoid drug free remission. The need for randomized, controlled trials remains necessary to further characterize the effectiveness of TNF inhibition in Takayasu arteritis. [20,21]

The end result is myofibroblastic proliferation, luminal stenosis, and tissue ischemia. Adaptive immune responses in the adventitia are triggered by a population of indigenous dendritic cells (DC) placed at the adventitia-media junction. These arterial DCs have a unique surface receptor profile, including a series of Toll-like receptors (TLR). These adventitial DCs produce chemo‐ kines (TNF alpha, IL-1B), recruit T cells and support their local activation. TNF is one of several

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The standard treatment for GCA is glucocorticoids, methotrexate might be used in some cases as a steroid sparing drug. Up to 60- 80% of treated patients ultimately develop serious adverse effects related to glucocorticoid therapy necessitating an alternative therapy. Tumor necrosis factor alpha inhibitors have been proposed as a therapeutic alternative. Randomized controlled trials studying the efficacy of infliximab in GCS revealed inflixi‐ mab to be non-superior to conventional therapy with insignificant difference in the percentage of patients successfully tapered off glucocorticoids (71-56%). Infliximab therapy was associated with significanlty higher rates of infections. The use of TNF-αI in GCA

cytokines linked to vascular injury in giant cell arteritis.

**Figure 5.** Identified Pathogenic Targets (black arrows) in Takayasu Arteritis.

remains a pit controversial awaiting further studies. [22-27]

#### *3.4.2. Giant Cell Arteritis — GCA*

Giant cell arteritis is a granulomatous vasculitis that affects predominantly large- to mediumsized arteries, including the aorta and its major branches with advancing concentric intimal hyperplasia and subsequent vascular occlusion. Figure 5 Experimental data support the concept that the disease is initiated in the most outer layer of the arterial wall, the adventitia. CD4 T cells are recruited to the adventitia, undergo local activation and subsequently orches‐ trate macrophage differentiation. T cells and macrophages infiltrate into all wall layers and acquire different effector functions dependent on cues in their immediate microenvironment.

**Figure 5.** Identified Pathogenic Targets (black arrows) in Takayasu Arteritis.

treated with either infliximab or etanercept for disease relapse with 93% of patients showing marked improvement with significant reduction in the dose of oral corticosteroids from a median dose of 20mg/day to 0 mg/day and 67% of patients sustaining up to 3 years glucocor‐ ticoid drug free remission. The need for randomized, controlled trials remains necessary to

Giant cell arteritis is a granulomatous vasculitis that affects predominantly large- to mediumsized arteries, including the aorta and its major branches with advancing concentric intimal hyperplasia and subsequent vascular occlusion. Figure 5 Experimental data support the concept that the disease is initiated in the most outer layer of the arterial wall, the adventitia. CD4 T cells are recruited to the adventitia, undergo local activation and subsequently orches‐ trate macrophage differentiation. T cells and macrophages infiltrate into all wall layers and acquire different effector functions dependent on cues in their immediate microenvironment.

further characterize the effectiveness of TNF inhibition in Takayasu arteritis. [20,21]

*3.4.2. Giant Cell Arteritis — GCA*

**Figure 4.** Mode of action of Tumor necrosis factor inhibitors.

244 Updates in the Diagnosis and Treatment of Vasculitis

The end result is myofibroblastic proliferation, luminal stenosis, and tissue ischemia. Adaptive immune responses in the adventitia are triggered by a population of indigenous dendritic cells (DC) placed at the adventitia-media junction. These arterial DCs have a unique surface receptor profile, including a series of Toll-like receptors (TLR). These adventitial DCs produce chemo‐ kines (TNF alpha, IL-1B), recruit T cells and support their local activation. TNF is one of several cytokines linked to vascular injury in giant cell arteritis.

The standard treatment for GCA is glucocorticoids, methotrexate might be used in some cases as a steroid sparing drug. Up to 60- 80% of treated patients ultimately develop serious adverse effects related to glucocorticoid therapy necessitating an alternative therapy. Tumor necrosis factor alpha inhibitors have been proposed as a therapeutic alternative. Randomized controlled trials studying the efficacy of infliximab in GCS revealed inflixi‐ mab to be non-superior to conventional therapy with insignificant difference in the percentage of patients successfully tapered off glucocorticoids (71-56%). Infliximab therapy was associated with significanlty higher rates of infections. The use of TNF-αI in GCA remains a pit controversial awaiting further studies. [22-27]

**Figure 6.** Pathogenesis of Giant Cell Arteritis. [22]

#### *3.4.3. Granulomatosis with polyangiitis GPA-(Wegener's granulomatosis)*

Granulomatosis with polyangiitis (Wegener's Granulomatosis) is a systemic inflammatory disorder characterized predominantly by the presence of a small vessel vasculitis with necrotizing granulomatous inflammation, primarily of the upper and lower respiratory tract. The pathogenetic pathways in ANCA associated vasculitis primarily involves neutrophil activation secondary to an infectious trigger. *S. aureus*-derived products (superantigens and peptidoglycans) stimulate APCs to produce IL-23, which induces proliferation of TH17 cells. IL-17 secretion from TH17 cells activates macrophages that, in turn, produce pro-inflammatory cytokines (IL-1β and TNF), which results in the priming of neutrophils. PR3 produced by neutrophils is processed by APCs, followed by presentation to TH cells. These cells provide help to B cells for the production of PR3-ANCAs, which interact with PR3 on the surface of primed neutrophils that are rolling on the endothelium. These neutrophils become firmly adhesive and produce ROS and NETs that cause necrotizing vasculitis. TH cells also differen‐ tiate into TEM cells that interact with the endothelium and participate in granuloma formation, resulting in granulomatous vasculitis. Figure 7

and release of enzymes including myeloperoxidase (MPO) and proteinase-3 (PR3) (a). Transient immune complexes are formed locally by binding of ANCA to PR3/MPO sticking to endothelial cells. Subsequently, complement is activated, which further promotes neutro‐ phil degranulation. This all adds to the development of necrotizing vasculitis. Whether this specific cascade is also applicable to disease pathogenesis in ANCA-negative AAV patients remains unclear. The expanded effector memory T cells (Tems) are not sufficiently regulated by regulatory T cells (Tregs, b). This virtually leads a state of imbalance between Tregs and Tems, resulting in further release of pro-inflammatory cytokines promoting neutrophil priming (a); moreover, ANCA production is enhanced by further T-cell/B-cell interaction. (c) Expanded circulating Tems migrate into target organs such as the lungs or the kidney. Within tissues, Tems drive granuloma formation, which is considered an 'executioner' of tissue destruction. Granulomas are composed of numerous cell types such as T cells, B cells, giant

**Figure 7.** Identified Pathogenic targets (red arrow heads) contributing to inflammation and tissue damage in anti-

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Randomized controlled trials studying the outcome of tumor necrosis factor inhibitors in ANCA associated vasculitis proved that anti TNF-α therapy was non-superior to conventional therapy in remission induction. The addition of infliximab to standard therapy did not confer

cells, and dendritic cells (DCs) with local ANCA production. [28]

clinical benefit for patients with active AAV [29, 30]

neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

Infection is the identified initial trigger with subsequent priming of neutrophils. Priming of neutrophils leads to a cascade of events including: (a) Up-regulation of adhesion molecules on endothelial cells, and expansion of circulating effector T cells. Primed neutrophils show increased surface expression of ANCA antigens and adhesion molecules. ANCA binding activates the neutrophil in the following ways: [1] enhancing vessel wall adherence and transmigration capacity; [2] production and release of oxygen radicals, and [3] degranulation

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**Figure 7.** Identified Pathogenic targets (red arrow heads) contributing to inflammation and tissue damage in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

*3.4.3. Granulomatosis with polyangiitis GPA-(Wegener's granulomatosis)*

resulting in granulomatous vasculitis. Figure 7

**Figure 6.** Pathogenesis of Giant Cell Arteritis. [22]

246 Updates in the Diagnosis and Treatment of Vasculitis

Granulomatosis with polyangiitis (Wegener's Granulomatosis) is a systemic inflammatory disorder characterized predominantly by the presence of a small vessel vasculitis with necrotizing granulomatous inflammation, primarily of the upper and lower respiratory tract. The pathogenetic pathways in ANCA associated vasculitis primarily involves neutrophil activation secondary to an infectious trigger. *S. aureus*-derived products (superantigens and peptidoglycans) stimulate APCs to produce IL-23, which induces proliferation of TH17 cells. IL-17 secretion from TH17 cells activates macrophages that, in turn, produce pro-inflammatory cytokines (IL-1β and TNF), which results in the priming of neutrophils. PR3 produced by neutrophils is processed by APCs, followed by presentation to TH cells. These cells provide help to B cells for the production of PR3-ANCAs, which interact with PR3 on the surface of primed neutrophils that are rolling on the endothelium. These neutrophils become firmly adhesive and produce ROS and NETs that cause necrotizing vasculitis. TH cells also differen‐ tiate into TEM cells that interact with the endothelium and participate in granuloma formation,

Infection is the identified initial trigger with subsequent priming of neutrophils. Priming of neutrophils leads to a cascade of events including: (a) Up-regulation of adhesion molecules on endothelial cells, and expansion of circulating effector T cells. Primed neutrophils show increased surface expression of ANCA antigens and adhesion molecules. ANCA binding activates the neutrophil in the following ways: [1] enhancing vessel wall adherence and transmigration capacity; [2] production and release of oxygen radicals, and [3] degranulation and release of enzymes including myeloperoxidase (MPO) and proteinase-3 (PR3) (a). Transient immune complexes are formed locally by binding of ANCA to PR3/MPO sticking to endothelial cells. Subsequently, complement is activated, which further promotes neutro‐ phil degranulation. This all adds to the development of necrotizing vasculitis. Whether this specific cascade is also applicable to disease pathogenesis in ANCA-negative AAV patients remains unclear. The expanded effector memory T cells (Tems) are not sufficiently regulated by regulatory T cells (Tregs, b). This virtually leads a state of imbalance between Tregs and Tems, resulting in further release of pro-inflammatory cytokines promoting neutrophil priming (a); moreover, ANCA production is enhanced by further T-cell/B-cell interaction. (c) Expanded circulating Tems migrate into target organs such as the lungs or the kidney. Within tissues, Tems drive granuloma formation, which is considered an 'executioner' of tissue destruction. Granulomas are composed of numerous cell types such as T cells, B cells, giant cells, and dendritic cells (DCs) with local ANCA production. [28]

Randomized controlled trials studying the outcome of tumor necrosis factor inhibitors in ANCA associated vasculitis proved that anti TNF-α therapy was non-superior to conventional therapy in remission induction. The addition of infliximab to standard therapy did not confer clinical benefit for patients with active AAV [29, 30]

The WGET (The Wegener Granulomatosis Etanercept Trial) was a double-blind study that aimed to assess the role of etanercept in the induction and maintenance of remission in 180 patients with GPA. In this trial the use of etanercept showed no impact on the rate of achieving sustained remission compared to placebo (69% for etanercept vs 75% for placebo) with a slight increase in the rate of solid tumors among patients receiving a combination therapy of cyclophosphamide and etanercept. A risk that proved to be insignificant. [31] Another open label phase II prospective study including 14 patients with acute flares of AASV either as first manifestation of disease or relapse demonstrated that the addition of adalimumab to predni‐ solone and cyclophosphamide for the treatment of severe ANCA associated systemic vasculitis (AASV) was associated with response rates and adverse events similar to standard therapy alone but with a reduced prednisolone exposure. [32]

the pathogenesis of such forms of vasculitis. [43] Reports support the benefit of infliximab in the treatment of some cases of deep cutaneous vasculitis as well as in difficult to treat cases who failed to respond to cyclophosphamide pulse therapy with successful tapering of oral corticosteroids [44, 45, 46] Cases of deep cutaneous vasculitis following infliximab therapy

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B lymphocytes are key players in immune mediated vasculitis representing the the humoral arm of the immune response. B cells produce pathogenic autoantibodies and because they have multiple effector functions, including antigen uptake and transport, antigen presentation and costimulation of T cells via membrane associated molecules, production of cytokines and chemokines migration to sites of inflammation. B lymphocytes arise from hematopoietic stem cells in the bone marrow. These cells mature independently of an antigen first into pro-B cells, then into pre-B cells and immature B cells. They subsequently enter the antigen-dependent phase in the peripheral lymphoid tissues, where mature-but-naive B cells, after encountering their antigen in the extrafollicular regions of the lymphoid organs, become activated B cells and migrate to the follicular regions. B lymphocytes then exit the follicular regions to differ‐ entiate into memory B cells, late plasmablasts and plasma cells. Specific markers, such as CD20, CD27, BAFF-R (B-cell-activating factor receptor), CD38 and CD138, identify the transitional

CD20 is a 297-amino acid activated glycosylated trans-membrane phosphoprotein specifically expressed on the surface of B cells, starting at the early pre-B cell stage and persists until the differentiation of B cells into plasma cells. CD-20 is not expressed on hematopoietic stem cells, pro-B cells, or normal plasma cells. Plasma-blasts and stimulated plasma cells may express CD20. CD20 is co-expressed on B cells with CD19, another B cell differentiation marker. CD20 appears to play a crucial role in B cell development, differentiation, proliferation and cell-cycle regulation events. B cell mediated disorders with clonal B cell expansion including lympho‐ mas, leukemias, autoimmune diseases were found to be associated with increasing expression

CD22 is a 135-kDa trans-membrane sialoglycoprotein, a member of the immunoglobulin superfamily. Its expression is restricted to lymphocytes of the B cell lineage and is highly

have also been reported. [47]

**5.1. The role of B cells in vasculitis**

**5. B cell targeted therapy in systemic vasculitis**

phases of B cells from stem cells to plasma cells. (Figure 7) [48, 49, 50]

**5.2. B cells surface target molecules in vasculitis**

of the CD-20 antigen in variable densities. [51, 52, 53, 54]

*5.2.1. CD-20 cell surface molecule*

*5.2.2. CD-22 cell surface molecule*

developmentally regulated.
