**2. Anti-CD20 monoclonal antibodies**

The CD20 (B1) antigen is a 33–35 kDa integral membrane protein expressed on the surfaces of non-malignant and most malignant B cells (Cragg, Walshe et al. 2005). The CD20 protein consists of cytoplasmic N- and C-termini and four hydrophobic regions for anchoring the molecule in the membrane (Robak 2008). The characteristics that make CD20 a good target

The Emerging Role of Monoclonal Antibodies in the Treatment of Systemic Lupus Erythematosus 257

treated with a 1 gm of rituximab, 750 mg of cyclophosphamide, and 100-250 mg of methylprednisolone, administered on 2 occasions 2 weeks apart (Lu, Ng et al. 2009). Twenty one patients (47%) reached partial remission after one cycle (mean followup 39.6 months). Treatment resulted in a decrease in median global BILAG scores from 12 to 5 (P < 0.0001) and median anti-double-stranded DNA antibody titers from 106 to 42 IU/ml (P < 0.0001). In addition an increase in the median C3 level from 0.81 to 0.95 mg/liter (P < 0.02) at 6 months

Willems et al. (Willems, Haddad et al. 2006) described the safety and efficacy of rituximab in 11 girls (mean age 13.9 years) with severe SLE including 8 girls with class IV or V lupus nephritis, 2 girls with severe autoimmune cytopenia and 1 girl with an antiprothrombin antibody. Patients received 2 to 12 intravenous infusions of rituximab (350-450 mg/m2/ infusion) with corticosteroids. Remission was achieved in 6 of 8 patients with lupus nephritis and in two patients with autoimmune cytopenia. However, severe adverse events

Looney et al. reported the results of the first dose escalation study of rituximab for the treatment of SLE (Looney, Anolik et al. 2004). The drug was added to ongoing therapy in 18 patients with moderately active SLE. Six patients received a single infusion of 100 mg/m2, six received one infusion of 375 mg/m2 and six patients received four weekly doses of 375 mg/m2. In this study, rituximab-induced B cell depletion was translated into a significant improvement in SLE disease activity even in the absence of substantial serologic responses. Most patients were able to decrease corticosteroid dose from 13 to 10 mg by the end of the study and three patients were able to discontinue concomitant immunosuppressives. The

Terrier et al. analyzed recently prospective data from the French AutoImmunity and Rituximab (AIR) registry, which includes data on patients with autoimmune disorders treated with rituximb (Terrier, Amoura et al. 2010). Overall response was observed in 80 of 113 patients (71%) by the SELENA-SLEDAI (SLE Disease Activity Index Score assessment). Efficacy was similar between patients receiving rituximab monotherapy and those receiving concomitant immunosuppressive agents. Articular, cutaneous, renal, and hematologic improvements were noted in 72%, 70%, 74%, and 88% of patients, respectively. In relapsed patients response was observed in 91% after retreatment with rituximab. Severe infections were observed in 12 patients (9%), corresponding to a rate of 6.6/100 patient-years. Most severe infections occurred within the first 3 months after the last rituximab infusion. Five patients died, due to severe infection (n = 3) or refractory autoimmune disease (n = 2). Merrill et al reported the results of the Exploratory Phase II/III SLE Evaluation of Rituximab (EXPLORER) trial, a placebo-controlled, double-blind, multicenter study of rituximab in patients with moderately-to-severely active extrarenal SLE (Merrill, Neuwelt et al. 2010). Patients were randomized at a 2:1 ratio to receive intravenous rituximab (1,000-mg) or placebo on days 1, 15, 168, and 182, which was added to prednisone. Of the 257 patients, 88 were assigned to receive placebo, and 169 were randomized to the rituximab arm. At week 52, no difference was observed in major clinical responses or partial clinical responses between the placebo group. Decreases in the level of anti-dsDNA autoantibodies and increases in complement C3 and C4 levels were greater in the rituximab group than in the placebo group. The overall response rate was 28.4% and 29.6%, respectively. The proportion of patients in whom serious infection developed was 17% in the placebo group and 9.5% in

was observed. Five serious adverse events were noted.

clinical response was most notable for rashes and arthritis.

occurred in 45% of the patients in this study.

the rituximab group.

antigen include its relatively high level of expression and close location of the extracellular epitopes to the cell surface. The intensity of antigen expression or the number of receptor sites on the cell surface appears to correlate with the clinical response. The cytotoxic activity of mAbs directed against CD20+ cells is thought to be based on antibody-dependent cellular cytotoxicity (ADCC) via natural killer (NK) cell responses, complement-dependent cytotoxicity (CDC), or by the induction of cell signaling followed by apoptosis. At present, rituximab is the most important mAb of clinical value in patients with autoimmune disorders and B-cell lymphoid malignancies. Over the last few years, new generations of anti-CD20 mAbs have been developed for potential benefits over rituximab (Robak and Robak 2011; Lim, Beers et al. 2010). They were engineered to have augmented antitumor activity by increasing CDC or ADCC activity and increased Fc binding affinity for the lowaffinity variants of the FcγRIIIa receptor (CD16) on immune effector cells. The secondgeneration mAbs are humanized or fully human to reduce immunogenicity, but with an unmodified Fc region. They include ofatumumab, veltuzumab, and ocrelizumab. The thirdgeneration mAbs are also humanized but in comparison with the second-generation mAbs they also have an engineered Fc region designed to increase their effector functions by increasing binding affinity for the FcγRIIIa receptor (Ruuls, Lammerts et al. 2008). Both polymorphisms in FcγRIIIa and structure of mAb Fc can impact on the affinity between FcγRIIIa and mAb. The third-generation mAbs include AME133v, Pro13192, and GA-101.

#### **2.1 Rituximab**

Rituximab is an IgG-1 immunoglobulin, containing murine light– and heavy–chain variable–region sequences and human constant region sequences. Rituximab is known as the first-generation mAb. Since approval in 1997, rituximab has become the standard of care in follicular B-cell lymphomas (FL), CLL, and aggressive lymphomas when combined with chemotherapy (Hauptrock and Hess 2008). Rituximab is administered as an intravenous infusion with a recommended dosage of 375 mg/m2 given once weekly for 4 weeks. Treatment with this agent is usually well tolerated. However, infusion-related reactions occur in the majority of patients. These adverse events are typically fever, chills, rigors and rare hypotension and bronchospasm, although the incidence of these side effects decreases with subsequent rituximab infusion. Moreover, prolonged impairment of antibody production causes the increased risk of viral and bacterial infections. It should be also remembered that rituximab is a human mouse chimeric antibody and hence treated patients may be susceptible to the development of human antichimeric antibodies, which can impact on responsiveness.

A recent study has shown that treatment with rituximab affects both the cellular and humoral arm of the immune system in patients with SLE (Lu, Ng et al. 2009).

A number of prospective studies and several retrospective cohort studies of rituximab in the treatment of SLE have been reported (Cambridge, Isenbergetal. 2008). In 2005 Leonardo et al. (Leandro, Cambridge et al. 2005) described female patients with SLE who were treated with combination of rituximab, CY and prednisolone. Each patient received two infusions of rituximab (500 mg/dose), two infusions of cyclophosphamide (750 mg/dose) and 60 mg prednisolone per day for five days. Five patients were analyzed and one patient was lost to follow up after 3 months. All five patients showed an improvement in British Isles Lupus Assessment Group (BILAG) scores from a median of 14 at baseline to a median of 6 at six months. Recently, the same group reported the results of 46 patients with active SLE were

antigen include its relatively high level of expression and close location of the extracellular epitopes to the cell surface. The intensity of antigen expression or the number of receptor sites on the cell surface appears to correlate with the clinical response. The cytotoxic activity of mAbs directed against CD20+ cells is thought to be based on antibody-dependent cellular cytotoxicity (ADCC) via natural killer (NK) cell responses, complement-dependent cytotoxicity (CDC), or by the induction of cell signaling followed by apoptosis. At present, rituximab is the most important mAb of clinical value in patients with autoimmune disorders and B-cell lymphoid malignancies. Over the last few years, new generations of anti-CD20 mAbs have been developed for potential benefits over rituximab (Robak and Robak 2011; Lim, Beers et al. 2010). They were engineered to have augmented antitumor activity by increasing CDC or ADCC activity and increased Fc binding affinity for the lowaffinity variants of the FcγRIIIa receptor (CD16) on immune effector cells. The secondgeneration mAbs are humanized or fully human to reduce immunogenicity, but with an unmodified Fc region. They include ofatumumab, veltuzumab, and ocrelizumab. The thirdgeneration mAbs are also humanized but in comparison with the second-generation mAbs they also have an engineered Fc region designed to increase their effector functions by increasing binding affinity for the FcγRIIIa receptor (Ruuls, Lammerts et al. 2008). Both polymorphisms in FcγRIIIa and structure of mAb Fc can impact on the affinity between FcγRIIIa and mAb. The third-generation mAbs include AME133v, Pro13192, and GA-101.

Rituximab is an IgG-1 immunoglobulin, containing murine light– and heavy–chain variable–region sequences and human constant region sequences. Rituximab is known as the first-generation mAb. Since approval in 1997, rituximab has become the standard of care in follicular B-cell lymphomas (FL), CLL, and aggressive lymphomas when combined with chemotherapy (Hauptrock and Hess 2008). Rituximab is administered as an intravenous infusion with a recommended dosage of 375 mg/m2 given once weekly for 4 weeks. Treatment with this agent is usually well tolerated. However, infusion-related reactions occur in the majority of patients. These adverse events are typically fever, chills, rigors and rare hypotension and bronchospasm, although the incidence of these side effects decreases with subsequent rituximab infusion. Moreover, prolonged impairment of antibody production causes the increased risk of viral and bacterial infections. It should be also remembered that rituximab is a human mouse chimeric antibody and hence treated patients may be susceptible to the development of human antichimeric antibodies, which can impact

A recent study has shown that treatment with rituximab affects both the cellular and

A number of prospective studies and several retrospective cohort studies of rituximab in the treatment of SLE have been reported (Cambridge, Isenbergetal. 2008). In 2005 Leonardo et al. (Leandro, Cambridge et al. 2005) described female patients with SLE who were treated with combination of rituximab, CY and prednisolone. Each patient received two infusions of rituximab (500 mg/dose), two infusions of cyclophosphamide (750 mg/dose) and 60 mg prednisolone per day for five days. Five patients were analyzed and one patient was lost to follow up after 3 months. All five patients showed an improvement in British Isles Lupus Assessment Group (BILAG) scores from a median of 14 at baseline to a median of 6 at six months. Recently, the same group reported the results of 46 patients with active SLE were

humoral arm of the immune system in patients with SLE (Lu, Ng et al. 2009).

**2.1 Rituximab** 

on responsiveness.

treated with a 1 gm of rituximab, 750 mg of cyclophosphamide, and 100-250 mg of methylprednisolone, administered on 2 occasions 2 weeks apart (Lu, Ng et al. 2009). Twenty one patients (47%) reached partial remission after one cycle (mean followup 39.6 months). Treatment resulted in a decrease in median global BILAG scores from 12 to 5 (P < 0.0001) and median anti-double-stranded DNA antibody titers from 106 to 42 IU/ml (P < 0.0001). In addition an increase in the median C3 level from 0.81 to 0.95 mg/liter (P < 0.02) at 6 months was observed. Five serious adverse events were noted.

Willems et al. (Willems, Haddad et al. 2006) described the safety and efficacy of rituximab in 11 girls (mean age 13.9 years) with severe SLE including 8 girls with class IV or V lupus nephritis, 2 girls with severe autoimmune cytopenia and 1 girl with an antiprothrombin antibody. Patients received 2 to 12 intravenous infusions of rituximab (350-450 mg/m2/ infusion) with corticosteroids. Remission was achieved in 6 of 8 patients with lupus nephritis and in two patients with autoimmune cytopenia. However, severe adverse events occurred in 45% of the patients in this study.

Looney et al. reported the results of the first dose escalation study of rituximab for the treatment of SLE (Looney, Anolik et al. 2004). The drug was added to ongoing therapy in 18 patients with moderately active SLE. Six patients received a single infusion of 100 mg/m2, six received one infusion of 375 mg/m2 and six patients received four weekly doses of 375 mg/m2. In this study, rituximab-induced B cell depletion was translated into a significant improvement in SLE disease activity even in the absence of substantial serologic responses. Most patients were able to decrease corticosteroid dose from 13 to 10 mg by the end of the study and three patients were able to discontinue concomitant immunosuppressives. The clinical response was most notable for rashes and arthritis.

Terrier et al. analyzed recently prospective data from the French AutoImmunity and Rituximab (AIR) registry, which includes data on patients with autoimmune disorders treated with rituximb (Terrier, Amoura et al. 2010). Overall response was observed in 80 of 113 patients (71%) by the SELENA-SLEDAI (SLE Disease Activity Index Score assessment). Efficacy was similar between patients receiving rituximab monotherapy and those receiving concomitant immunosuppressive agents. Articular, cutaneous, renal, and hematologic improvements were noted in 72%, 70%, 74%, and 88% of patients, respectively. In relapsed patients response was observed in 91% after retreatment with rituximab. Severe infections were observed in 12 patients (9%), corresponding to a rate of 6.6/100 patient-years. Most severe infections occurred within the first 3 months after the last rituximab infusion. Five patients died, due to severe infection (n = 3) or refractory autoimmune disease (n = 2).

Merrill et al reported the results of the Exploratory Phase II/III SLE Evaluation of Rituximab (EXPLORER) trial, a placebo-controlled, double-blind, multicenter study of rituximab in patients with moderately-to-severely active extrarenal SLE (Merrill, Neuwelt et al. 2010). Patients were randomized at a 2:1 ratio to receive intravenous rituximab (1,000-mg) or placebo on days 1, 15, 168, and 182, which was added to prednisone. Of the 257 patients, 88 were assigned to receive placebo, and 169 were randomized to the rituximab arm. At week 52, no difference was observed in major clinical responses or partial clinical responses between the placebo group. Decreases in the level of anti-dsDNA autoantibodies and increases in complement C3 and C4 levels were greater in the rituximab group than in the placebo group. The overall response rate was 28.4% and 29.6%, respectively. The proportion of patients in whom serious infection developed was 17% in the placebo group and 9.5% in the rituximab group.

The Emerging Role of Monoclonal Antibodies in the Treatment of Systemic Lupus Erythematosus 259

with rheumatoid arthritis (RA), and 25 (0.26%) with other connective tissue diseases (CTDs) (Molloy and Calabrese 2009). Additional controlled studies with new designs are needed to

Over the last few years, new generations of anti-CD20 monoclonal antibodies have been developed for potential benefits over the classical, first-generation mAb rituximab. Compared with rituximab, new mAbs have enhanced antitumor activity resulting from increased CDC and ADCC, and increased Fc binding affinity for the low-affinity variants of

Ofatumumab (HuMax-CD20; Arzerra™, GlaxoSmithKline plc/Genmab A/S) is a secondgeneration, fully human, anti-CD20, IgG1 mAb in phase I, II and III trials for hematological malignancies and autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis. Ofatumumab specifically recognizes an epitope encompassing both the small and large extracellular loops of CD20 molecule, and is more effective than rituximab at CDC induction and killing target cells. In April 2010, the European Medicines Agency granted a conditional marketing authorization for ofatumumab, for the treatment of fludarabinerefractory CLL patients. It has been reported recently that ofatumumab, administered as 2 i.v. infusions at doses 300 mg, 700 mg, or 1,000 mg is clinically effective in patients with active RA (Østergaard , Baslund et al. 2010). Rapid and sustained peripheral B cell depletion was noted in all dose groups. Overall, 70% of patients receiving ofatumumab had a moderate or good response according to the European League Against Rheumatism

Veltuzumab (IMMU-106, hA20; Immunomedics Inc., Morris Plains, NJ) is a secondgeneration, type 1, humanized, anti-CD20, IgG1 mAb with complementarity-determining regions (CDRs) similar to rituximab (Goldenberg, Rossi et al. 2009). This mAb is generated using the same human immunoglobulin as epratuzumab and has a >90% humanized framework. It is also very similar to rituximab in terms of antigen binding, specificity binding, and dissociation constant. Veltuzumab differs from rituximab by one amino acid (Asp101 instead of Asn101) in the CDR 3 of the variable heavy chain. Smaller murine regions may reduce infusion reactions, infusion times, and immunogenicity. This antibody has enhanced binding avidities and a stronger effect on CDC compared with rituximab in selected cell lines. Veltuzumab is safe and active agent in NHL. B cells were depleted after the first infusion of all tested doses, including dose levels less than those typically used with

Ocrelizumab (Genentech Inc/Biogen Idec Inc/Chugai Pharmaceutical Co Ltd/Roche Holding Ag) is a second-generation, type 1, humanized, anti-CD20, IgG1 mAb with modifications of the Fc region that lead to enhanced ADCC and reduced CDC activities compared with rituximab (Kausar, Mustafa et al. 2009).This agent has the potential for enhanced efficacy compared with rituximab due to increased binding affinity for the low-

the FcγRIIIa receptor (CD16) on immune effector cells (Czuczman & Gregory 2010).

define the place of rituximab in the therapeutic arsenal for SLE.

**2.2 New generations of Anti-CD20 monoclonal antibodies** 

**2.2.1 Ofatumumab** 

(EULAR) criteria at week 24.

rituximab (Morschhauser, Leonard et al. 2009).

**2.2.2 Veltuzumab** 

**2.2.3 Ocrelizumab** 

Rituximab is also an active treatment agent in patients with lupus nephritis and central nervous system (CNS) involvement. Sfikakis et al. reported clinical response in 80% and sustained complete response in 40% of patients with class III and IV nephritis treated with rituximab and moderate doses of corticosteroids (Sfikakis, Boletis et al. 2005). In the study of Ng et al. 21 patients with renal involvement were treated with rituximab and cyclophosphamide (Ng, Cambridge et al. 2007). They had a decrease in median urinary protein creatinine ratio (PCR) from 446 to 190 mg/mmol 6 months. More recently, Pepper et al. treated 18 patients with class III/IV/V lupus nephritis with rituximab. All patients were on steroids prior to the development of lupus nephritis (Pepper, Griffith et al. 2009). The patients received mycophenolate mofetil maintenance therapy. Fourteen of 18 patients achieved complete or partial remission with a sustained response of 67% at 1 year. In addition, serum albumin increased from a mean of 29 g/L at presentation to 34 g/L at 1 year (P = 0.001). Importantly, following treatment with rituximab, 6 patients stopped prednisolone, 6 patients reduced their maintenance dose and 6 patients remained on the same dose (maximum 10 mg). No severe infections were observed.

The study performed by Tokunaga et al. showed marked improvement following rituximab therapy in patients with neuropsychiatric SLE (Tokunaga, Saito et al. 2007). A monoclonal antibody was administered at doses of 375 mg/m2 once weekly for four weeks or 1000 mg once weekly for two weeks in 10 patients with refractory neuropsychiatric SLE. Treatment resulted in rapid improvement of CNS–related manifestations, particularly acute confusional state. Rituximab also improved cognitive dysfunction psychosis and seizure and reduced the SLEDAI on day 28 in all 10 patients. These effects lasted for more than a year in 5 patients. In another study, Smith et al. (Smith, Jones et al. 2006) evaluated prospectively the effects of rituximab treatment for refractory SLE and vasculitis. Patients received four weekly infusions of rituximab at a dose of 375 mg/m2. Intravenous cyclophosphamide (500 mg) was administered along with the first infusion in an effort to achieve early disease control. Remission followeing rapid B cell depletion was achieved in all 11 patients including 6 complete responses and 5 partial responses. Moreover, a renal response occurred in all 6 patients with lupus nephritis. Clinical improvement was accompanied by a significant reduction in the daily dose of prednisone. Seven of 11 patients experienced a relapse, a median of 12 months after treatment. After relapse, six patients with SLE were retreated with rituximab and all achieved remission and did so more quickly than after the primary treatment.

Rituximab is generally well tolerated. Even fewer adverse events have been observed in patients treated for SLE than in the lymphoma patients (Tokunaga, Saito et al. 2007). The most common adverse events during or following rituximab therapy are infusion related symptoms, typically fever, chills, rigors and hypotension. In patients who receive premedication consisting of antipyretic and antihistaminic drugs together with corticosteroids, infusion-related side effects are usually only mild or moderate and do not require discontinuation of rituximab administration. Occasionally, serious infections were also reported. However, these may have been related to the underlying disease and/or concomitant therapy with other immunosuppressive agents. In 2006, an FDA alert was reported after two SLE patients treated with rituximab had died from progressive multifocal leukoencephalopathy (PML) (Ermann and Bermas 2007). However, both patients had received additional treatment with cyclophosphamide. At present, it is difficult to estimate the risk of this complication in SLE patients treated with rituximab. In recent analysis, among the rheumatic diseases, 43 cases of PML (0.44%) were associated with SLE, 24 (0.25%) with rheumatoid arthritis (RA), and 25 (0.26%) with other connective tissue diseases (CTDs) (Molloy and Calabrese 2009). Additional controlled studies with new designs are needed to define the place of rituximab in the therapeutic arsenal for SLE.

### **2.2 New generations of Anti-CD20 monoclonal antibodies**

Over the last few years, new generations of anti-CD20 monoclonal antibodies have been developed for potential benefits over the classical, first-generation mAb rituximab. Compared with rituximab, new mAbs have enhanced antitumor activity resulting from increased CDC and ADCC, and increased Fc binding affinity for the low-affinity variants of the FcγRIIIa receptor (CD16) on immune effector cells (Czuczman & Gregory 2010).
