OP0302 Significant reductions of pathogenic autoantibodies by synergetic rituximab and belimumab treatment effectively inhibits neutrophil extracellular traps in severe, refractory sle - the synbiose study

Abstract

INTRODUCTION AND AIMS: Neutrophil extracellular traps (NETs) are extracellular, decondensed DNA strands covered with antimicrobial proteins that are part of the first-line defence against pathogens. However, in SLE, overall release of NETs is increased and degradation of NETs is impaired leading to a high amount of extracellular nuclear material, potentially leading to the formation of SLE-specific antibodies. These pathogenic autoantibodies deposit in glomeruli in lupus nephritis (LN) and perpetuate autoimmunity by inducing more NETs. The present study hypothesized that combining anti-CD20 mediated B-cell depletion with BAFF (B-cell activating factor) inhibition can target autoreactive plasma cells and thereby effectively reduce pathogenic autoantibodies and NET induction in severe SLE. The present study aimed to investigate whether Rituximab (RTX) + Belimumab (BLM) affected pathogenic antibodies in relation to NET induction in severe refractory SLE. METHODS: As part of a phase 2 proof-of-concept study, the SynBioSe study, serum levels of anti-DNA autoantibodies were measured in severe, refractory SLE patients before and after treatment with RTX following BLM. Additionally, ex vivo NET induction was assessed before and after treatment with a novel highly sensitive method based on 3D confocal laser scanning microscopy. In this assay, healthy neutrophils are incubated with 10% serum of patients and healthy controls. NET induction is depicted as the fold increase compared to healthy controls. Furthermore, we investigated whether NET induction was mediated by immune complexes. RESULTS: The study included 10 severe, refractory SLE patients with lupus nephritis and 1 patient with neuropsychiatric lupus. NET induction was found to be high at baseline with a median fold induction of 4.5 [range 2.6-11.7]. After 24 weeks, NET induction was significantly decreased (median fold NET induction of 1.6 [0.4-6.1], p=0.01). In addition, treatment with RTX+BLMled to a significant reduction in antidsDNA antibodies at week 24 with a median of 35 IU/ml [range 10-374] compared to 120 [18-505] at baseline (p=0.012). Total immunoglobulin levels temporarily declined but returned to screening levels at week 24. NET induction correlated significantly with anti-dsDNA antibody levels (r=0.42, p=0.03) and also with SLEDAI scores (r=0.53, p=0.003). Therefore, we examined whether the observed induction of NETs could be explained by circulating immune complexes (ICx). ICx were degraded by preincubating anti-dsDNA positive SLE sera with nuclease, resulting in a significant decrease in NET induction (median % decrease of 91.7 [range 67.6-98.1]). In addition, depletion of IgG from anti-dsDNA positive SLE sera resulted in significantly lower NET induction. Finally, immobilized IgG isolated from anti-dsDNA positive SLE sera, but not of control serum, resulted in a significant NET induction. CONCLUSIONS: Within refractory SLE patients, RTX + BLM resulted in concordant reductions in pathogenic anti-dsDNA antibodies and NET-inducing capacity. This study strongly suggests that NET induction in SLE is mediated by immune complexes, providing a possible explanation underpinning the clinical benefits of RTX+BLM in SLE.