Effect of long-term belimumab treatment on B cells in systemic lupus erythematosus: extension of a phase II, double-blind, placebo-controlled, dose-ranging study

Abstract

Objective: To understand the effects of long-term BLyS inhibition in human systemic lupus erythematosus (SLE).

Methods: Seventeen patients with SLE who were enrolled in a clinical trial of belimumab, a BLyS-specific inhibitor, plus standard of care therapy were studied. Phenotypic analysis of lymphocytes was performed using flow cytometry. Circulating antibody-secreting cells were enumerated using enzyme-linked immunospot assay. Serum was analyzed by enzyme-linked immunosorbent assay using an antibody that recognizes products of the V(H)4-34 gene. Lymphocyte counts, Ig levels, and anti-double-stranded DNA antibody levels were available as part of the clinical trial analyses.

Results: Samples were collected on days 0, 84, 168, 365, and 532 and after day 730. The total number of B cells started to decrease from baseline between days 84 and 168. This was due to a decrease in naive and transitional B cells. CD27+IgD+ memory B cells and plasmablasts decreased only after 532 days, whereas CD27+IgD- memory B cells were not affected, and there were no changes in T cells. Serum IgM levels began to decline between days 84 and 168, but there were no changes in serum levels of IgG, IgG anti-DNA antibodies, or V(H)4-34 antibodies during the study. SLE patients had more IgM-, IgG-, and autoantibody-producing B cells than did normal controls on day 0. There was only a modest decrease in the frequency of total IgM-producing, but not IgG-producing, cells on days 365 and 532, consistent with the phenotypic and serologic data.

Conclusion: Our data confirm the dependence of newly formed B cells on BLyS for survival in humans. In contrast, memory B cells and plasma cells are less susceptible to selective BLyS inhibition.

Trial registration: ClinicalTrials.gov NCT00071487.

Figures

Figure 1

Chart of belimumab treatment regimens of patients in the primary study and after rollover into the extension study at Day 392. At week 80, all patients rolled over to 10mg/kg. The color code of each patient used in Figures 2, 3, 5 and 6 is included. Patient demographic data are shown in the table. Age is shown in years and starting prednisone dose in mg/day.

Figure 2

Serologic data over time in 17 belimumab treated patients. Each patient is color coded according to Figure 1. Decreased levels of IgM are observed over time (A). Statistically significant changes (compared to Day 0) are marked with an asterix (*p

Figure 3

Analysis of antibody-forming cells by ELISpot. A: Frequency of total and autoreactive IgM- and IgG- producing cells in the peripheral blood of belimumab-treated patients at their first pre-treatment visit and 31 normal controls. B–E. Absolute number of antibody- and autoantibody- forming cells/ml over time in 17 belimumab-treated patients. Each patient is color-coded according to Figure 1. p values compared with Day 0 are shown for each time. Data for Days 84 and 168 are shown together with data shown for Day 84 only for those patients for whom data from Day 168 was unavailable. p values compared with Day 0 are shown for each time. Statistically significant changes (compared to Day 0) are marked with an asterix (*p730; D: p = 0.09 for Days 562 and >730.

Figure 4

Absolute number of B and T cells over time. A decrease in total B cells became significant at Day 365 (p=0.008) and remained significant over months (Day 532, p=0.001 and p=0.002 at or after day 730) whereas T cell count did not change significantly throughout the study.

Figure 5

B cell subset analysis. A–D: Gating strategy of different B cell subsets. Live CD19+ gated B cells are shown. A: Two years after initiation of belimumab therapy a predominance of Ig-class switched memory B cells and plasmablasts was observed in a representative example (62%, CD27+/++IgD−). Other B cell subsets analyzed were CD27+IgD+ pre-switched memory B cells (10%) and double negative CD27−IgD− B cells (14%). B: Naive (CD27−IgD+CD10−) and transitional (CD27−IgD+CD10+) B cells were diminished (12% and 1%, respectively). Naïve cells were calculated by subtracting the percent transitional cells from the percent CD27−IgD+ cells (lower right quadrant in panel A). C: Using a monoclonal antibody to CD38, plasmablasts or plasma cells (CD27++CD38++) could be differentiated from CD27+IgD− post-switched memory B cells. D: Phenotype of all subsets analyzed.

Figure 6

Absolute number of B cell subsets over time. Cells were gated on CD19 and then as shown in Figure 5. Data for Days 84 and 168 are shown together with data shown for Day 84 for those patients for whom data from Day 168 was unavailable (p values for naïve B cells Day 84 p