Tocilizumab in systemic lupus erythematosusData on safety preliminary efficacy and impact on circulating plasma cells from an open-label phase I dosage-escalation study.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 62, No. 2, February 2010, pp 542–552 DOI 10.1002/art.27221 © 2010, American College of Rheumatology Tocilizumab in Systemic Lupus Erythematosus Data on Safety, Preliminary Efficacy, and Impact on Circulating Plasma Cells From an Open-Label Phase I Dosage-Escalation Study Gabor G. Illei,1 Yuko Shirota,1 Cheryl H. Yarboro,2 Jimmy Daruwalla,2 Edward Tackey,2 Kazuki Takada,2 Thomas Fleisher,3 James E. Balow,4 and Peter E. Lipsky2 curred in 11 patients; none was associated with neutropenia. Disease activity showed significant improvement, with a decrease of >4 points in the modified Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index score in 8 of the 15 evaluable patients. Arthritis improved in all 7 patients who had arthritis at baseline and resolved in 4 of them. Levels of anti–double-stranded DNA antibodies decreased by a median of 47% in patients in the 4 mg/kg and 8 mg/kg dosage groups, with a 7.8% decrease in their IgG levels. These changes, together with a significant decrease in the frequency of circulating plasma cells, suggest a specific effect of tocilizumab on autoantibody-producing cells. Conclusion. Although neutropenia may limit the maximum dosage of tocilizumab in patients with SLE, the observed clinical and serologic responses are promising and warrant further studies to establish the optimal dosing regimen and efficacy. Objective. To assess the safety of interleukin-6 receptor inhibition and to collect preliminary data on the clinical and immunologic efficacy of tocilizumab in patients with systemic lupus erythematosus (SLE). Methods. In an open-label phase I dosageescalation study, 16 patients with mild-to-moderate disease activity were assigned to receive 1 of 3 doses of tocilizumab given intravenously every other week for 12 weeks (total of 7 infusions): 2 mg/kg in 4 patients, 4 mg/kg in 6 patients, or 8 mg/kg in 6 patients. Patients were then monitored for an additional 8 weeks. Results. The infusions were well tolerated. Tocilizumab treatment led to dosage-related decreases in the absolute neutrophil count, with a median decrease of 38% in the 4 mg/kg dosage group and 56% in the 8 mg/kg dosage group. Neutrophil counts returned to normal after cessation of treatment. One patient was withdrawn from the study because of neutropenia. Infections ocClinicalTrials.gov identifier: NCT00046774. Supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH. This study was performed under a Collaborative Research and Development Agreement with Chugai Pharmaceutical Company, Ltd. 1 Gabor G. Illei, MD, PhD, MHS, Yuko Shirota, MD, PhD: National Institute of Arthritis and Musculoskeletal and Skin Diseases, and National Institute of Dental and Craniofacial Research, NIH; 2Cheryl H. Yarboro, RN, BSPA, CCRC, Jimmy Daruwalla, BSc, Edward Tackey, MD, Kazuki Takada, MD, Peter E. Lipsky, MD: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH; 3Thomas Fleisher, MD: Department of Laboratory Medicine, Clinical Center, NIH; 4James E. Balow, MD: National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland. Address correspondence and reprint requests to Gabor G. Illei, MD, PhD, MHS, Head, Sjögren’s Syndrome Clinic, National Institute of Dental and Craniofacial Research, National Institutes of Health, 10 Center Drive, Room 1N114, Bethesda, MD 20892. E-mail: firstname.lastname@example.org. Submitted for publication October 20, 2008; accepted in revised form October 16, 2009. Autoantibody production, complement activation, immune complex deposition, and leukocyte infiltration of target organs are key immunopathogenic events in systemic lupus erythematosus (SLE). Multiple cytokines have been implicated in the regulation of disease activity or organ involvement in SLE. Among these, interleukin-6 (IL-6), which exerts pleiotropic effects on numerous cell types (1), is thought to play an important role. In murine models of lupus, an ageassociated increase in serum levels of IL-6 and abnormal expression of the IL-6 receptor have been described (2–4). The addition of exogenous IL-6 was shown to increase autoantibody production and accelerate the progression of glomerulonephritis (5,6), whereas block542 IL-6 RECEPTOR BLOCKADE IN SLE ade of IL-6 or its receptor was shown to prevent increased anti–double-stranded DNA (anti-dsDNA) antibody levels and progression of proteinuria and to improve mortality rates (7–9). Lupus patients have elevated serum levels of IL-6 (10–13), which were found to be correlated with disease activity or with anti-dsDNA levels in some, but not all, studies. Moreover, neutralization of IL-6 led to a significant decrease in the spontaneous production of immunoglobulin (12) and anti-dsDNA in vitro (14). Several studies have demonstrated increased urinary excretion of IL-6 in patients with active proliferative lupus nephritis (13,15,16). IL-6 excretion was found to be decreased following cyclophosphamide treatment, suggesting that IL-6 may play an important role in lupus nephritis. Based on these data, we hypothesized that blocking the effect of IL-6 may be beneficial in SLE. Tocilizumab, a humanized monoclonal antibody against the ␣-chain of the IL-6 receptor, prevents the binding of IL-6 to membrane-bound and soluble IL-6 receptor (17). The safety and efficacy of tocilizumab have been evaluated in clinical trials in patients with rheumatoid arthritis (RA), juvenile idiopathic arthritis, and Castleman’s disease (18). Herein, we report the findings of a pilot clinical study of tocilizumab treatment in patients with SLE. PATIENTS AND METHODS Study design. This was an open-label phase I dosageescalation pilot study to evaluate the safety and tolerability of tocilizumab in patients with SLE and to obtain preliminary evidence of its potential efficacy in this disease. The study was approved by the Institutional Review Board of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health (NIH). All patients signed informed consent forms. Patient selection. Between 2003 and 2005, 16 adult patients (age ⬎18 years) who fulfilled the American College of Rheumatology classification criteria for SLE (19,20) and attended the NIH Clinical Center were enrolled in the study. All patients had moderately active lupus, as defined by 1 of the following 2 sets of criteria. Criteria set 1 consisted of the presence of chronic glomerulonephritis, with an inadequate response to at least 6 months of adequate immunosuppressive therapy (with either methylprednisolone pulse doses, cyclophosphamide, azathioprine, cyclosporine, mycophenolate mofetil, high-dose daily corticosteroids, methotrexate, or intravenous immunoglobulin [IVIG]), plus the following 4 features: less than a 30% increase in serum creatinine levels as compared with the lowest level achieved during treatment; proteinuria at levels ⱕ1.5 times the value at baseline (before treatment); ⱕ2⫹ cellular casts in the urinary sediment; and extrarenal disease activity not exceeding a score of 10 on the 543 nonrenal components of the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (21). Criteria set 2 consisted of moderately active extrarenal lupus, which was defined as an extrarenal SELENA–SLEDAI score in the range of 3–10. The SELENA–SLEDAI score must have been stable for at least 2 weeks prior to screening. Because 2 of the main effects of IL-6 are on inflammatory responses and antibody production, we required the presence of at least 1 serologic marker of autoantibody production or systemic inflammation. Thus, 1 or more of the following 4 features had to be present in eligible patients: a serum anti-dsDNA antibody level ⱖ30 IU, an IgG anticardiolipin antibody level ⱖ20 IgG phospholipid units/ml, a C-reactive protein level (CRP) ⬎0.8 mg/dl, or an erythrocyte sedimentation rate (ESR) ⬎25 mm/hour in men and ⬎42 mm/hour in women. Patients who were taking prednisone (or equivalent) were required to have been taking a stable dosage of ⱕ0.3 mg/kg/day for at least 2 weeks before the first dose of study medication was administered. Patients were required to use an effective form of contraception throughout the study. The main exclusion criteria included pregnancy; any therapy with human or murine antibodies or any experimental therapy within 3 months; therapy with cyclophosphamide; pulse methylprednisolone or IVIG within 4 weeks; or azathioprine, mycophenolate mofetil, cyclosporine, or methotrexate within 2 weeks of the first dose of study medication. Patients with any of the following abnormal findings on laboratory tests were also excluded: serum creatinine level ⬎3.0 mg/dl, white blood cell count ⬍3,500/ l, absolute neutrophil count ⬍3,000/l, absolute lymphocyte count ⱕ500/l, hemoglobin value ⬍8.0 gm/dl, platelet count ⬍50,000/l, alanine aminotransferase and/or aspartate aminotransferase (AST) level ⬎1.5 times the upper limit of normal, alkaline phosphatase level ⬎1.5 times the upper limit of normal, or ⬎1,000 Epstein-Barr virus genome equivalents/106 peripheral blood mononuclear cells. Study medication protocol. The tocilizumab was provided by Chugai Pharmaceutical (Tokyo, Japan). Study subjects were assigned to receive 1 of the following 3 doses of tocilizumab: group 1 (n ⫽ 4) received 2 mg/kg, group 2 (n ⫽ 6) received 4 mg/kg, and group 3 (n ⫽ 6) received 8 mg/kg. Tocilizumab was administered intravenously every 2 weeks for 12 weeks, for a total of 7 infusions. Concomitant immunosuppressive therapy. Patients who were taking prednisone (or equivalent) at study entry were required to be receiving a dosage of ⱕ0.3 mg/kg/day. The prednisone dosage was kept stable during the first 7 weeks of treatment. After that time, a gradual tapering of the dosage to 0.15 mg/kg/day was allowed. Temporary increases in the prednisone dosage were allowed based on disease activity. Patients had to be withdrawn from the study if a prednisone dosage ⱖ0.5 mg/kg/day was required or if there was no response to a 2-week course of prednisone (or equivalent) at a dosage of ⱕ0.5 mg/kg/day. The following medications were allowed, provided that they were administered at stable dosages for at least 2 weeks before, as well as during, the study: hydroxychloroquine, nonsteroidal antiinflammatory drugs, angiotensin-converting enzyme inhibitors, and angiotensin receptor antagonists. 544 Safety and toxicity analyses. Patients were monitored during the infusions of study medication and every 2–4 weeks for the 5-month study period. Safety assessments included unexpected toxicities, adverse events encountered during or after the drug infusion, changes in vital signs, and changes in clinical laboratory data. Adverse events were graded according to the National Cancer Institute Common Toxicity Criteria (CTC) guidelines. Upon occurrence of a grade 3 or 4 adverse event, which was at least possibly related to the study drug, tocilizumab was discontinued unless the event resolved spontaneously within 9 days. Patients who were withdrawn from treatment received 8 weeks of followup after withdrawal. Efficacy analysis. Clinical and laboratory data were collected for preliminary evaluation of the potential activity of tocilizumab on the SLE as well as the measures of inflammation. Parameters used for efficacy measures were assessed at the initial screening, prior to selected study drug infusions (weeks 0, 2, 4, 6, and 12), and during followup visits (weeks 14, 16, and 20). Lupus activity indices and serologic markers of disease activity and inflammation were used to assess efficacy. Lupus activity indices consisted of the Systemic Lupus Activity Measure (SLAM) (22) and the modified SELENA–SLEDAI (mSELENA–SLEDAI). This latter index was modified to omit 1 of the standard parameters (“low complement”) because tocilizumab has been shown to lower complement levels in RA patients independently of disease activity (Tocilizumab Investigator’s Brochure). The following serologic markers of SLE were determined: anti-dsDNA antibodies, anticardiolipin antibodies, antinuclear antibodies, anti–extractable nuclear antigen. In addition, quantitative assessments of immunoglobulins (IgG, IgA, and IgM) and markers of inflammation (CRP, ESR, fibrinogen, and ferritin) were performed. Measurement of complement activation products (CAP). Complement activation products iC3b, C4d, and C5b–9 were measured in plasma by enzyme-linked immunosorbent assay (Quidel, San Diego, CA) according to the manufacturer’s instructions. Flow cytometry. Flow cytometric analysis was performed using a FACSCalibur flow cytometer with CellQuest Pro software (both from Becton Dickinson, Mountain View, CA). Statistical analysis. The safety analysis included every patient who received ⱖ1 infusion of the study drug. The efficacy analysis included patients who received at least 3 infusions of tocilizumab. Continuous variables were summarized, with means, standard deviations, medians, and ranges reported. Categorical variables were summarized by frequencies and counts of patients in the corresponding categories. All statistical tests were performed at the 0.05 significance level, and all confidence intervals were 95% 2-sided intervals. No formal sample size calculation was performed, since this study was designed predominantly to explore the tolerability and safety of these regimens. The choice of 16 patients was based on the experience with similar studies in the past. The study was not powered to detect differences between groups or to confirm efficacy. Therefore, there is a risk of false-positive results because of the large number of end points being considered. As a consequence, results of efficacy analyses comparing the dosage groups are exploratory in nature. ILLEI ET AL RESULTS Characteristics of the SLE patients at baseline. Sixteen SLE patients (13 women and 3 men) from various ethnic backgrounds and with moderate disease activity were enrolled in the study. The baseline demographic and clinical characteristics are shown in Table 1. The mean mSELENA–SLEDAI score was 11.5, 10, and 8 in the 2 mg/kg, 4 mg/kg, and 8 mg/kg dosage groups, respectively. All but 1 of the patients were taking prednisone. All 12 of the patients who were taking hydroxychloroquine had been receiving stable dosages for at least 3 months prior to the first dose of study medication. Fifteen patients received 7 infusions of tocilizumab (4 at 2 mg/kg, 6 at 4 mg/kg, and 5 at 8 mg/kg). One patient in the 8 mg/kg dose group was withdrawn after the first infusion because of grade 3 neutropenia. Findings of the safety analyses. The infusions were well tolerated, without any clinically significant infusion reactions. All patients experienced 1 or more tocilizumab treatment-related adverse events; however, most of these were mild and resolved spontaneously. Serious adverse events. There were 2 serious adverse events, both of which occurred in the same patient; this patient was in the group receiving 8 mg/kg of tocilizumab. The first serious adverse event was a hospitalization for acute gastroenteritis 10 days after the second infusion. A detailed diagnostic evaluation failed to identify any specific cause. The patient recovered spontaneously and completed the treatment course. At 8 weeks after the last tocilizumab dose, this same patient developed acute pyelonephritis, which responded well to treatment with intravenous antibiotics. In addition to these 2 serious adverse events, 2 patients in the group receiving 8 mg/kg experienced severe neutropenia, with an absolute neutrophil count of 500–1,000/l (CTC grade 3). Infections. Eleven patients (4 receiving 2 mg/kg of tocilizumab, 4 receiving 4 mg/kg, and 3 receiving 8 mg/kg) experienced a total of 16 infections between the start of study treatment and the end of the followup period (Table 2). Two patients experienced repeated infections. The majority of these were upper respiratory tract (n ⫽ 5) or urinary tract (n ⫽ 3) infections. Ten of the 16 episodes of infection were treated with systemic antibiotics or antivirals. No infection led to withdrawal from the study, but the next tocilizumab infusion was delayed by 2 weeks when 1 patient in the 2 mg/kg dosage group experienced herpes zoster keratitis. This responded to treatment with antiviral agents and did not IL-6 RECEPTOR BLOCKADE IN SLE 545 Table 1. Baseline characteristics of the SLE patients in the safety and efficacy analyses, by tocilizumab dosage group* Tocilizumab dose All patients Safety analysis No. of patients Age, median (range) years No. (%) female Ethnicity, no. (%) Caucasian African American Other† Duration of SLE, median (range) years Efficacy analysis No. of patients‡ No. (%) with renal involvement mSELENA–SLEDAI, median (range) score§ SLAM, median (range) score Anti-dsDNA No. (%) positive Median (range) level, IU/ml‡ Prednisone dosage, median (range) mg/day 2 mg/kg 16 36.5 (23–54) 13 (81.3) 4 mg/kg 4 34.5 (23–37) 4 (100) 9 (56.3) 5 (31.3) 2 (12.4) 14.5 (1.3–23.6) 1 (25) 1 (25) 2 (50) 11 (1.3–22.8) 15 5 (33.3) 8 (4–15) 8 (2–12) 4 2 (50) 11.5 (4–15) 8 (4–12) 13 (86.7) 192 (0–1,849) 7.5 (0–20) 8 mg/kg 6 47.5 (26–54) 4 (66.7) 6 38.0 (28–47) 5 (83.3) 4 (66.7) 2 (33.3) 0 (0) 16.7 (6.5–23.6) 4 (66.7) 2 (33.3) 0 (0) 15.6 (4.9–19.9) 6 1 (16.7) 10 (4–14) 8 (2–9) 4 (100) 124 (37–457) 7.5 (1.25–10) 4 (67) 121.5 (0–872) 6.25 (2.5–15) 5 2 (40) 8 (6–10) 7 (2–10) 5 (100) 201 (51–1,849) 10 (0–20) * Tocilizumab was administered every other week for 12 weeks (total of 7 infusions). SLE ⫽ systemic lupus erythematosus; SLAM ⫽ Systemic Lupus Activity Measure; anti-dsDNA ⫽ anti–double-stranded DNA. † One of these 2 patients was Asian, and the other was Hispanic. ‡ One African American man assigned to receive 8 mg/kg of tocilizumab was withdrawn after the first infusion because of grade 3 neutropenia. § Hypocomplementemia was excluded from scoring on the modified Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index (mSELENA–SLEDAI). See Patients and Methods for details. recur after tocilizumab was resumed. All other patients continued their tocilizumab therapy uninterrupted while being treated for their infection. Changes in blood chemistry parameters. A slight increase in the mean AST level, but within the range of normal, was seen during the study period. The mean levels of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides increased slightly during the early treatment period, but there were no clinically or statistically signif- Table 2. Treatment-emergent infections observed during the study period* Tocilizumab dose 2 mg/kg Patient Patient Patient Patient Patient Patient Patient 4 mg/kg Patient Patient Patient Patient 8 mg/kg Patient Patient Patient Patient Patient Infection Time point (week) SAE 001 002 002 002 002 003 004 Urinary tract infection Folliculitis Upper respiratory tract infection Otitis media Urinary tract infection Sinusitis Upper respiratory tract infection 14 2 5 5 20 16 13 No No No No No No No Oral antibiotics Topical antibiotics Oral antibiotics Oral antibiotics Oral antibiotics Oral antibiotics Oral antibiotics 005 007 Upper respiratory tract infection Herpes zoster keratitis 15 5 No No 009 010 Upper respiratory tract infection Oral candidiasis 8 2 No No Oral antibiotics Tocilizumab infusion delayed by 2 weeks; antiviral Symptomatic Topical antifungal 013 013 013 014 020 Urinary tract infection Labial herpes simplex Acute pyelonephritis Upper respiratory tract infection Fungal vaginosis 12 13 20 1 3 No No Yes No No Oral antibiotics None Intravenous antibiotics Symptomatic Oral antifungal Treatment * Tocilizumab was administered every other week for 12 weeks (total of 7 infusions). SAE ⫽ serious adverse event. 546 icant changes by the end of treatment (data not shown). The albumin levels increased from a mean of 3.45 mg/dl to 3.77 gm/dl (P ⫽ 0.001 by Student’s t-test). Changes in hematologic parameters. The mean platelet counts decreased slightly with treatment, but the changes were not clinically significant. Conversely, the hematocrit and hemoglobin values showed gradual and sustained increases over the treatment period, with a maximum mean increase in the hemoglobin value of 1.54 gm/dl at week 16 in the group receiving 8 mg/kg of tocilizumab. (Data from the assessment of hematologic parameters over the course of the study are available upon request from the corresponding author.) White blood cell counts decreased in all dosage groups, largely because of the dose-related reduction in absolute neutrophil counts. Most of the change in neutrophil counts occurred following the first dose, reaching a maximum at the end of the treatment phase and returning to baseline levels by week 20 of followup (Figures 1a and b). The median decrease at 14 weeks (2 weeks after the last treatment) was 2% (range 0–15%) in those receiving 2 mg/kg of tocilizumab, 38% (range 7–42%) in those receiving 4 mg/kg, and 56% (range 48–72%) in those receiving 8 mg/kg. Two patients in the 8 mg/kg dosage group had CTC grade 3 neutropenia (absolute neutrophil count ⬍1,000/l). One of these patients was withdrawn from the study after the first dose of tocilizumab. In the other patient, the neutropenia occurred 2 weeks after the last infusion of tocilizumab. Neither of these patients had any infections or other clinical consequences of the neutropenia, and their neutrophil counts recovered spontaneously off study drug treatment. Findings of the efficacy analyses. Fifteen patients were included in the preliminary efficacy analysis. All of these patients had completed the 7 infusions of study drug as well as the followup assessments. Changes in markers of inflammation. The levels of all acute-phase reactants decreased promptly and significantly upon tocilizumab treatment, confirming the biologic activity of tocilizumab. Most changes occurred after the first dose and were maintained during the treatment period, particularly the ESR and fibrinogen levels (P ⬍ 0.001 at 6 and 14 weeks for both acute-phase reactants). (Data from the assessment of the ESR and fibrinogen levels over the course of the study are available upon request from the corresponding author.) Serologic response. There were clear dose-related decreases in the levels of complement C3 and C4. Since a decrease in complement levels can be caused by either decreased production or increased consumption, we measured the levels of the complement activation prod- ILLEI ET AL Figure 1. Changes in absolute neutrophil counts and complement products in patients with systemic lupus erythematosus receiving treatment with 2 mg/kg, 4 mg/kg, or 8 mg/kg of tocilizumab from baseline to week 14 of study. a and b, There was a dose-dependent decrease in the absolute neutrophil count (ANC), including the number of neutrophils per microliter (a) and the proportional change from baseline (b), during treatment. These values returned to baseline during the followup period after treatment. c, Tocilizumab led to a similar decrease in the levels of complement C3 and of the complement activation products iC3b and C5b–9 (terminal membrane attack complex), all of which returned to baseline during the followup period after treatment. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.005 for the whole cohort at the indicated time points as compared with baseline, by repeated-measures analysis of variance. ucts iC3b, C4d, and C5b–9 in 10 patients in the 4 and 8 mg/kg dosage group. All of these complement activation products showed decreased absolute levels or decreased IL-6 RECEPTOR BLOCKADE IN SLE 547 Figure 2. Serologic changes in patients with systemic lupus erythematosus receiving treatment with 2 mg/kg, 4 mg/kg, or 8 mg/kg of tocilizumab from baseline to week 14 of study. a, Levels of anti–doublestranded DNA (anti-dsDNA) antibodies decreased during treatment in all tocilizumab dosage groups (left), but mainly in the combined group of patients receiving the medium and high dosages (right). b, The proportional change in anti-dsDNA levels was substantially higher than the small, not statistically significant, decrease in IgG levels in all tocilizumab dosage groups (left), especially in the combined group of patients receiving the medium and high dosages (right). Only patients who had anti-dsDNA antibodies at baseline (n ⫽ 13 in the entire cohort; n ⫽ 9 in the medium/high dose only group) were included in these analyses. Data are shown as box plots. Each box represents the 25th to 75th percentiles, the lines within the boxes represent the median (50th percentile), and the lines outside the boxes represent the 10th and 90th percentiles. Solid circles indicate outliers. P values comparing the end of treatment data with the baseline data were determined by Wilcoxon’s signed rank test. levels relative to C3 (Figure 1c) or C4, with the levels of the terminal membrane attack complex decreasing most significantly. There were no significant changes in IgA and IgM levels, but by the end of the treatment period, IgG levels had decreased by a median of 130 mg/dl (minimum –660, maximum ⫹230 mg/dl; P ⫽ 0.04 by Wilcoxon’s signed rank test) as compared with baseline. Next, we evaluated the effect of tocilizumab on anti-dsDNA levels. Thirteen patients had detectable anti-dsDNA antibodies at baseline, ranging from 37 IU/ml to 1,849 IU/ml (normal ⬍25). The median decrease in anti-dsDNA antibody levels at week 14 was ⫺9 IU/ml (minimum ⫺417, maximum ⫹125; P ⫽ 0.03 by Wilcoxon’s signed rank test) (Figure 2a). To adjust for the wide range of baseline values, we also evaluated the proportional change in IgG and anti-dsDNA levels in these 13 patients and found that the relative change in anti-dsDNA levels was twice as much (median ⫺17.6%) as the relative change in IgG levels (median ⫺7.91%) (Figure 2b). In contrast to the findings in the 4 mg/kg and 8 mg/kg dosage groups, where we did not observe any increase in anti-dsDNA antibodies, 2 of 4 patients in the 2 mg/kg dosage group had an increase level of antidsDNA antibodies at week 14. Therefore, we performed a subgroup analysis of the 9 patients in the combined medium- and high-dose groups (4 and 8 mg/kg) who had anti-dsDNA antibodies at baseline (range 51–1,849 IU/ ml). In these patients anti-dsDNA antibody levels decreased by a median of ⫺113 IU/ml (minimum ⫺417, maximum ⫹1; P ⫽ 0.01 by Wilcoxon’s signed rank test) 548 ILLEI ET AL Figure 3. Changes in circulating plasma cells in patients with systemic lupus erythematosus (SLE) receiving treatment with 2 mg/kg, 4 mg/kg, or 8 mg/kg of tocilizumab from baseline to week 14 of study. a, Percentages of plasma cells in healthy control subjects (n ⫽ 14) versus SLE patients (n ⫽ 13) at baseline. Patients had a significantly higher proportion of plasma cells at baseline as compared with healthy controls (determined by Student’s t-test). Each data point represents a single subject; the open circles joined with a line show the mean of each group. b, Flow cytometry showing the response to tocilizumab at baseline and week 12 in a representative SLE patient. There was a change in circulating CD38⫹⫹⫹IgD– plasma cells before and after therapy. Values shown in each compartment are percentages. Cell subsets in the lower compartments are defined at the bottom. c, Frequency of plasma cells in the SLE patients at baseline, week 6, week 14 (end of tocilizumab treatment), and week 20 (end of followup). The frequency of plasma cells decreased significantly with tocilizumab treatment. Values are the mean ⫾ SEM. P value was calculated by repeated-measures analysis of variance, with the Bonferroni/Dunn correction for multiple comparisons. at week 14 (Figure 2a). Analysis of the proportional changes in these patients showed a small, but statistically significant, change in the IgG levels (median change –7.8% [minimum –32.7%, maximum 0.0%]; P ⫽ 0.01) at the end of treatment (week 14). However, this was much smaller than the –46.8% change in anti-dsDNA levels (minimum –62.7%, maximum 0.52%; P ⫽ 0.01) (Figure 2b). In fact, 5 of 9 patients had a ⬎45% decrease in the anti-dsDNA antibody level. The levels of other autoantibodies we measured, such as antinuclear antibodies, anti-SSA, anti-SSB, and anticardiolipin antibodies, did not change significantly. Changes in peripheral plasma cell frequency. There were no significant changes in the total number of lymphocytes or in the overall T lymphocyte or B lymphocyte counts. The frequency of CD38highCD19lowIgD– plasma cells was significantly expanded in SLE patients at baseline as compared with healthy controls (mean 5.3% versus 1.2%) (Figure 3a). The response to tocilizumab by the end of treatment in a representative SLE patient is shown in Figure 3b. For the entire cohort, a significant reduction in the percentages of plasma cells was seen as early as 6 weeks (mean 3.05%) (Figure 3c). This was maintained throughout the treatment (mean 3.43% at 14 weeks) and followup (mean 3.46% at 20 weeks) periods. Clinical response. Disease activity showed a modest but significant improvement by the end of the treatment period (Figure 4). Mean SLAM scores decreased from 7.1 at baseline to 5.0 at week 14 (P ⫽ 0.002), and mean mSELENA–SLEDAI scores decreased from 9.5 to 5.5 (P ⫽ 0.001). The decrease in the SLAM score was mainly related to improvement in the ESR, the degree of fatigue, and the hematocrit value, whereas the decrease in the mSELENA–SLEDAI score was mainly because of improvement in arthritis and rash. A clinically significant improvement in the mSELENA– SLEDAI score was predefined as a decrease of ⱖ4 points from baseline. This was achieved by 8 of 15 patients (3 each in the 2 mg/kg and 4 mg/kg groups and 2 in the 8 mg/kg group). Five of these 8 patients also achieved the recently proposed (23) criteria of an improvement of ⱖ7 points. Using a similar approach for the SLAM scores, 5 patients had an improvement of ⱖ4 points (1 in the 2 mg/kg group and 2 each in the 4 mg/kg and 8 mg/kg groups), and none had an improvement of ⱖ7 points. There was a significant decrease from baseline in both the physician’s and the patient’s global assessment of disease activity (mean ⫾ SD change from baseline –1.7 ⫾ 1.2 for the physician’s global assessment and –1.7 ⫾ 1.7 for the patient’s global assessment; P ⬍ 0.002 for both comparisons). Interestingly, both the IL-6 RECEPTOR BLOCKADE IN SLE Figure 4. Clinical efficacy of treatment with 2 mg/kg, 4 mg/kg, or 8 mg/kg of tocilizumab from baseline to week 14 in patients with systemic lupus erythematosus (SLE). a, Swollen joint counts at baseline, week 6, week 14 (end of tocilizumab treatment), and week 20 (end of followup) in 7 SLE patients who had arthritis at baseline. In all 7 patients, there was an improvement in arthritis, as determined by the swollen joint count, with tocilizumab treatment, with complete resolution in 4 patients. Data are shown as box plots. Each box represents the 25th to 75th percentiles, the lines within the boxes represent the median (50th percentile), and the lines outside the boxes represent the 10th and 90th percentiles. Solid circles indicate outliers. P values comparing the end of treatment data with the baseline data were determined by Wilcoxon’s signed rank test. b, Scores on the modified Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index (mSELENA–SLEDAI) and the Systemic Lupus Activity Measure (SLAM) at baseline, weeks 6, week 14 (end of tocilizumab treatment), and week 20 (end of followup) in the 15 SLE patients evaluated for efficacy. There was improvement in overall disease activity over the course of treatment, with the mSLEDAI and SLAM scores showing a significant decrease at the end of treatment. Values are the mean and SD. P values were determined by repeated-measures analysis of variance. physician’s and the patient’s global assessments decreased significantly in the 2 mg/kg and 4 mg/kg groups, but not in the 8 mg/kg group. 549 Seven patients (4 receiving 4 mg/kg of tocilizumab and 3 receiving 8 mg/kg) had arthritis at baseline. The mean number of swollen joints improved from 7.7 at baseline to 5.4 at 6 weeks and 1.1 at the end of the treatment period, with complete resolution of arthritis in 4 patients. At the last followup, 5 of the 7 patients had a reactivation of their arthritis (mean swollen joint count 5.4). Six patients had an inflammatory rash at the start of tocilizumab treatment, which resolved in 3 of them between weeks 2 and 6. Fatigue, as assessed by the SLAM, was present in 7 patients at baseline and resolved in 6 of them. There were 3 patients without fatigue at baseline who reported having fatigue on at least 1 occasion during the treatment period; in none of these patients was the fatigue sustained. Five patients had renal disease at baseline. All had moderate proteinuria, 4 had pyuria, and 3 had hematuria. None had cellular casts. Other causes of these abnormalities, such as infection, diabetes mellitus, and uncontrolled hypertension, were excluded before these abnormalities were attributed to the SLE. There was no significant change in the mean level of proteinuria during the study. The number of patients with active urinary sediment was too low to assess efficacy, but 2 of 2 patients with significant hematuria (urinary red blood cell count ⬎10 per high-power field) at baseline (determined as the average of the screening and pretreatment values) and 3 of 4 with significant pyuria (urinary white blood cell count ⬎10 per high-power field) had ⬎50% improvement at the end of treatment (average of week 12 and week 14 values). Most patients (12 of 15) were taking ⱕ10 mg of prednisone (or equivalent) daily which was kept stable during the study. The prednisone dosage was successfully decreased by an average of 7.5 mg/day in the 3 patients who were taking either 15 mg/day (n ⫽ 1) or 20 mg/day (n ⫽ 2) at study entry. There was no SLE flare during the treatment period. One patient receiving 4 mg/kg of tocilizumab experienced a flare 2 weeks after her last dose and required an increase in her prednisone dosage from 7.5 mg/day to 15 mg/day. Two other patients in the group receiving 2 mg/kg of tocilizumab required an increase in the prednisone dosage at the last followup visit; one of them also received methotrexate for the treatment of arthritis and rash. DISCUSSION Our data provide the first evidence that blocking IL-6 receptors with tocilizumab has an acceptable safety 550 profile and suggest a possible immunologic and clinical benefit in SLE. Patients tolerated the infusions well, with no infusion reactions. Infections were the most common adverse events, with two-thirds of the patients experiencing at least 1 infection during the 5-month study period. Although the rate of infections was higher than expected, consistent with other studies of tocilizumab (24–26), most of these infections were mild and resolved with or without antibiotics during continued tocilizumab therapy. Larger controlled studies will be necessary to obtain a better estimate of the risk of infection associated with tocilizumab as compared with the risk with other drugs used to treat SLE. Liver enzyme abnormalities and increases in serum lipid levels have been described with tocilizumab therapy (25–27). We did not observe any clinically important abnormal findings on liver function tests because patients with significant elevations in liver enzyme levels and those taking potentially hepatotoxic medications were excluded from the study. The main adverse event in this study was doserelated neutropenia. Absolute neutrophil counts decreased by a median of 38% and 56% in the 4 mg/kg and 8 mg/kg tocilizumab groups, respectively. Most of the changes occurred after the first dose and remained relatively stable during the treatment. Similar decreases in neutrophil counts have been observed in other studies. In the OPTION (Tocilizumab Pivotal Trial in Methotrexate Inadequate Responders) study, one-third of the patients with RA who were treated with 8 mg/kg of tocilizumab every 4 weeks in combination with stable dosages of methotrexate had an absolute neutrophil count below the lower limit of normal at least once during the study (24). This is comparable to what we have observed in lupus patients who received 4 mg/kg of tocilizumab every 2 weeks. It is very important to note that neutropenia did not temporally correlate with the occurrence of infections in either study. The cause of the decline in the neutrophil count is not clear and requires further investigation. It may be related to decreased production or, more likely, to changes in neutrophil trafficking, or both. IL-6 plays a major role in regulating the acutephase responses of inflammation, and blocking IL-6 is expected to reverse these effects. Accordingly, elevated levels of markers of inflammation decreased promptly following the first dose and remained stable during the treatment phase. The antiinflammatory effect of tocilizumab was further supported by an improvement in hemoglobin values and serum albumin levels. In this ILLEI ET AL study, we did not see a significant improvement in proteinuria, and therefore, the improvement in albumin levels is a reflection of a decrease in systemic inflammation. The most common form of anemia in SLE is anemia of chronic inflammatory disease. Since red blood cells play a major role in the clearance of immune complexes, correcting the anemia may increase the clearance of circulating immune complexes and may thus contribute to a decrease in disease activity (28) in addition to improving oxygen-carrying capacity and tissue oxygenation. Tocilizumab treatment was associated with hypocomplementemia in previous studies, but it was not known if this was explained by a decreased production or an increased consumption of complement components. Our data showing that complement activation products decreased to a similar or higher degree as the C3 and C4 levels provide the first evidence that the tocilizumabassociated hypocomplementemia represents decreased production rather than increased activation. Since hypocomplementemia can also herald active lupus, complement activation products, rather than common complement components, should be used as markers of lupus activity in future studies of tocilizumab. Fifteen patients were available for evaluation of efficacy. Although the number of patients was too small for a formal comparison among the 3 dosage groups, we did not detect any obvious differences in clinical outcomes among them. The findings in the 4 patients receiving 2 mg/kg were similar to those in the 11 patients receiving 4 mg/kg and 8 mg/kg, with more individual variations and a delayed onset of response. Overall disease activity, as measured by 2 disease activity indices and both the patient’s and the physician’s global assessments, improved by the end of the treatment period. Although the improvement was modest, it was statistically significant and was consistent across all measures. We defined a priori a decrease of ⱖ4 points in the mSELENA–SLEDAI score as a clinically important change. This was achieved by more than one-half of the patients (8 of 15). One-third of the patients also met the more stringent criteria of a decrease of ⱖ7 points, which was recently proposed by a consensus panel (23). The slightly better response based on the mSELENA– SLEDAI scores as compared with the SLAM scores reflects differences in how the 2 indices weigh certain manifestations of lupus, such as complete or partial resolution of arthritis and resolution of (even mild) hematuria or pyuria. Most of the changes occurred during the first 6 weeks of the study and reached IL-6 RECEPTOR BLOCKADE IN SLE maximum levels by 12 week, with an increase in disease activity occurring 4–8 weeks after the last treatment. Of the individual manifestations of SLE, arthritis improved the most, with complete resolution in 4 of the 7 patients who had arthritis at baseline. Five of the patients enrolled in the study had renal involvement. All had chronic glomerulonephritis characterized mainly by proteinuria; this did not change during the study. Proteinuria is multifactorial at this stage, with a significant contribution of noninflammatory processes, such as hemodynamic abnormalities and fibrosis, which may not be altered by IL-6 receptor blockade or may require more time to respond. Although we have seen some improvement in urinary sediment, the number of patients with active sediment at baseline and the short duration of the study prevent our drawing any conclusion about the potential efficacy of tocilizumab in lupus nephritis. One of the major biologic actions of IL-6 is its ability to stimulate B lymphocyte differentiation into immunoglobulin-secreting cells. Notably, we observed a statistically significant decrease in circulating plasma cells during treatment, as well as a significant decline in anti-dsDNA antibody levels. This change seems to be specific, since levels of other autoantibodies did not change and since there was only a small, albeit statistically significant, decrease in total IgG levels. The clinical significance of the decrease in anti-dsDNA antibody levels is unclear, but these results are consistent with previous ex vivo observations that IL-6 blockade may alter the B cell abnormalities found in lupus. There are limitations to our study. First, the 3-month treatment period is too short to allow an estimation of the long-term toxicities of tocilizumab in SLE. Second, we excluded patients who were receiving concomitant immunosuppressive agents, and thus, this study may not reflect everyday practice. Third, given the open-label design and the lack of a control group, all efficacy data should be considered preliminary. Fourth, because of the small number of patients, we could not make formal comparisons among the 3 dosage groups. However, our data suggest that neutropenia is more severe in those taking 8 mg/kg of tocilizumab; therefore, future studies should consider evaluating doses equivalent to 4 mg/kg administered every 2 weeks. This pilot study provides the first data that tocilizumab can effectively block IL-6 in patients with SLE. The improvements in markers of inflammation as well as in the clinical and serologic manifestations of lupus activity are encouraging and should be explored further in controlled studies of tocilizumab. 551 ACKNOWLEDGMENTS We would like to thank Drs. Larissa Lapteva and Sarah Okada for their assistance with patient evaluations, Ms Margaret Brown and Tuyet-Hang Pham for their technical assistance, and the Nursing Staff of the NIH Clinical Center 5SW Day Hospital for their dedicated care of the patients. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Illei had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. 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