B cells have been targeted in the treatment of systemic lupus erythematosus (SLE) andrheumatoid arthritis (RA) owing to the central role they play in the pathogenesis ofthese disorders. These cells play a critical role in host defence through theirmaturation into antibody-secreting plasma cells, secretion of proinflammatory cytokines,antigen presentation and co-stimulatory support for T cells. However, dysfunctionalrecognition of self-antigens as nonself-antigens results in autoantibody production,sustained by plasma cells derived from the B-cell lineage that survive for prolongedperiods in the lymphoid tissues. B cells also participate in inflammatory reactionsthrough antibody-independent mechanisms by acting as antigen-presenting cells andco-stimulation of T cells and other inflammatory cell types, although as yet there areno validated biomarkers that distinguish pathogenic from protective B-cell subsets.Reagents that specifically target pathogenic B-cell subsets are therefore not likely tobe available in the near future. This reality provides the rationale for targeting Bcells in patients with SLE, RA and other autoimmune diseases [15].

B-cell-targeted immunotherapy was initially developed for the treatment ofB-cell-related malignancies, which are associated with poor prognosis despite aggressivecytotoxic therapies. Of the many surface-expressed antigens on B cells studied aspossible targets, CD20 - a transmembrane phosphoprotein expressed in normal B cells aswell as 90% of lymphomas - is not shed or modulated, making it an attractive target. In1994, Reff and colleagues reported a major (95%) and sustained (up to 90 days) B-celldepletion using a murine mAb (2B8) that targeted CD20 on B cells in nonhuman primates [6]. In 1997, a landmark study reported on both the safety and efficacy ofrituximab, a chimeric (mouse-human) mAb directed against CD20, for the treatment ofrelapsed, refractory low-grade or follicular lymphoma [7]. In November 1997, rituximab was licensed for this indication. Rituximab isnow a part of the standard therapeutic regimen in the management of B-cell malignanciesand remains among the most successful therapeutic mAbs. Interestingly, the response rateis variable amongst individuals with the same histological type of lymphoma as well asthe overall response rate between different histological types [8]. This suggests that B-cell depletion is not uniform across patients or indeeddiseases for reasons yet to be fully understood, but Fcγ receptor function appearsimportant with enhanced Fcγ receptor IIb expression being associated with reducedrituximab efficacy in lymphoma [9]. Intriguingly, polymorphisms of this receptor are associated with SLE,although their precise role in the disease and potential for targeted therapeuticintervention is not understood.

In 1999, Professor Edwards' group at University College London treated a small number ofpatients with refractory RA using rituximab, having been encouraged by the safety andefficacy profile of induced transient depletion of B cells in haematologicalmalignancies. This study and subsequent studies of rituximab in RA, including a largephase II randomised controlled trial, indicated that the treatment was potentially safeand effective [1013]. The regimen in these studies utilised two doses (1,000 mg) of rituximabgiven 2 weeks apart, with premedication including a single 100 mg intravenous dose ofmethylprednisolone and 10 mg chlorphenamine. In the original study, patients alsoreceived a course of high-dose prednisolone (60 mg for up to 3 weeks and then taperingover the next 3 to 4 weeks or maintaining at 5 mg a day). Responding patients wereretreated at or just before predicted relapse. Initially, intravenous cyclophosphamidewas used to accompany the rituximab [10]. The phase II study showed that cyclophosphamide could be replaced bymethotrexate or rituximab on its own, although the response rates were better whenrituximab was used in combination with methotrexate. Further, the assigned dose ofprednisolone was reduced to 60 mg/day oral prednisone on day 2 and days 4 to 7 and 30mg/day oral prednisone on days 8 to 14 [11].

Clinical experience of rituximab in SLE

The first open, uncontrolled study of rituximab for patients with SLE, by ProfessorIsenberg's group at University College London, showed improvements in both clinical andlaboratory features of disease following treatment with rituximab in refractory SLE [14]; these observations have been supported by the publication of many othersimilar open, nonrandomised studies [1519] (Table 1). The University College London regimenemployed premedication with 100 mg intravenous methylprednisolone in addition to 750 mglow-dose intravenous cyclophosphamide (for renal manifestations) 1 day prior to thefirst of two doses of rituximab, given 2 weeks apart. More recently just one dose ofcyclophosphamide has been used, and any subsequent need for immunosuppressive therapy isadjusted based on the merits of clinical response and disease manifestation activitythat can be assessed using well-validated tools such as the British Isles LupusAssessment Group (BILAG) 2004 index (for example, using the BLIPS computer softwareprogram; LIMATHON, Sheffield, UK).

Table 1 Reported efficacy of rituximab in nonrandomised trials of systemic lupuserythematosus

Appreciating this robust clinical management focused on the individual patient -potentially involving multi-disciplinary expert opinion, including rheumatologists,dermatologists and renal physicians - is important when comparing the results with thosefrom large multicentre randomised controlled trials with variable quality observationsin a broad population.

Worthy of note is that the indication for rituximab at Professor Isenberg's centre is acombination of active disease (renal or nonrenal) (assessed by the BILAG 2004 index)poorly controlled despite at least two standard immunosuppressive agents (not includingcorticosteroids) used for sufficient time at optimal doses. To date, 100 patients havebeen treated at University College London with at least one cycle of rituximab and morethan 30 patients have received repeated treatment. Although involving only smallnumbers, the observations from repeating the regimen showed that improvements indisease, including remission rates, were sustained in patients who responded to theinitial treatment [20]. This same group has previously demonstrated following B-cell depletiontherapy (BCDT) that anti-double-stranded DNA (anti-dsDNA) and anti-nucleosome antibodiesreduce to 30 to 40% of baseline, whereas other autoantibodies such as anti-Ro andantibodies to pneumococcal polysaccharide (protective) remain unaltered. Thisobservation would suggest that rapidly proliferating clones of B cells may give rise toshort-lived plasma cells that produce these anti-dsDNA, anti-cardiolipin andanti-nucleosome antibodies and appear preferentially affected by BCDT [21], whereas other autoantibodies such as anti-Ro and anti-RNP or protectiveantibodies, which develop following immunisation and are thought to be produced bylong-lived plasma cells, remain unaltered.

In line with this experience, anti-dsDNA antibody levels tend to fall but not tonormalise and these antibodies are probably produced by a combination of short-lived andlong-lived plasma cells. Similar to these findings, a post-hoc analysis of theEXPLORER trial focusing on the biological effects of rituximab revealed a significantreduction in the levels of anti-dsDNA and anti-cardiolipin antibodies and a significantincrease in complement levels and serum BAFF in the rituximab-treated group versusplacebo. Analysis of the repopulation dynamics of subsets of B cells identifiednaïve cells as the primary phenotype detected first in circulation; however, thephenotype analysis was limited in that CD27- memory cells were not examinedin this study [22]. The changes in biological effects did not translate into clinical benefitsat 1 year. Whether a long-term follow up with more detailed phenotype analysis atvarious time points would help predict response to rituximab therapy is not known.However, designing clinical trials to define the precise relationship between thebiological effects that occur following BCDT and the clinical response in the long term(typically, 2 to 5 years) would be met with the potential challenge of maintainingremission in the placebo group with conventional immunosuppressants alone. The effectsextend to global disease control including an improvement in lipid profile [23], but such benefits are not necessarily captured in randomised controlledtrials with a short duration of follow-up.

Recently, following the approach by a group at Imperial College (see later) in a pilotstudy, eight patients with active disease were treated at diagnosis with rituximab in anattempt to avoid the use of corticosteroids. Using this approach it was possible toreduce the cumulative dose of steroids substantially in five of the eight patients [24], a major long-term advantage.

A recent review of the rituximab experience in approximately 200 patients withrefractory SLE, from open studies and real clinical experience, indicated that manywould respond at least partially to B-cell depletion [25]. Differences in determining endpoints for these studies make it difficult toestablish formal median and range of improvements. In a phase I/II dose-escalation trialof the safety and efficacy of rituximab in addition to ongoing therapy in 18 patientswith SLE, three dosing regimens of rituximab were studied as follows: six patientsreceived a low dose, a single infusion of 100 mg/m2; six patients received anintermediate dose, a single infusion of 375 mg/m2; and five patients receiveda high dose, four infusions of 375 mg/m2 administered 1 week apart. There wasa significant improvement in the disease activity, as measured by systemic lupusactivity measure scores, in all patients by 2 months, which persisted at 12 monthsregardless of a change in anti-dsDNA antibody and complement levels. Six of 17 patientsdeveloped human anti-chimeric antibodies, resulting in reduced serum rituximab levelsand inefficient B-cell depletion and less impressive efficacy. Importantly, there wereno significant adverse events [26]. The UK-BIOGEAS registry study of 164 patients with refractory or relapsinglupus nephritis reported a 67% partial or complete response rate to rituximab usingstandardised response criteria [27].

Clinicians therefore continue to use rituximab for refractory lupus nephritis as well asnonrenal manifestations including haematological, skin and central nervous systemmanifestations where clinically useful responses have been reported [28, 29]. There is thus extensive nonrandomised and retrospective experience ofrituximab in the treatment of refractory SLE. A role for rituximab for this indicationis supported by the consistency of the reports of improvement but differences inregimens, concomitant medications and endpoints remain, making it difficult to assessthe extent of effectiveness of B-cell depletion accurately. Additionally, there isuncertainty as to how to reduce relapse risk after rituximab, and an unqualifiedrecommendation for rituximab in refractory SLE will require higher quality evidence.

Safety and efficacy in clinical trials

To evaluate the safety and efficacy of rituximab in SLE in a clinical trial setting, twodouble-blind, randomised, placebo-controlled trials (DBRCTs) investigating renal (LUNARstudy) and nonrenal (EXPLORER study) manifestations were undertaken (Table 2). Both trials addressed the hypothesis that the addition ofrituximab to the standard of care, corticosteroids and immunosuppressants was superiorto addition of placebo for the control of SLE activity.

Table 2 Summary of the randomised-controlled trials of rituximab therapy in systemic lupuserythematosus

In the EXPLORER study, the safety and efficacy of rituximab in moderate-to-severe activenonrenal SLE was evaluated [30] (Figure 1). This study included 257 patients with≥1 BILAG A score (>50% of patients at entry) or ≥2 BILAG B scores despiteongoing stable-dose immunosuppressant therapy with either azathioprine (100 to 250mg/day), mycophenolate (1 to 4 g/day) or methotrexate (7.5 to 25 mg/week), which wascontinued during the trial. Background immunosuppressive therapy was evenly distributed.A key feature of treatment in this study was the additional course of high-dosecorticosteroids patients received early in the study. Corticosteroids were given atinitial doses of 0.5 mg/kg, 0.75 mg/kg or 1 mg/kg depending on severity (by BILAG score)and type of disease manifestations, followed by a taper regimen. Of the overallpopulation, >50% were classed as steroid dependent, and ≥60% of patientsreceived an average 45.9 ± 16.4 mg prednisolone and then attempted to reduce to atarget dose of <10 mg/day over the 10-week taper period and ≤5 mg/day at week52.

Figure 1
figure 1

Treatment protocol of the BELONG study. AZT, azathioprene; CYC,cyclophosphamide; EL, EUROLUPUS; LN, lupus nephritis; MMF, mycophenolate mofetil;OCR, ocrelizumab; ORR, overall renal response; PBO, placebo.

Patients were randomised at a ratio of 2:1 to receive rituximab (1,000 mg) or placebo.Eighty-eight patients received placebo and 169 patients received rituximab (two dosesgiven 14 days apart) on days 1, 15, 168, and 182. The majority (≥50%) of patientsin both groups had musculoskeletal and mucocutaneous disease.

The primary endpoint of the EXPLORER study was stringent, with complete and partialresponse definitions as follows.

To classify as a complete/major response, at week 24 an improvement in all organ systemswith a BILAG C score or better was required. Further, this response was to be sustainedat week 52, without experiencing a severe or moderate/severe flare during the period toweek 24 and week 52, respectively. A severe flare was defined as a BILAG A score or astwo new domains with BILAG B scores [31].

Patients were considered to have attained a partial response if: there was animprovement in all organ systems with a BILAG C score or better, which was sustained for16 consecutive weeks; a BILAG B score in no more than one organ system at week 24without a new BILAG A or BILAG B score to week 52 was achieved; and, at week 24, no morethan two BILAG B scores were achieved without new BILAG A or BILAG B scores provided thebaseline BILAG score was one A score plus ≥2 B scores, ≥2 A scores, or≥4 B scores.

The secondary endpoints included the time-adjusted area under the curve minus thebaseline BILAG score over 52 weeks, the proportion of patients who achieved a major andpartial clinical response, the proportion of patients who achieved a BILAG C score inall organ systems at week 24, the time to the first moderate to severe disease flare,improvement in quality of life as measured by the Lupus Quality of Life, and theproportion of patients who achieved a major clinical response with a prednisolone dose<10 mg/day from week 24 to week 52. In addition, serological activity parametersincluding levels of autoantibodies, complement, immunoglobulins, T-cell and B-cellcounts and human anti-chimeric antibody were monitored.

In the intent-to-treat analysis of 257 patients, approximately 70% of patients completedthe study in both arms and the safety and tolerability was similar in both groups. Therewas no difference between the addition of placebo and rituximab to the standard of carein the primary and secondary efficacy endpoints, including the BILAG-defined response,in terms of both area under the curve and other analyses.

A preplanned subgroup analysis, however, detected a beneficial effect of rituximab inthe primary endpoint in the African American and Hispanic patients, a major clinicalresponse in 13.8% and a partial response in 20% when compared with 9.4% and 6%,respectively. Notably, these patients had more active disease and more refractorydisease as previously reported [32]. There were significant biological effects in the rituximab-treated group,with greater falls in anti-dsDNA levels and rises in complement levels compared withplacebo. Interestingly, up to 9.5% of patients did not achieve complete B-celldepletion, but analysis without these patients did not change the primary outcome. Thisphenomenon has been observed in autoimmune prone mice [33, 34]. A recent study investigating the role of highly sensitive flow cytometrydetected a correlation between clinical response and B-cell numbers [35].

The LUNAR study investigated the safety and efficacy of 2 × 1,000 mg rituximab, atboth 0 and 6 months, as compared with placebo in addition to background therapy withhigh-dose glucocorticoids and mycophenolate mofetil 3 g/day in 144 patients withproliferative lupus nephritis, classes III and IV (Figure 2).

Figure 2
figure 2

Treatment protocols of the EXPLORER and LUNAR studies. (a) EXPLORERstudy. (b) LUNAR study. BILAG, British Isles Lupus Assessment Group;ISN/RPS, International Society of Nephrology/Renal Pathology Society; MMF,mycophenolate mofetil; MTX, methotrexate; Rx-AZA, treatment with azathioprine.

The primary endpoint of the study was the proportion of patients with a complete orpartial remission of nephritis at 12 months. Complete response was defined as, at week52: serum creatinine improving from abnormal to normal level or from normal to≤115% of baseline normal; a fall in the urine protein-creatinine ratio to <0.5;and urine sediment containing <5 red blood cells in a high-power field without casts.Patients who did not meet complete response were considered to have achieved a partialresponse if: serum creatinine reduced to ≤115% of abnormal baseline; the number ofred blood cells/high-power field reduced to ≤50% baseline without red blood cellcasts; and a reduction in urine protein-creatinine ratio from ≥3.0 to ≤3.0or to <1 from ≤3.0.

The secondary endpoints were: complete renal response sustained from week 24 to week 52;time to first complete renal response; and, at week 52, the urine protein-creatinineratio improving from >3 to <1, the time-adjusted area under the curve minus theBILAG global score, and a change in the physical function of SF-36 health survey. As inthe EXPLORER study, serological indices, human anti-chimeric antibodies and B-celldepletion were monitored.

The response rates for rituximab and placebo were 26% and 30% for complete renalresponse and 30% and 15% for partial renal response, respectively. At week 52, morepatients in the placebo arm (8 patients vs. 0 patients) received rescue cyclophosphamidetherapy. Improvement in proteinuria was 32% and 9% for rituximab and placebo,respectively. Analogous to the findings in the EXPLORER study and the ALMS trial, agreater proportion of black patients responded favourably, although this was notstatistically significant. There was a greater reduction in anti-dsDNA levels in therituximab-treated group. Whether the response noted in patients of African ancestry isattributable to the disease severity alone or whether there are potential differences inB-cell responsiveness to rituximab therapy in these patients is as yet unclear. In thisrespect, it is worth noting that ethnicity might influence the clinical response totreatment even with conventional immunosuppressants as noted in the ALMS study. Our owndata (D Isenberg, unpublished observations) has not indicated a clearly differentoutcome at 12 months post BCDT comparing Caucasians, Afro-Caribbean or Asian patients.Drawing any firm conclusions based on the disease severity alone would therefore bedifficult.

However, overall this was a negative study in that there was no significant differencebetween the rituximab group and the placebo group. The absolute difference in responsewas 11%, with 54% and 43% responding in the rituximab and placebo groups, respectively [36]. This value was less than the planned 23% difference, which in retrospectlooks over-optimistic especially considering the analysis at only 12 months in thispopulation. Again, differences in serological markers between groups were found and asubsequent analysis found greater falls of proteinuria in the rituximab group. MoreAfrican patients in the rituximab group responded and cyclophosphamide rescue wasrequired more frequently in the placebo group. Therefore, despite some clear signals ofefficacy and safety, this study did not meet its primary or secondary endpoints.

Why did these two DBRCTs fail to meet their endpoints? As discussed earlier, there areseveral confounding factors that may have masked the ability to accurately quantify anysignificant clinically meaningful beneficial effects of rituximab (Table 3), perhaps the most important being the aggressive backgroundimmunosuppressive therapy in the placebo and rituximab-treated groups. High-dosecorticosteroids, in particular, may have prevented the full extent of efficacy ofrituximab becoming evident, a factor that warrants due consideration in the design offuture clinical trials for any investigational agent. The dilemma for trial designers ishow rapidly to reduce glucocorticoid in patients with organ-threatening SLE. Trials withduration beyond 12 months would have greater chance of demonstrating the specifictreatment effect that could be attributed to rituximab if corticosteroids are reduced tolow levels during the first 6 months. Corticosteroid dosing could also be included inthe threshold for response in trial endpoints. For example, standard treatment shouldallow low-dose prednisolone and the proportion of patients requiring >7.5 mg/dayprednisolone could be classed as a failure. In the open studies, response was definedwith such stringent criteria. Furthermore, applying such criteria would not detectorgan-specific improvement; for example, a significant sustained improvement in a severehaematological abnormality but concurrent minor or moderate flare in skin ormucocutaneous disease would be classified as a failure.

Table 3 Potential explanations for the apparent discrepancy in clinical response reportedin clinical experience and DBRCTs

The planned efficacy margin in the LUNAR study was influenced by the 55% complete andpartial response rate in the ALMS trial at 6 months using either mycophenolate orcyclophosphamide and corticosteroids. This suggested that 45% did not respond tostandard of care; however, reasons for failure in the ALMS trial included death, severeadverse events, drug intolerance and patient/physician preference. One can estimate thattrue treatment failure was closer to 25% than 45%. A further factor in nephritis trialsis the delayed response of the outcome measure, proteinuria, to reduction inhistological activity in the kidney. The true time to remission of proteinuria is up to2 years. Had the LUNAR trial aimed for a 12% efficacy difference and involved a 2-yearduration, the study may have met its endpoint despite a small sample size.

One should also note that to date there is insufficient evidence to support the routineuse of rituximab therapy for patients with specific neuropsychiatric manifestations.However, in a study of 10 patients with a range of neuropsychiatric manifestations(including cognitive dysfunction, psychosis and seizures) refractory to conventionalimmunosuppressants, including intravenous cyclophosphamide, there was a significantimprovement, measured by the Systemic Lupus Erythematosus Disease Activity Index scoreat 28 days after treatment with rituximab, in all patients - and in five patients theresponse lasted for more than 1 year [28].

The other anti-CD20 mAb investigated in clinical trials for SLE is ocrelizumab (ahumanised anti-CD20 mAb). In rheumatoid arthritis, ocrelizumab (two regimens used: 200mg and 500 mg ×2 every 6 months) was effective in reducing signs and symptoms andjoint damage when added to a stable dose of methotrexate [37, 38]. However, a detailed analysis of results from four DBRCTs investigating thesafety and efficacy of ocrelizumab for RA indicated that an increase in seriousinfections associated with ocrelizumab compared with placebo were dose dependent andoccurred more frequently in Asia (particularly Japan) [39].

Two simultaneous clinical trials were initiated to study the safety and efficacy inlupus. Ocrelizumab was dosed differently from the RA and the rituximab SLE studies, ateither 400 or 1,000 mg intravenously ×2 at entry with repeat, single dosing every 4months. This regimen was designed to induce and maintain B-cell depletion throughout thetrial periods. The BEGIN study for nonrenal SLE was cancelled early. The BELONG studyfor proliferative lupus nephritis compared 1,000 mg or 400 mg ocrelizumab at 1 day and15 days, then repeated with a single dose every 4 months on a background of high-doseglucocorticoids and either mycophenolate mofetil or cyclophosphamide dosed according tothe EUROLUPUS protocol (Figure 1). Although the study was designedto continue for to at least 2 years, the primary endpoint was the proportion of patientsachieving partial or complete nephritis remission at 48 weeks. A total of 381 patientswere recruited before the trial was stopped early due to an imbalance in the rate ofserious infections in the ocrelizumab patients receiving mycophenolate. The 221 patientswho had passed the 32-week treatment point were assessed. The absolute difference inrenal response was 12%, with 63% and 51% for the combined ocrelizumab and placebo groupsprospectively. However, it is worth noting that in the subgroup analysis there was agreater treatment effect of ocrelizumab when combined with the EUROLUPUScyclophosphamide regime (renal response of 65.7% for ocrelizumab vs. 42.9% for EUROLUPUSalone) than with mycophenolate mofetil (renal response of 67.9% for ocrelizumab vs.61.7% for mycophenolate alone), which was largely explained by a higher response rate ingeneral with mycophenolate mofetil whilst perhaps again reflecting the outcome seen withrituximab in the LUNAR study [40] (Table 4).

Table 4 Safety and efficacy of ocrelizumab in lupus nephritis: design and results of theBELONG study

Efficacy of the BCDT has also been demonstrated in another autoimmune condition,relapsing-remitting multiple sclerosis. A recent phase II randomised clinical trialinvestigating the safety and efficacy of ocrelizumab (given together with pre-infusionsteroids only) in multiple sclerosis showed a significant reduction in neurologicallesions compared with placebo as assessed by gadolinium-enhanced magnetic resonanceimages. Serious adverse events occurred in three of 55 patients receiving 2,000 mgocrelizumab (one of 55 patients receiving 600 mg ocrelizumab, and two of 54 patientseach in the placebo group and the IFNβ-1a group) [41]. These results also support the notion that treatment regimens of BCDTcontinue to have the potential to be safe in the wider context of treatment for chronicrefractory autoimmune diseases.

Although not the principal focus of this review, it is notable in two trials involving>800 patients in each trial that belimumab (Benlysta), an anti-BLyS antibody, met itsprimary endpoint with a 10% and 14% absolute response difference over placebo [42, 43]. The primary endpoint was a composite score, the SLE Responder Index,comprising a fall in Systemic Lupus Erythematosus Disease Activity Index of 4 points, nonew BILAG A or B scores, and no change in the physician's global assessment. Thecomparisons were made at the start of the study, and at 52 or 76 weeks. These studiesdemonstrate: the need for larger trials looking for small but meaningful treatmenteffects; the potential efficacy of B-cell-targeted therapy; a similar magnitude ofresponse to that seen in the LUNAR and BELONG studies, which collectively raises thequestion of defining a clinically meaningful treatment effect in SLE trials; and a newapproach to defining a primary endpoint, the SLE Responder Index.

Lessons learned so far and future clinical trial design - how to get it right?

The failure of clinical trials in SLE has introduced palpable uncertainty whilstproviding some invaluable lessons regarding expectations for potential new therapies,carefully planned trial designs and appropriate endpoints for the particularagent/regimen in question. It is relevant to note that most preliminary data usedrituximab for refractory SLE when standard agents had failed. This is in contrast to therandomised trials, which added rituximab on top of standard therapy for nonrefractorypatients. Several factors specific to SLE increase the complexity in designingsuccessful trials. RA is a less heterogeneous disease and is much better understood whencompared with SLE and when arthritis is the main manifestation, despite the potentialfor other organs to be involved. Moreover, there exists a good deal more standardisationfor clinical trials including validated endpoints - for example, Disease Activity Index,28-joint Disease Activity Score. Conducting large-scale studies in a relatively shortperiod of time is therefore possible - particularly as RA is more common and patientaccess is better, making statistically powered studies of relatively short durationfeasible. For lupus, including nephritis, we are still some distance from achieving thesame level of understanding and standardisation in the clinical trial setting.

In an attempt to improve the lupus patient's great unmet need, the European LeagueAgainst Rheumatism has made a few suggestions to help researchers design successfultrials [44]. The main points for the future design of clinical trials are to use strictlyevaluated (a surrogate of therapeutic success against mortality or end-organ failure)outcome measures, including the disease activity indices, and to follow a standardisedapproach towards recording adverse events that could be used to measure benefit-to-riskratios from interventions, comparable between trials. Increasingly important in futuretrials, when comparing the interventional drugs, is the real difference there may be intheir potential to cause harm in the long term.

The aims of randomised controlled trials are to be defined to test robust hypothesesgenerated based on the available evidence from the open studies and clinical experience.Further, careful attention needs be paid when considering important factors, patientselection and sample size, the therapeutic agent or regimen and its potentialeffectiveness (and meaningful treatment delta vs. control), the disease outcome measuresand disease activity indices, adequate follow-up and the adverse events (Tables 5 and 6). These variable factors contribute toa great element of uncertainty in predicting the probability of the success of clinicaltrial design in SLE.

Table 5 Adverse events reported in published studiesa during or afterrituximab-induced B-cell depletion therapy
Table 6 Challenging areas in trial design and possible options

Patient selection and sample size

From a clinical trial design point of view, there are important differences in thepatient cohort, the treatment regimen and the outcome measures used in open studiesand real clinical experience when compared with the DBRCTs.

Firstly, the patient cohort in open studies and in clinic experience, at the time ofrituximab treatment, had moderate-to-severe disease activity and most had failedconventional immunosuppressants (standard of care). In contrast, patientsparticipating in the two DBRCTs (EXPLORER and LUNAR studies) had active disease, butpatients who had failed conventional therapy (cyclophosphamide and calcineurininhibitors) were excluded. Further, patients with central nervous systemmanifestations and severe organ-threatening conditions were excluded - situations inwhich rituximab has demonstrated a favourable record in the open studies [28, 4547]. Capturing the variability in organ-specific outcomes for differentinterventions tested is important. For example, rituximab may be a better choice thanother conventional immunosuppressants when both renal and haematologicalabnormalities co-exist. A favourable clinical response is more likely in seropositivepatients. However, we have previously noted that anti-Sm positivity and/or a low C3level at the time of treatment is associated with a reduction in the likelihood ofsustained benefit from B-cell depletion, and again suggest there is much work to bedone to understand lupus disease and factors that may influence the design,population and, ultimately, the outcome of clinical trials [48].

The therapeutic agent and the regimen

Rituximab has been mainly been used to achieve B-cell depletion in two regimens,either as two doses of 1,000 mg given 2 weeks apart (two-dose regime, commonly usedin SLE and RA) or as four doses of 375 mg/m2 (four-dose regime, mostcommon regime used in lymphoma, paediatric autoimmune diseases) given 1 week apart(ocrelizumab in SLE moved on from this to initial doses 2 weeks apart followed by asingle infusion every 4 months to achieve and sustain B-cell depletion). Notably, asystematic review of the clinical experience of rituximab for the treatment ofrefractory SLE suggests that the lymphoma regimen (four doses, 375 mg/m2,given 1 week apart) may be more effective in achieving an improvement in disease thanthe two-dose regimen (two doses given 2 weeks apart) [49]. Based on this review alone, however, it is difficult to draw firmconclusions about the relative efficacy of either regimen. Catapano and colleagues,using both regimens of rituximab for the treatment of refractory SLE, although not ina formal comparative setting, did not detect a significant difference in either thedegree of B-cell depletion or clinical outcomes [19]. The two-dose regimen, more convenient for patients requiring just the twohospital infusion visits, is therefore preferred.

Defining standard treatment used in the comparative arm is important, because notdoing so would allow generous use of other immunosuppressants - particularlycorticosteroids, which are highly effective but associated with unacceptable adverseeffects in the long term, not necessarily identified in clinical trials withshort-term follow-up.

It would be interesting to take a treatment-to-target approach to achieve an adequatedegree of B-cell depletion and clinical response. For example, evidence suggests thatthe efficacy depends on the extent of B-cell depletion in RA [50]. Several research groups have noted that the degree of B-cell depletion isvariable in SLE and that early repopulation is common in patients with a poorresponse to rituximab [35]. The underlying reasons for the variability in B-cell depletion remainelusive. A polymorphism in Fcγ receptor IIIa has been shown to be important inachieving an adequate degree of B-cell depletion, in favour of the high-affinitygenotypes Fcγ receptor IIIa V158F (V, valine; F, phenylalanine) [51]. Treatment-to-target would therefore seem a rational approach to take inan attempt to improve the major clinical response. However, some patients willprobably require more frequent doses than others. One approach could be tocounterbalance this variation using alternative dose regimes; for example, using two500 mg doses given 2 weeks apart, as in a recent trial in RA that reported equalefficacy, safety and tolerability between the two regimes using 500 mg or 1 g,provided adequate depletion was achieved [50, 52]. Different dosing regimens could potentially have considerableimplications: first, patient convenience, with a four-dose regimen requiring morehospital visits; second, a very-low dose regimen has been associated with thedevelopment of anti-drug antibodies in SLE while a medium dose (500 mg rituximab×2) has been shown to be adequate in a number of patients with RA [50]; and, finally, cost-effectiveness of BCDT. In this respect, it has beennoted that rituximab might be rapidly consumed in some patients, more frequently inSLE than RA [53]. This consumption would consequently reduce serum rituximab levels and mayreduce clinical efficacy.

Taking experience from ocrelizumab therapy in lupus, careful consideration is alsonecessary when designing studies to test the safety and efficacy of B-cell-targetedapproaches, including depletion in patients with active disease also takingmycophenolate. A combination of ocrelizumab and recently commenced mycophenolate doesnot appear to result in a meaningful additive response and results in an increasedrisk of infection adverse events (whether the combined impact on the B-cellpopulation of anti-CD20 and mycophenolate was a contributory factor is notunderstood), whereas this was not the case when used in combination with theEUROLUPUS cyclophosphamide followed by azathioprine regimen.

Defining the standard of care in the placebo arm is important to allow detection ofthe efficacy for the intervention tested. For example, in the placebo arm a patientwith disease activity requiring >7.5 mg prednisolone being classed as a failurewill allow detecting the steroid-sparing effect of the intervention, a majoradvantage in the long term. The question has been raised as to whether to userituximab in combination with cyclophosphamide, azathioprine or mycophenolate, butthere are some conflicting data [19, 54]. The definitive answer is therefore awaited.

Another conundrum not yet fully resolved is whether there really is added benefit inusing repeated rituximab infusions on a regular basis (that is, maintenance therapy)or whether it is preferable to repeat B-cell depletion only when the patientsrelapse. A concern about repeated infusion is the potential occurrence ofhypogammaglobulinaemia. Information from studies in patients with RA (J Edwards,personal communication) suggests that many patients begin to drop their IgG levelsafter annual rituximab infusions, particularly in patients with low baseline IgGlevels [55]. Comparative data for SLE patients are awaited.

Clinical evidence for rituximab use - early disease or chronic refractorydisease?

Limited evidence from two studies is worth considering. Firstly, as discussed, whenused early in conventional immunosuppressive naïve disease, rituximab seems tobe effective and has a steroid-sparing effect [24]. Further, Pepper and colleagues have prospectively analysed the responseto rituximab for biopsy-proven lupus nephritis, where a total of 14/18 (78%) patientsachieved a complete or partial remission with a sustained response in 12/18 at 1 year(67%), with two patients having a relapse with an increase in proteinuria. There wasa reduction in prednisolone usage from a mean of 10 mg to 5 mg at 2 years, sixpatients stopped, six patients managed to reduce the dose and the remaining weremaintained on the same dose. Five patients required a temporary increase forextra-renal manifestations [56].

Defining the outcome measures and clinical response

Clinical outcome measures are to be defined based on evidence, taking into accountthe probability of detecting change given the expected natural progress of theorgan-specific disease manifestations in an appropriate time-frame (potentially incontrast to the artificial time points used in clinical trials). In parallel, it isimportant to include the biomarkers that predict disease activity and outcomes inSLE. For example, there are a few validated outcome measures that predict end-stagerenal disease; it has been shown that doubling of serum creatinine [57, 58] and persistently elevated serum creatinine at 48 weeks [58] is predictive of end-stage renal disease. Another routinely availablebiomarker in clinical practice is urinary protein and an improvement in proteinuriaat 1 year [59] and a decrease in serum creatinine or proteinuria at 6 months [60], whilst it may also be reasonably expected that renal response maycontinue to improve beyond the first year of treatment and may be relevant toconsider when identifying the maximal treatment difference for a clinical trial.However, there is limited evidence of reliable predictors of long-term outcome fornonrenal SLE. For reasons discussed earlier, steroid-sparing effect is an importantfactor when deciding the immunosuppressant of choice [56].

What disease assessment index to use?

Disease activity indices have been developed with a view to assess either diseaseactivity or damage. The proposed SLE Responder Index, although used in the belimumabstudies [61, 62], has never been validated or shown to be reliable or sensitive to changeor appropriate for wide use when evaluating efficacy with other investigationalagents. The key problem with global score indices is that they do not capture partialimprovement and/or deterioration.

The definitions of treatment failure and flare remain variable between studies, whichlimit direct comparison of efficacy of different therapeutic agents. To facilitate abetter comparison between studies, therefore, it is important to standardise thedefinition of a flare and treatment failure.

Adequate follow-up period to detect significant change in the disease activity anddisease damage

Allowing an adequate follow-up period to detect clinically meaningful effects is veryimportant. For example, haematological abnormalities such as anaemia and autoimmunethrombocytopaenia and skin changes such as vasculitic rash improve rapidly; incontrast, response in nephritis may take much longer to detect. Other importantfactors such as the effects of long-term accruement of organ damage and drug-relatedadverse effects could only be detected after many years.

Defining the adverse effects

Adverse events recorded in the clinical trials in SLE have not been adequatelystandardised to allow comparison between trials. In chronic disease such as SLE wherea number of treatments have proved to have modest efficacy, adverse effectsassociated with treatment have a significant influence on the choice of treatment. Asdiscussed, achieving primary and secondary endpoints of efficacy at the expense ofunacceptable adverse events has proven unfruitful in the case of the anti-CD20(ocrelizumab) in RA [39] whilst the BELONG lupus nephritis trial was stopped early due to animbalance of infectious adverse events. This finding does raise the question ofwhether the screening and monitoring criteria can be applied more stringently for thedetection of risk or actual opportunistic infections prior to inclusion in the study,particularly when recruiting patients residing in areas endemic for opportunisticinfections as mycobacteria or hepatitis. Also, another important question remainingunanswered is whether the adverse effects of biological agents are influenced byother identifiable factors such as disease history and treatment as well as apatient's immunology or indeed ethnicity. A robust definition of categories ofadverse events therefore needs to be tested in clinical trials to understand andcompare the safety of interventions in clinical trials. For example, is mycophenolatesafe to use following rituximab induction therapy? Does the dose of mycophenolateneed to be modified to a low-dose regime or should an alternative less potentimmunosuppressant such as azathioprine be used? Further, the dose of drug may bebetter adjusted based on patient characteristics; for example, a dose defined by theweight of the patient rather than a predefined dose (that is, 2 to 3 g). This factoris especially important when considering the use of mycophenolate in patients withlow body mass index; for these patients, even 2 g may be a relatively high dose,especially when used in the maintenance regime following rituximab induction therapy.The recording of adverse events in clinical trials and open studies could bestandardised adhering to rheumatology-specific criteria such as the OMERACT [63].

Key messages

  • B-cell depletion with rituximab continues to be used in clinicalpractice for the treatment of refractory SLE, on the basis of a considerable number ofpublications describing the safety and efficacy data from small open studies andclinical experience whilst noting that it has not been approved by health authoritiesfor the treatment of lupus.

  • Contributing features that may have led to the failure of DBRCTs withanti-CD20-mediated B-cell depletion or at least identifying any true treatment effectsize probably include concomitant use of high-dose steroids, stringent andnonorgan-specific clinical response criteria, too short a follow-up, and, from astatistical perspective, the sample size. However, the trials confirm the safety ofrepeated treatment with rituximab.

  • A better response to rituximab detected in patients ofAfrican-American and Hispanic ancestry highlights the importance of preplanned subgroupanalysis and the need to better understand the potential disease drivers of a treatmenteffect when compared with a standard-of-care regimen in a trial setting.

  • The significant biological effects seen with rituximab need to bemonitored to assess clinical benefit and risk in the long term.

  • Future clinical trial design in SLE and lupus nephritis may be guidedby the key working groups of experts, including the European League Against Rheumatismtask force, in order to achieve standardisation and to continually apply lessons fromboth clinical and trial experience.


This article has been published as part of Arthritis Research & Therapy Volume 15 Supplement 1, 2013: B cells in autoimmune diseases: Part 2. Thesupplement was proposed by the journal and content was developed in consultation withthe Editors-in-Chief. Articles have been independently prepared by the authors and haveundergone the journal's standard peer review process. Publication of the supplement wassupported by Medimmune.