B-cell depletion in the treatment of patients with systemic lupus erythematosus: a longitudinal analysis of 24 patients

M. J. Leandro, G. Cambridge, J. C. Edwards, M. R. Ehrenstein and D. A. Isenberg

Centre for Rheumatology, University College London, London, UK.

Correspondence to: D. Isenberg, Centre for Rheumatology, University College London, 4th Floor, Arthur Stanley House, 40–50 Tottenham Street, London W1T 4NJ, UK. E-mail: d.isenberg{at}ucl.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 References
 
Objectives. To assess the clinical and basic serological consequences of B-cell depletion with rituximab in the treatment of patients with systemic lupus erythematosus (SLE) who have failed conventional immunosuppression.

Methods. An open study of 24 patients with severe SLE followed for a minimum of 3 months is reported. In the majority of patients (19 out of 24), 6 months follow-up data are described. Disease activity in these patients was assessed every 1–2 months using the British Isles Lupus Assessment Group (BILAG) system and estimates of anti-double-stranded DNA antibodies and serum C3 levels. During the follow-up period, significant side-effects were sought and the reduction in oral prednisolone was recorded. It was our general practice to stop concomitant immunosuppression (e.g. azathioprine, mycophenolate) when B-cell depletion was given (in most cases in the form of two 1 g intravenous infusions of rituximab 2 weeks apart accompanied by two 750 mg intravenous cyclophosphamide infusions and two methylprednisolone infusions of 250 mg each).

Results. Twenty-two patients were female and two male. At the time of B-cell depletion, the mean age was 28.9 yr (range 17–49) and the mean disease duration was 7.9 yr (range 1–18). The global BILAG score (P < 0.00001), serum C3 (P < 0.0005) and double-stranded DNA binding (P < 0.002) all improved from the time of B-cell depletion to 6 months after this treatment. Only one patient failed to achieve B-lymphocyte depletion in the peripheral blood. The period of B-lymphocyte depletion ranged from 3 to 8 months except for one patient who remains depleted at more than 4 yr. Analysis of the regular BILAG assessments showed that improvements occurred in each of the eight organs or systems. The mean daily prednisolone dose fell from 13.8 mg (S.D. 11.3) to 10 mg (S.D. 3.1).

Conclusion. In this open study of patients who had failed conventional immunosuppressive therapy, considerable utility in the use of B-cell depletion has been demonstrated. Our data provide strong support for the performance of a full double-blind control trial.

KEY WORDS: SLE, Rituximab, B-cell depletion


    Introduction
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 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 References
 
The past 50 yr have seen a substantial improvement in the mortality and morbidity figures for patients with systemic lupus erythematosus (SLE). In the 1950s, the 4-yr survival for lupus was thought to be approximately 50% [1], whereas most recently published series [reviewed in 2] now estimate a 15-yr survival rate of the order of 80%. This major improvement has been brought about by the introduction of corticosteroids, other immunosuppressive drugs, such as azathioprine and cyclophosphamide, and adjunctive therapies, including dialysis and renal transplantation. Although the more recent figures are most encouraging, a cohort of patients continues to suffer from very aggressive disease which, even with full compliance, continues to cause worrying morbidity and, sadly, mortality. In our own group of over 400 patients under follow-up, 15% have died at an average age of 51 yr [3]. Part of the ongoing problem is the severity of the disease in some patients, while in other cases the side-effects of the currently available immunosuppressives, notably the increased risk of infection, continue to make a major contribution to this increased mortality.

There is therefore a continuing need to identify new, more effective therapies for severely affected patients, with fewer side-effects. As reviewed elsewhere, recent interest has focused on the utility of removing C20-positive B lymphocytes in patients with SLE with rituximab, a chimeric monoclonal antibody directed to CD20 that is very effective in depleting normal and malignant B lymphocytes in vivo [4, 5]. Some encouraging early results have now been published [6, 7].

The notion that B-cell depletion might be useful as a form of treatment for patients with an autoimmune rheumatic disease, rheumatoid arthritis, was first demonstrated in an open study [8, 9]. A large double-blind multicentre controlled trial has recently confirmed the value of B-cell depletion in rheumatoid arthritis [10]. These early results were obtained by using a combination of rituximab, a chimeric monoclonal antibody recognizing the CD20 marker, together with corticosteroids (orally or intravenously) and intravenous cyclophosphamide or oral methotrexate.

In our first report of six patients with SLE given rituximab, intravenous cyclophosphamide and corticosteroids [6], we described both clinical improvement and a lowering of double-stranded DNA (dsDNA) antibody levels and an increase in C3 in some of the patients. We now report a long-term follow-up study of up to 4 yr in 24 patients treated with B-cell depletion.


    Patients, materials and methods
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 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 References
 
All of the patients included in the study met four or more of the revised classification criteria for the American College of Rheumatology [11]. Most patients were treated on an urgent basis because of clinical need as they had failed conventional immunosuppressive therapy. Local hospital ethical committee approval and patients’ informed consent was obtained. Patients’ baseline characteristics are summarized in Table 1.


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TABLE 1. Patients' baseline characteristics

 
Treatment protocols were based on rituximab. The first six patients (patients 1–6) were treated with two infusions of 500 mg rituximab (half dose) and two infusions of 750 mg cyclophosphamide under oral steroid cover (2 x prednisolone 60 mg for 5 days) given 2 weeks apart. Subsequently the patients were treated with a similar protocol but with full-dose rituximab (2 x 1000 mg) and intravenous methylprednisolone (2 x 250 mg) instead of oral steroids. Two patients received rituximab monotherapy: one had an allergy to cyclophosphamide (patient 12) and the other patient refused to have the drug (patient 9). Patients were allowed to continue prednisolone and hydroxychloroquine. Other immunosuppressives were stopped at baseline except for two patients who continued their azathioprine (patient 15 started at baseline on 100 mg; patient 16 continued on 150 mg). All except three patients had previously been treated with intravenous cyclophosphamide (Table 1).

The clinical assessment of these patients, most of whom were followed every 1–2 months in our lupus clinic, was undertaken using the British Isles Lupus Assessment Group (BILAG) disease activity index [12]. This index distinguishes disease activity in eight organs or systems using the principle of the physician's intention to treat. A combination of clinical features and a small number of blood test results allows the distinction of patients from the most active, grade A, to the never active, grade E, in that organ or system. A global BILAG numerical score can be derived using the following notation: A = 9 points, B = 3 points, C = 1 point, and D/E = 0 points. The patients’ routine full blood count, serum creatinine, C3 (laser nephelometry), total immunoglobulin and dsDNA binding (Shield Diagnostics, Dundee, UK; normal <50 units/ml) and urine protein to creatinine ratio were tested in the hospital routine laboratories. Paired t-tests or Wilcoxon matched-pairs signed-rank tests were used to compare global BILAG scores and key laboratory test results prior to and 3 months and 6 months after B-cell depletion.


    Results
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 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 References
 
Twenty-four patients were treated (22 female, two male). At the time of B-cell depletion, the mean age was 28.9 yr (range 17–49) and mean disease duration was 7.9 yr (range 1–18). Data at baseline and 3 months include all patients. Data at 6 months include 19 of the 24 patients. For patients 1, 18, 19 and 22, data at 6 months are not available. Patient 1 was lost to follow up at 3 months. Patient 18 did not deplete and other treatment options had to be considered. Patient 19 repopulated at 3 months and was retreated. Patient 22 died at approximately 5 months with pancarditis.

Only one patient (patient 18) failed to achieve B-lymphocyte depletion in the peripheral blood (i.e. CD 19 count <0.005 x 109/l). The period of B-lymphocyte depletion ranged from 3 to 8 months, except for one patient who remains depleted at more than 4 yr (patient 3).

The mean BILAG global score fell from a mean of 13.9 (S.D. 5.8) at baseline to 6.8 (S.D. 4.7) at 3 months and 5.0 (S.D. 2.3) at 6 months. Table 2 shows more detailed data on sustained improvement of at least two BILAG grades, i.e. A -> D, A -> C and B -> D, and also B -> C in each of the eight organs or systems. It is clear that B-cell depletion was able to improve some aspects of lupus activity in every organ and system. In contrast, no lupus patient developed any major sustained deterioration (i.e. new A scores or D -> B scores) before B-cell repopulation of the peripheral blood occurred. Manifestations such as fatigue, arthralgia/arthritis, serositis, nephritis, thrombocytopenia and haemolytic anaemia responded particularly well to the treatment. It should be noted that relatively few patients with major CNS disease were treated (n = 3). No clear differences in outcome were detected between patients treated with different protocols. The mean daily dose of prednisolone before treatment was 13.8 mg (S.D. 11.3) and for those completing 6 months of follow-up (n = 21) it had fallen to 10 mg (S.D. 3.1).


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TABLE 2. Changes in detailed BILAG scores 6 months after B-cell depletion (n = 19 patients)

 
Mean total serum immunoglobulin levels decreased mildly to moderately but remained within the normal range. Figure 1 shows the global BILAG score, clinical and laboratory parameters before and 6 months after B-cell depletion. It is notable that the fall in global BILAG score (P<0.00001), the fall in serum anti-dsDNA antibody levels (P<0.002) and the rise in serum C3 levels (P<0.0005) are highly statistically significant. The protein:creatinine ratio decreased following treatment but the decrease did not reach statistical significance (P<0.08).



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FIG. 1. BILAG global scores, serum levels of C3 and anti-dsDNA antibodies, and urine protein:creatinine ratio at baseline and 6 months after B-cell depletion (n = 19 patients; median ± S.E.M.).

 
One patient (patient 9) had a severe reaction to the second rituximab infusion, with thrombocytopenia followed by pancytopenia, from which he recovered completely. One patient (patient 22) died approximately 5 months after B-cell depletion. She had had very aggressive disease with major skin, renal, joint and neuropsychiatric disease. Her BILAG score improved from 45 at baseline to 25 after 3 months but she later developed a fatal pancarditis. B-cell repopulation of the peripheral blood was already detectable before she developed sepsis. No other serious adverse events, including serious or opportunistic infections, have been observed. In particular, one patient (patient 3) remains depleted more than 4 yr after treatment. She has not had any serious or opportunistic infections.

The mean follow-up is now 23 months (range 3–51). At present, 13 patients (patients 2, 3, 6, 8, 9, 13, 14, 15, 16, 17, 21, 23 and 24) remain well enough not to have had to restart other immunosuppressive therapy (follow-up ranging from 7 to 51 months). Seven patients have been retreated (eight treatments). One patient failed to deplete on retreatment due to a documented specific human anti-chimeric antibody (HACA) response (described elsewhere [13]).


    Discussion
 Top
 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 References
 
This report significantly extends both the clinical follow-up and serological analysis following the use of B-cell depletion in a group of patients with SLE whose disease has remained active in spite of intensive immunosuppression. The results of this open study conducted over a period of up to 4 yr provide further encouragement for the view that B-cell depletion may be a very useful addition to the range of therapies available for patients with very active lupus. In a group of 24 patients with active, refractory SLE, all 23 patients who showed significant depletion improved following treatment. Encouragingly, we have seen significant clinical improvement in virtually all of the organs and systems commonly affected by lupus. The possible exception to this is patients with major CNS lupus, for whom we have insufficient data as yet to make any tenable claim.

Whilst B-cell depletion using a combination of rituximab, cyclophosphamide and prednisolone is unlikely to be a cure for lupus, the fact that the majority of our patients were substantially improved in spite of having failed years of conventional immunosuppression is notable. Our key observations are that: the treatment regimens we have used almost always achieve a substantial depletion of B cells in the peripheral blood (>99%); the overwhelming majority of patients in whom this depletion is achieved improved clinically, some going into remission; the periods of B-cell depletion in the peripheral blood ranged from 3 to 11 months, except for one patient who remains depleted at more than 4 yr; disease flare does not occur until or a variable period of time after the CD20-positive B cells return to the peripheral blood; and the treatment was well tolerated in the majority of patients. We did, however, note that one patient had a severe infusion reaction and that another patient with very aggressive disease died of pancarditis 5 months after being treated. B-cell repopulation of the peripheral blood was already detectable before she died. In this small, heterogeneous group of patients, we have not been able to detect clear differences between the different protocols. We would advise against using a smaller dose of rituximab (2 x 500 mg) as this might predispose to the development of HACAs. In our experience, the period of B-cell depletion in the peripheral blood in patients with SLE is frequently shorter than in patients with rheumatoid arthritis treated with the same protocol or than what has been reported for patients with lymphoma. It is likely that these differences are accounted for by variation in the pharmacokinetics and effectiveness of the mechanisms of depletion of CD20-positive B cells by rituximab.

The mean daily dose of prednisolone before treatment was 13.8 mg (S.D. 11.3) and for those completing 6 months of follow-up (n = 21) it had fallen to 10 mg (S.D. 3.1). Oral prednisolone was only decreased slowly and progressively once clinical improvement was clear. Patients who remained well after 6 months continued to decrease the dose of prednisolone further.

The serological data are also of great interest. We have shown in this report that antibodies to dsDNA and serum C3 levels are significantly improved following B-cell depletion. This improvement matches the global and individual BILAG organ/system scores. Looney et al. [14] also reported a significant improvement in the global disease activity of 11 out of 17 of the patients they treated (using the SLAM activity index) in the absence of a significant change in anti-dsDNA antibody or complement levels. In the same trial these authors report a significant decrease in anti-dsDNA antibody levels in four out of eight patients in whom effective B-cell depletion was obtained and in whom elevated levels of anti-DNA antibodies at baseline were noted, even though a significant decrease was not found in the group as a whole [15]. It should be noted that this was a phase I/II dose escalation trial and patients were given three different doses of rituximab, two below the recommended dose, and unaccompanied by cyclophosphamide or methylprednisolone. Their results also confirm the safety of B-cell depletion as a therapeutic approach.

In summary, these encouraging long-term results on 24 patients with lupus severely active despite years of immunosuppression strongly encourage the view that removal of CD20-positive B cells may be a very useful way of treating patients with active lupus. It is now timely to undertake a large double-blind randomized control trial of this treatment.

D.A.I. declares that in lieu of payment for a consultancy undertaken for Roche, they have paid a fee into a back arthritis charity of which he is one of the trustees. The other authors have declared no conflict of interest.


    References
 Top
 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 References
 

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  11. Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7.[ISI][Medline]
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Submitted 26 April 2005; revised version accepted 19 July 2005.



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