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

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

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

Infliximab at a dose of 5mg/kg, showed efficacy in cases with refractory uveo-retinitis with improvement of visual acuity, healing of oro-genital ulcers, healing of intestinal lesions and remission. It has also been disclosed that infliximab is a rapid and effective therapy for sightthreatening panuveitis in Behçet's disease. Infliximab administration thus leads to a rapid and effective suppression of acute ocular inflammation, and the remission of the uveitis remained for as long as 28 days after infliximab administration in all five patients. Etanercept is also now being used in refractory Behçet's disease. [33, 34, 35, 36, 37] Despite that Tumor necrosis factor therapy is not an approved therapeutic alternative in Behcet's syndrome due to lack of randomized controlled studies, yet TNF inhibition might provide a valuable alternative in Behçet's syndrome patients who were proven refractory to more commonly used treatments.

TNF has been suggested as an important cytokine in the active phase of coronary disease in mouse models of Kawasaki disease.However, the current use of anti TNF therapy in Kawasaki

Cutaneous vasculitis, is a disease with an annual incidence rate ranging from 39.6 to 59.8 per million, can be classified as primary or idiopathic; or secondary, when it presents as a mani‐ festation of connective tissue diseases, infections, drug reactions or malignancies. [41, 42] Most of the idiopathic cases are self-limited and responsive to supportive measures (limb elevation, warming, avoid standing) and nonsteroidal anti-inflammatory drugs, potent immunesuppressants are sometimes required for the management of the refractory situations. [42]

The high serum levels of the pro-inflammatory cytokines TNF-*α*, IL-1*β* observed in sera of patients with small vessels cutaneous vasculitis supports a potential role of these cytokines in

remains restricted only to cases with immunoglobulin resistant disease. [38,39]

**4.3. Deep idiopathic small vessels cutaneous vasculitis**

alone but with a reduced prednisolone exposure. [32]

248 Updates in the Diagnosis and Treatment of Vasculitis

**4.1. Behcet's syndrome**

**4.2. Kawasaki disease**

**4. TNF-α inhibitors in other forms of systemic vasculitis**

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

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

#### *5.2.1. CD-20 cell surface molecule*

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

#### *5.2.2. CD-22 cell surface molecule*

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 developmentally regulated.

CD22 is present in the cytoplasm of pro- and pre-B cells and becomes detectable on the cell surface only at mature stages of B cell differentiation. Cell surface expression is lost during terminal differentiation into plasma cell and after B cell activation. CD22 is also expressed by the vast majority of B cell NHLs. The CD22 molecule has multiple ligands because it binds to α2–6-linked sialic acid residues present on glycoproteins expressed by activated T and B cells, monocytes, neutrophils, erythrocytes, and activated endothelial cells. Although its function is not yet well understood, CD22 appears to be involved in the regulation of B cell activation through BCR signaling, (demonstrating both positive and negative roles in vitro) as well as in cell adhesion. In vivo, the important biological functions of this receptor have been demon‐ strated by genetic disruption of CD22. CD22-deficient mice have a shorter life span, a reduced number of mature B cells in the bone marrow and in circulation, and a chronic exaggerated antibody response to antigen and develop elevated levels of autoantibodies, suggesting a key role for CD22 in B cell development, survival, and function. [55-60]

**5.3. B-cell targeted therapy**

*5.3.1. The B-cell depletion therapy*

*5.3.1.1. Rituximab*

54] Figure 9.

the use of B cell depletion therapy in vasculitis.

**Figure 9.** Mechanism of B cell depletion with anti-CD20 therapy [54]

Currently available B cell targeted biologic drugs can be classified into two different mecha‐ nisms, the first mechanism is the B cell depletion therapy, the second mechanism acts via inhibition of B cell maturation. Different trials and case reports showed promising results with

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A chimeric human/mouse IgG1 antibody directed at human CD20, which is found on only pre-B and mature B cells. B-cell depletion with rituximab might be useful for patients with autoimmune diseases driven by autoantibody production. Rituximab (anti-CD-20 therapy) showed efficacy in autoimmune disorders including refractory systemic lupus with nephritis and vasculitis. The types of vasculitis investigated in this regard include cryoglobulinemic vasculitis, ANCA associated vasculitis, cutaneous vasculitis with connective tissue diseases like rheumatoid arthritis, systemic lupus erythematosus and Sjogren's syndrome. [51, 52, 53,

#### **5.3. B-cell targeted therapy**

CD22 is present in the cytoplasm of pro- and pre-B cells and becomes detectable on the cell surface only at mature stages of B cell differentiation. Cell surface expression is lost during terminal differentiation into plasma cell and after B cell activation. CD22 is also expressed by the vast majority of B cell NHLs. The CD22 molecule has multiple ligands because it binds to α2–6-linked sialic acid residues present on glycoproteins expressed by activated T and B cells, monocytes, neutrophils, erythrocytes, and activated endothelial cells. Although its function is not yet well understood, CD22 appears to be involved in the regulation of B cell activation through BCR signaling, (demonstrating both positive and negative roles in vitro) as well as in cell adhesion. In vivo, the important biological functions of this receptor have been demon‐ strated by genetic disruption of CD22. CD22-deficient mice have a shorter life span, a reduced number of mature B cells in the bone marrow and in circulation, and a chronic exaggerated antibody response to antigen and develop elevated levels of autoantibodies, suggesting a key

role for CD22 in B cell development, survival, and function. [55-60]

**Bone marrow Antigen Independent**

250 Updates in the Diagnosis and Treatment of Vasculitis

**Pro‐B cells**

**Serum BAFF and CD38**

**Pre‐B cells**

**Immature B cells**

**Mature B cells**

**Cell Surface Antigens**

**Figure 8.** Surface molecules and receptors involved in B cell Maturation, the black bullet refers to identified therapeu‐

**Short lived Plasma cells**

> **Active B cells**

**Memory B cells**

**Peripheral Lymphoid tissue Antigen Dependent Phase**

> **Late plasma‐ blast cells**

**Plasma cells**

**Early Plasma‐blast CD20 CD27 CD40L**

**Stem cells**

**CD19 CD20 CD27**

**CD138**

tic targets [51]

Currently available B cell targeted biologic drugs can be classified into two different mecha‐ nisms, the first mechanism is the B cell depletion therapy, the second mechanism acts via inhibition of B cell maturation. Different trials and case reports showed promising results with the use of B cell depletion therapy in vasculitis.

#### *5.3.1. The B-cell depletion therapy*

#### *5.3.1.1. Rituximab*

A chimeric human/mouse IgG1 antibody directed at human CD20, which is found on only pre-B and mature B cells. B-cell depletion with rituximab might be useful for patients with autoimmune diseases driven by autoantibody production. Rituximab (anti-CD-20 therapy) showed efficacy in autoimmune disorders including refractory systemic lupus with nephritis and vasculitis. The types of vasculitis investigated in this regard include cryoglobulinemic vasculitis, ANCA associated vasculitis, cutaneous vasculitis with connective tissue diseases like rheumatoid arthritis, systemic lupus erythematosus and Sjogren's syndrome. [51, 52, 53, 54] Figure 9.

**Figure 9.** Mechanism of B cell depletion with anti-CD20 therapy [54]

#### *5.3.1.2. Epratuzumab*

Epratuzumab is a humanized monoclonal antibody targeting CD22 receptors on B lympho‐ cytes. Two multicenter, placebo-controlled, randomized, double-blind studies (EMBODY™ 1 and EMBODY™ 2), designed to evaluate the efficacy, safety, tolerability, and immunogenicity of Epratuzumab in patients with moderate to severe SLE, are being designed and processed, each will enroll 780 subjects and will last a maximum of 54 weeks. Ongoing experimental studies addressing the role of anti-CD22 in ANCA vasculitis are being run with promising results, whilst randomized controlled trials are still lacking. [61-65]

genotype 1, and interferon induced many side effects. On the other hand, treatments such as cyclophosphamide and plasmapheresis are generally reserved for life- or organ-threatening

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Rituximab showed efficacy in cases of HCV-mixed cryoglobulinemia. The rationale behind anti-B-cell therapy in mixed cryoglobulinemic vasculitis includes the presence immune complex deposition as the cause of symptoms in hepatitis C virus extra-hepatic syndrome, in which chronic stimulation by hepatitis C virus induces the production of cryoglobulins by infected B cells. [75, 76] Rituximab was successfully used in combination with antiviral agents as well as mono-therapy in HCV cryoglobulinemic vasculitis. Rituximab combined with Peg-IFN-α/ribavirin delete both virus-dependent and -independent B-cell clones. Antiviral therapy alone decreased the memory B cells; whereas in association with rituximab, naive B cells are the main depleted population. The observable delay in B-cell reconstitution after rituximab plus Peg-IFN-α/ribavirin stresses the synergistic action of rituximab and antiviral therapy at the immunologic level. Rituximab shortens the therapeutic interval required for achieving a complete clinical response. Clonal expansion of marginal zone–like IgM+ CD27+ B cells (VH1-69 clonal B) has been recently observed in certain HCV-MC patients. Rituximab with Peg-IFN and ribavirin exerts a synergistic effect on polyclonal B lymphocyte expansion. Rituximab plus Peg-IFN-α/ribavirin was more efficient to suppress both memory and VH1-69 clonal B cells compared with Peg-IFN-α/ribavirin alone. A standard therapeutic dose of 375 mg/m2 weekly for 4 weeks is effective, well tolerated and induces a significant and rapid improvement of clinical signs (purpura, arthralgia, peripheral neuropathy) with a decline of cryocrit in most patients with mixed cryoglobulinemia even in cases resistant to IFN therapy. Rituximab trials emphasized the benefit of the drug in inducing remission in cutaneous vasculitis, cryoglobulinemic glomerulonephritis, cryoglobulinemic neuropathy and in

Complete clinical remission was associated with a significant reduction of RF activity and anti-HCV antibody titers. Relapse might occur in up to 36.1%.Complete immunologic response was higher with the combination of rituximab plus Peg-IFN-α/ribavirin. Rituximab treatment of a renal-transplant patient with de novo HCV-related type III cryoglobulinemic MPGN resulted in clearance of cryoglobulinemia, a decrease in proteinuria without a change in serum creatinine or HCV RNA. [69, 77, 78, 79] Rituximab can cause serum sickness, serum sickness like disease, neutropenia and increased risk of infections, pneumopathy, varicella zoster infection, erysipelas, thrombosis of the retinal artery and cold agglutinin disease, occasional flare of vasculitis. Factors that were found to be associated with increased risk of side effects include high complement activation, higher rituximab doses and elevated levels of cryoglo‐ bulins. An increase of viremia might be observed in responders with rituximab monotherapy with insignificant variation of transaminases or deterioration of liver disease. [80, 81] Table 1

Standard treatment of ANCA-associated vasculitic syndromes (AAVS) is composed of remission induction regimen that involves the use of cyclophosphamide and high-dose glucocorticoids, followed by a remission maintenance regimen using methotrexate, azathio‐

underlying malignant lymphoproliferative disorder. [74, 76, 77, 78]

*5.3.2.2. ANCA-Associated Systemic Vasculitis (AASV)*

disease.

#### *5.3.2. The role of B cells targeted therapy in systemic vasculitis*

#### *5.3.2.1. Cryoglobulinemic vasculitis*

Mixed cryoglobulinemia (MC) is a chronic immune complex (IC) mediated systemic small vessel vasculitis, characterized by immune complex deposits (cryo-deposits) and frequent visceral involvement. A frequent synonym of this disease is "cryoglobulinemic vasculitis". [66] The term cryoglobulinemia refers to the presence of a single or monoclonal immunoglobulin Ig or more polyclonal immunoglobulins. Cryoglobulins are classified on the basis of their Ig components. These cryoglobulins precipitate at temperatures below 37°C giving rise to high molecular weight aggregates and re-dissolve on rewarming. Cryoglobulins are found in small quantities in normal serum and are present in variable concentrations in many pathological conditions, including myeloproliferative disorders, autoimmune disorders and several infectious diseases. They are classified into: Type I cryoglobulins (10-15% of cryoglobulins) is comprised simply of monoclonal immunoglobulins, typically IgM but less frequently IgG, IgA, or serum light chains. Individuals with Type I cryos typically have a paraproteinemia (e.g., myeloma, Waldenstrom's macroglobulinemia). Type II cryoglobulins (50-60% of cryoglobu‐ lins) occurs when a monoclonal Ig M recognizes and binds to polyclonal IgG's, accordingly, type II cryos are typically IgM-IgG complexes.Type III cryoglobulins (30-40% of cryoglobulins) are composed of polyclonal Ig M that binds to polyclonal IgG. Type II & III cryoglobulinemia are referred to as "Mixed Cryoglobulinemia", these two types are most commonly associated with hepatitis C virus infection. [67]. The term essential cryoglobulinemia was used to describe cryoglobulinemia without identifiable underlying disease, currently it is clear that most of the patients with essential mixed cryoglobulinemia are chronically infected with HCV [68] The classic pathology in cryoglobulinemic vasculitis is leukocytoclastic vasculitis. The disease presents by Meltzer triad which is a triad of purpura, weakness, arthralgia and/or arthritis in 25-30% of cases, thereafter, a series of multisystem pathologies follow involving peripheral nervous system and the kidneys. Widespread vasculitis involving medium-small sized arteries, capillaries and venules with multiple organ involvement may develop in a small proportion of patients [69-72].

The incidence of hepatitis C infection in mixed cryoglobulinemia ranges from 40 to 90%. [69, 73, 74] The hepatitis C virus infects B cells, resulting in clonal expansion and stimulation of autoantibody production. Eradication of hepatitis C with interferon and ribavirin has been tried, but is often ineffective for controlling extra-hepatic disease,[ <sup>45</sup> ] particularly with genotype 1, and interferon induced many side effects. On the other hand, treatments such as cyclophosphamide and plasmapheresis are generally reserved for life- or organ-threatening disease.

*5.3.1.2. Epratuzumab*

252 Updates in the Diagnosis and Treatment of Vasculitis

Epratuzumab is a humanized monoclonal antibody targeting CD22 receptors on B lympho‐ cytes. Two multicenter, placebo-controlled, randomized, double-blind studies (EMBODY™ 1 and EMBODY™ 2), designed to evaluate the efficacy, safety, tolerability, and immunogenicity of Epratuzumab in patients with moderate to severe SLE, are being designed and processed, each will enroll 780 subjects and will last a maximum of 54 weeks. Ongoing experimental studies addressing the role of anti-CD22 in ANCA vasculitis are being run with promising

Mixed cryoglobulinemia (MC) is a chronic immune complex (IC) mediated systemic small vessel vasculitis, characterized by immune complex deposits (cryo-deposits) and frequent visceral involvement. A frequent synonym of this disease is "cryoglobulinemic vasculitis". [66] The term cryoglobulinemia refers to the presence of a single or monoclonal immunoglobulin Ig or more polyclonal immunoglobulins. Cryoglobulins are classified on the basis of their Ig components. These cryoglobulins precipitate at temperatures below 37°C giving rise to high molecular weight aggregates and re-dissolve on rewarming. Cryoglobulins are found in small quantities in normal serum and are present in variable concentrations in many pathological conditions, including myeloproliferative disorders, autoimmune disorders and several infectious diseases. They are classified into: Type I cryoglobulins (10-15% of cryoglobulins) is comprised simply of monoclonal immunoglobulins, typically IgM but less frequently IgG, IgA, or serum light chains. Individuals with Type I cryos typically have a paraproteinemia (e.g., myeloma, Waldenstrom's macroglobulinemia). Type II cryoglobulins (50-60% of cryoglobu‐ lins) occurs when a monoclonal Ig M recognizes and binds to polyclonal IgG's, accordingly, type II cryos are typically IgM-IgG complexes.Type III cryoglobulins (30-40% of cryoglobulins) are composed of polyclonal Ig M that binds to polyclonal IgG. Type II & III cryoglobulinemia are referred to as "Mixed Cryoglobulinemia", these two types are most commonly associated with hepatitis C virus infection. [67]. The term essential cryoglobulinemia was used to describe cryoglobulinemia without identifiable underlying disease, currently it is clear that most of the patients with essential mixed cryoglobulinemia are chronically infected with HCV [68] The classic pathology in cryoglobulinemic vasculitis is leukocytoclastic vasculitis. The disease presents by Meltzer triad which is a triad of purpura, weakness, arthralgia and/or arthritis in 25-30% of cases, thereafter, a series of multisystem pathologies follow involving peripheral nervous system and the kidneys. Widespread vasculitis involving medium-small sized arteries, capillaries and venules with multiple organ involvement may develop in a small

The incidence of hepatitis C infection in mixed cryoglobulinemia ranges from 40 to 90%. [69, 73, 74] The hepatitis C virus infects B cells, resulting in clonal expansion and stimulation of autoantibody production. Eradication of hepatitis C with interferon and ribavirin has been

particularly with

tried, but is often ineffective for controlling extra-hepatic disease,[ <sup>45</sup> ]

results, whilst randomized controlled trials are still lacking. [61-65]

*5.3.2. The role of B cells targeted therapy in systemic vasculitis*

*5.3.2.1. Cryoglobulinemic vasculitis*

proportion of patients [69-72].

Rituximab showed efficacy in cases of HCV-mixed cryoglobulinemia. The rationale behind anti-B-cell therapy in mixed cryoglobulinemic vasculitis includes the presence immune complex deposition as the cause of symptoms in hepatitis C virus extra-hepatic syndrome, in which chronic stimulation by hepatitis C virus induces the production of cryoglobulins by infected B cells. [75, 76] Rituximab was successfully used in combination with antiviral agents as well as mono-therapy in HCV cryoglobulinemic vasculitis. Rituximab combined with Peg-IFN-α/ribavirin delete both virus-dependent and -independent B-cell clones. Antiviral therapy alone decreased the memory B cells; whereas in association with rituximab, naive B cells are the main depleted population. The observable delay in B-cell reconstitution after rituximab plus Peg-IFN-α/ribavirin stresses the synergistic action of rituximab and antiviral therapy at the immunologic level. Rituximab shortens the therapeutic interval required for achieving a complete clinical response. Clonal expansion of marginal zone–like IgM+ CD27+ B cells (VH1-69 clonal B) has been recently observed in certain HCV-MC patients. Rituximab with Peg-IFN and ribavirin exerts a synergistic effect on polyclonal B lymphocyte expansion. Rituximab plus Peg-IFN-α/ribavirin was more efficient to suppress both memory and VH1-69 clonal B cells compared with Peg-IFN-α/ribavirin alone. A standard therapeutic dose of 375 mg/m2 weekly for 4 weeks is effective, well tolerated and induces a significant and rapid improvement of clinical signs (purpura, arthralgia, peripheral neuropathy) with a decline of cryocrit in most patients with mixed cryoglobulinemia even in cases resistant to IFN therapy. Rituximab trials emphasized the benefit of the drug in inducing remission in cutaneous vasculitis, cryoglobulinemic glomerulonephritis, cryoglobulinemic neuropathy and in underlying malignant lymphoproliferative disorder. [74, 76, 77, 78]

Complete clinical remission was associated with a significant reduction of RF activity and anti-HCV antibody titers. Relapse might occur in up to 36.1%.Complete immunologic response was higher with the combination of rituximab plus Peg-IFN-α/ribavirin. Rituximab treatment of a renal-transplant patient with de novo HCV-related type III cryoglobulinemic MPGN resulted in clearance of cryoglobulinemia, a decrease in proteinuria without a change in serum creatinine or HCV RNA. [69, 77, 78, 79] Rituximab can cause serum sickness, serum sickness like disease, neutropenia and increased risk of infections, pneumopathy, varicella zoster infection, erysipelas, thrombosis of the retinal artery and cold agglutinin disease, occasional flare of vasculitis. Factors that were found to be associated with increased risk of side effects include high complement activation, higher rituximab doses and elevated levels of cryoglo‐ bulins. An increase of viremia might be observed in responders with rituximab monotherapy with insignificant variation of transaminases or deterioration of liver disease. [80, 81] Table 1

#### *5.3.2.2. ANCA-Associated Systemic Vasculitis (AASV)*

Standard treatment of ANCA-associated vasculitic syndromes (AAVS) is composed of remission induction regimen that involves the use of cyclophosphamide and high-dose glucocorticoids, followed by a remission maintenance regimen using methotrexate, azathio‐


studies demonstrated successful treatment of the ANCA-associated vasculitides with rituxi‐ mab. [85-88] These early successes led to two landmark studies that support the use of B-cell therapies for these diseases. The efficacy of rituximab in ANCA associated vasculitis was further studied in 2010 in two randomized controlled trials. Both trials examined the use of

A multicentered randomized, double-blind, non-inferiority study of patients with severe GPA and microscopic polyangiitis. In this trial, 197 patients with either new or relapsing disease were randomized in a 1:1 ratio to receive remission-induction therapy with either oral

received the same glucocorticoid regimen (i.e., up to 3 pulses of 1 g of intravenous methyl‐ prednisolone, and then prednisone 1 mg/kg/day, followed by a protocolized taper). Approx‐ imately 50% of subjects enrolled in this trial had significant renal disease and 28% had alveolar hemorrhage at trial entry, although patients were not eligible for this trial if their serum creatinine exceeded 4 mg/dl or if the patient required mechanical ventilation during the study period. The primary end point of this study was achievement of remission at 6 months in the

The RAVE trial showed that rituximab was non-inferior to cyclophosphamide in inducing remission especially in cases with alveolar haemorrhage and/or glomerulonephritis. Rituxi‐ mab was proven superior to cyclophosphamide in the treatment of relapsing disease in AASV. Rituximab therapy was associated with sustained remission over a period of 6 months with encouragingsteroidsparingeffect.Ingeneral,the frequencyof adverse eventswas the same and correlated significantly with the early use of high doses of glucocorticoids in both groups.

*5.3.2.2.2. The Randomized Trial of Rituximab versus Cyclophosphamide in ANCA-Associated*

A randomized open-label study that looked at the effectiveness of rituximab for the treatment of 44 patients with newly diagnosed ANCA-associated glomerulonephritis. Subjects were randomized in a 3:1 ratio, stratified by age, diagnosis and baseline renal function. The rituximab group (33 patients) received standard dosing of rituximab (375 mg/m2 weekly for 4 doses) as well as two intravenous cyclophosphamide pulses (15 mg/kg) with the first and third rituximab doses. A third dose of cyclophosphamide was permitted if remission had not been achieved after 6 months of therapy. No maintenance therapy was given in this group. The control group (11 patients) received intravenous cyclophosphamide monthly for 3–6 months followed by azathioprine for remission maintenance. Some subjects also underwent plasma‐ pheresis. Remission was defined as the absence of disease activity for 2 months and relapse was defined as any disease activity after remission had been attained. In this trial, 76% of patients in the rituximab group had a sustained remission at 12 months as opposed to 82% in the cyclophosphamide group. Severe adverse effects were noted 42% of the rituximab patients and 36% of patients in the control group. A total of eight patients died, six of whom had been

weekly for 4 weeks). Both groups

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rituximab for patients with ANCA-associated vasculitis.

cyclophosphamide (2 mg/kg/day) or rituximab (375 mg/m2

absence of glucocorticoids.

*Vacsulitis (RITUXVAS) Trial*

*5.3.2.2.1. The Rituximab for ANCA-associated Vasculitis (RAVE) trial*

**Table 1.** Rituximab treatment in Patients with HCV cryoglobulinemic vascultis.CNI = Calcineurin inhibitor; HCV = Hepatitis C virus; HSV = Herpes simplex virus; MMF = Mycophenolate mofetil; NR = Not reported; S = Steroids.↑: Increase; ↓: Decrease; =: No change. [69]

prine or other antimetabolite therapy. Patients who develop undetectable ANCA titers after treatment are less likely to experience disease relapse than patients who remain ANCA positive. Conventional immunosuppression led to a dramatic improvement in the prognosis of patients with increasing remission reaching up to 70%. Conventional immunosuppression might fail to achieve remission in a substantial minority of patients (25-30%). [82] With the tumor necrosis factor inhibition therapy clearly proven to be non-superior to conventional lines of therapy alternative biologic targets are being extensively investigated in AAVS. [31]

B-cell activation is believed to play an important role in the pathogenesis of the ANCAassociated vasculitis concerning antigen presentation, activation of T cell differentiation to Th1 and Th17 cell types and activating TNF-primed neutrophils, leading to premature degranu‐ lation and resultant endothelial damage. [83] Given the role of B cells in the pathogenesis of this disease, B cell depletion and interruption of B cell dependent T cell regulator function with rituximab represents a potentially attractive treatment alternative. [84] Open label and cohort studies demonstrated successful treatment of the ANCA-associated vasculitides with rituxi‐ mab. [85-88] These early successes led to two landmark studies that support the use of B-cell therapies for these diseases. The efficacy of rituximab in ANCA associated vasculitis was further studied in 2010 in two randomized controlled trials. Both trials examined the use of rituximab for patients with ANCA-associated vasculitis.

#### *5.3.2.2.1. The Rituximab for ANCA-associated Vasculitis (RAVE) trial*

A multicentered randomized, double-blind, non-inferiority study of patients with severe GPA and microscopic polyangiitis. In this trial, 197 patients with either new or relapsing disease were randomized in a 1:1 ratio to receive remission-induction therapy with either oral cyclophosphamide (2 mg/kg/day) or rituximab (375 mg/m2 weekly for 4 weeks). Both groups received the same glucocorticoid regimen (i.e., up to 3 pulses of 1 g of intravenous methyl‐ prednisolone, and then prednisone 1 mg/kg/day, followed by a protocolized taper). Approx‐ imately 50% of subjects enrolled in this trial had significant renal disease and 28% had alveolar hemorrhage at trial entry, although patients were not eligible for this trial if their serum creatinine exceeded 4 mg/dl or if the patient required mechanical ventilation during the study period. The primary end point of this study was achievement of remission at 6 months in the absence of glucocorticoids.

The RAVE trial showed that rituximab was non-inferior to cyclophosphamide in inducing remission especially in cases with alveolar haemorrhage and/or glomerulonephritis. Rituxi‐ mab was proven superior to cyclophosphamide in the treatment of relapsing disease in AASV. Rituximab therapy was associated with sustained remission over a period of 6 months with encouragingsteroidsparingeffect.Ingeneral,the frequencyof adverse eventswas the same and correlated significantly with the early use of high doses of glucocorticoids in both groups.

#### *5.3.2.2.2. The Randomized Trial of Rituximab versus Cyclophosphamide in ANCA-Associated Vacsulitis (RITUXVAS) Trial*

prine or other antimetabolite therapy. Patients who develop undetectable ANCA titers after treatment are less likely to experience disease relapse than patients who remain ANCA positive. Conventional immunosuppression led to a dramatic improvement in the prognosis of patients with increasing remission reaching up to 70%. Conventional immunosuppression might fail to achieve remission in a substantial minority of patients (25-30%). [82] With the tumor necrosis factor inhibition therapy clearly proven to be non-superior to conventional lines of therapy alternative biologic targets are being extensively investigated in AAVS. [31] B-cell activation is believed to play an important role in the pathogenesis of the ANCAassociated vasculitis concerning antigen presentation, activation of T cell differentiation to Th1 and Th17 cell types and activating TNF-primed neutrophils, leading to premature degranu‐ lation and resultant endothelial damage. [83] Given the role of B cells in the pathogenesis of this disease, B cell depletion and interruption of B cell dependent T cell regulator function with rituximab represents a potentially attractive treatment alternative. [84] Open label and cohort

**Study**

Sansonno *et al. 2003*

Zaja *et al.* 2003

Roccatello *et al.* ,2004

Quartuccio *et al.,* 2006

Basse *et al.* 2005

Visentini *et al.* 2007

**Patients (number with nephritis)**

254 Updates in the Diagnosis and Treatment of Vasculitis

20 (1)

15 (2)

6 (5)

5 (5)

7 (7) (postkidney transplant)

6 (2)

Increase; ↓: Decrease; =: No change. [69]

**Rituximab dose**

375 mg/m2 weekly × 4 weeks

375 mg/m2 weekly × 4 weeks

375 mg/m2 weekly × 4 weeks; 375 mg/m2 monthly × 2 months

375 mg/m2 weekly × 4 weeks

375 mg/m2 weekly × 2–4 weeks

250 mg/m2 weekly × 2 weeks

**Other treatments**

S (low doses)

S (<0.5 mg/ kg/day)

S (one case)

CNI, MMF and S

**Remission overall (nephritis)**

> 16/20 (1/1)

> 13/14 (1/2)

> > 5/5

5/5 (5/5)

7/7

S 4/6 (1/2) 4/5 (2/2)

**Table 1.** Rituximab treatment in Patients with HCV cryoglobulinemic vascultis.CNI = Calcineurin inhibitor; HCV = Hepatitis C virus; HSV = Herpes simplex virus; MMF = Mycophenolate mofetil; NR = Not reported; S = Steroids.↑:

**Remission Purpura Neuropathy**

> 12/14 (6/12)

12/12 (5/5)

4/4 (5/6)

4/4 (1/2) **Side effects HCV viral load**

↑ responders = nonresponders

> ↑ 2/8 ↑ 1/8 = 5/8

4 unchanged

NR

↓ 2/5 = 3/5

NR NR

Septic fever (1)

Retinal thrombosis (1)

Transient bradycardia (2)

Transient neutropenia (1)

Lethal infection (2, fungal and HSV)

Lethal intestinal infarction

**Relapse (number of cases)**

> 4/16 (>7 months)

6 (3–6 months)

2 (>12 months)

3 (>5, >7 and >12 months)

NR

A randomized open-label study that looked at the effectiveness of rituximab for the treatment of 44 patients with newly diagnosed ANCA-associated glomerulonephritis. Subjects were randomized in a 3:1 ratio, stratified by age, diagnosis and baseline renal function. The rituximab group (33 patients) received standard dosing of rituximab (375 mg/m2 weekly for 4 doses) as well as two intravenous cyclophosphamide pulses (15 mg/kg) with the first and third rituximab doses. A third dose of cyclophosphamide was permitted if remission had not been achieved after 6 months of therapy. No maintenance therapy was given in this group. The control group (11 patients) received intravenous cyclophosphamide monthly for 3–6 months followed by azathioprine for remission maintenance. Some subjects also underwent plasma‐ pheresis. Remission was defined as the absence of disease activity for 2 months and relapse was defined as any disease activity after remission had been attained. In this trial, 76% of patients in the rituximab group had a sustained remission at 12 months as opposed to 82% in the cyclophosphamide group. Severe adverse effects were noted 42% of the rituximab patients and 36% of patients in the control group. A total of eight patients died, six of whom had been randomized to receive rituximab therapy. Adverse events were not lower in the rituximab group as had been expected. [90]


Abbrevaitions: Antineutrophil cytoplasmic autoantibodies; RAVE: Rituximab for ANCA-associated vasculitis; RITUXVAS: Rituximab versus cyclophosphamide in ANCA-associated vasculitis. [55,56]

> of B cells to BCR activation. TACI and BCMA signal through the classic NF-κB pathway and through the Mek (mitogen-activated protein extracellular signal-related kinase) pathway to up-regulate anti-apoptotic and down-regulate pro-apoptotic pathways, and through JNK/p38 (c-Jun N-terminal kinase) to drive class-switching. Survival and reactiva‐ tion of B cell memory is BAFF-independent. Plasma cells express TACI and/or BCMA and their survival can be supported by either BAFF or APRIL. In contrast, B1 cells do not require BAFF or APRIL for survival. BAFF plays an important role in humoral immuni‐ ty. T cell-independent type II responses require the interaction of BAFF 60-mer or membrane BAFF with TAC. This interaction is vital for T cell-dependent immunoglobu‐ lin (Ig)M responses. BAFF is also an essential component of the innate immune response and is induced in myeloid DC by type I interferons (IFNs). BAFF up-regulates Toll-like receptor (TLR) expression, promotes B cell survival and, together with IL-6, promotes Ig class-switching and plasma cell differentiation. Soluble BAFF and APRIL are expressed at high levels in the serum and in the target organs of individuals with established anti‐ body dependent autoimmune diseases. Therapeutic antagonism of BAFF and its homo‐ logue APRIL (a proliferation-inducing ligand) targets an important homeostatic signal for B cell survival and selection (Figure 10). Belimumab and atacicept are two potential therapeutic anatgonists to the BAFF-APRIL pathway for B cell activation that are current‐

Recent Advances in the Management of Refractory Vasculitis

http://dx.doi.org/10.5772/54517

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**Figure 10.** B cells maturation and antibody production in autoimmune diseases.

ly being investigated. [92-105]

**Table 2.** Demographics of patients enrolled in RAVE and RITUXVAS.

Rituximab may be an effective alternative treatment in newly diagnosed as well as refractory ANCA-associated vasculitis. Wegener's Granulomatosis (granulomatosis with polyangiitis) patients with retro-orbital granulomas tend to be less responsive to rituximab therapy. [91] The great limitation of rituximab is some cases with GPA or microscopic polyangiitis may need to be retreated following initial treatment. New anti-CD20 agents, or agents that attack different B-cell precursors, may overcome this hurdle, and may enable even longer periods of remission.
