POS0680 BELIMUMAB ADD-ON THERAPY MOBILISES MEMORY B CELLS INTO THE CIRCULATION OF PATIENTS WITH SLE

Abstract

Background:Belimumab (BLM), a recombinant human monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with active severe systemic lupus erythematosus (SLE) and lupus nephritis (LN). There is clinical evidence that combining BLM with B cell depleting therapy can ameliorate disease activity in severe, refractory SLE patients1. Although BLM is a B cell directed therapy and has been shown to significantly decrease total B cells, flow cytometry observations suggest a rapid increase of circulating memory B cells (MBC)2.Objectives:To investigate dynamics of B-cell subsets in SLE patients treated with or without BLM, with a focus on assessing MBC characteristics.Methods:Extensive B cell subset phenotyping was performed by high-sensitivity (HS) flow cytometry (acquisition of 107 leukocytes; per EuroFlow protocols3) on samples from active LN or SLE patients with major organ involvement treated with standard of care (SOC) consisting of high dose steroids and mycophenolate mofetil combined with or without the addition of BLM. MBC gene expression profiles were characterized with single-cell RNA and V(D)J sequencing (ScRNA-SEQ).Results:By employing HS flowcytometry, we established that the absolute increase in circulating MBC in SLE and LN patients was significant for patients who initiated BLM (Figure 1). The increase was observed in a broad range of MBC subsets (Unswitched, IgG1+, IgG2+, IgA1+, IgA2+) at 2 and 4 weeks following initiation of BLM treatment. This rise in MBC could hypothetically be attributed to either proliferation of blood MBC, BLM induced migration of tissue-resident MBCs or BLM related retention of tissue-destined MBC in the blood. ScRNA-SEQ analysis of cell cycle gene-expression was performed and established in both groups a non-proliferating phenotype [in approximately ~94%] of MBC post-treatment, including absence of MKI67 as active proliferation marker. Clonal diversity analysis comparing week 2 with baseline revealed an unexpected decrease of the largest MBC clones in BLM, whereas no change in clonality was observed with SOC alone. Together these data indicate that proliferation is unlikely to be responsible for the observed increase in MBC by BLM. Furthermore, a clear difference was found in gene-expression levels between both treatment groups: BLM was responsible for the upregulation of 72 vs 10 genes in SOC, likewise 162 vs 32 genes were downregulated. Most importantly, a significant downregulation of the migration genes SELL (CD62L), CCR7, ITGB1, RAC2 and ICAM2, were specifically seen in BLM treated patients. This may reflect disrupted lymphocyte trafficking, preventing MBCs from transmigrating from the blood into tissue owing to reduced migration molecules, or preventing MBCs from being retained at the tissue level owing to reduction in tissue adhesion proteins.Conclusion:The addition of BLM to SOC significantly increases MBCs in patients with SLE independently of proliferation, accompanied by a strong modulation of gene expression, including reduced expression of migration markers pointing towards disrupted lymphocyte trafficking. These data may have important implications for improving treatment strategies in patients with LN or severe SLE, as a deeper depletion of autoreactive MBCs could be established by adding B-cell-depleting therapy after the initiation of BLM.Figure 1.Change in pre-germinal center and memory B cell counts from baseline to week 4 of patients with SLE or LN treated with SOC (n=8) or SOC+BLM (n=11).