S267: PROPERDIN-BLOCKING ANTIBODIES ATTENUATE COMPLEMENT ALTERNATIVE PATHWAY ACTIVATION TRIGGERED BY CELL-FREE HEME IN SICKLE CELL DISEASE MODELS

Abstract

Background: Complement activation may play an important role in sickle cell disease (SCD) pathophysiology. Heme has been identified as a trigger of complement activation in SCD patients and is present in excess during severe vaso-occlusive crises. Heme is also a DAMP (damage associated molecular pattern) and induces thrombo-inflammation through tissue factor expression. ALXN1820 is a bi-specific VHH nanobody that simultaneously binds human albumin and properdin, thereby effectively and selectively inhibiting alternative pathway (AP) activation and formation of C3 and C5 convertases. The safety of ALXN1820 has been assessed in non-human primates and is currently being tested in a phase 1 study in healthy participants. Aims: 1) To assess if complement activation and heme are functionally linked; 2) To investigate the therapeutic potential of ALXN1820 in in vitro SCD models and in in vivo SCD models using a mouse properdin-blocking monoclonal antibody (mAb), 14E1. Methods:In vitro, complement inactivated C3b (iC3b) and C5b-9 deposition on red blood cells (RBCs) from patients with SCD and complement iC3b and C5b-9 deposition on the endothelial cell line HMEC-1 were assessed by flow cytometry after exposure to heme +/- ALXN1820. In vivo, in Townes SCD mice, vaso-occlusion and hyper-hemolysis were induced by intravenous heme injection (50 µmol/kg) or hypoxia-reoxygenation (8% O2 for 3h then 21% O2 for 1h) in the presence and absence of 14E1 (40 mg/kg), a mouse-specific properdin inhibitor. Vaso-occlusion was measured by staining RBCs with immunofluorescence-labeled Ter-119 antibodies on lung and liver sections. C3b and C5b-9 deposition on SCD RBCs was assessed by flow cytometry. Markers of hemolysis (bilirubin, lactate dehydrogenase, free hemoglobin, free heme) were quantified in plasma using commercial assays. Results: In RBCs from patients with SCD, cell-free heme triggered marked iC3b and C5b-9 deposition (Table). Deposition was blocked in the presence of ALXN1820 by >95% for iC3b and by >85% for C5b-9. HMEC-1 cells exposed to heme showed marked deposition of iC3b and C5b-9. In the presence of ALXN1820, deposition was blocked by >70% for iC3b and >85% for C5b-9. Pretreatment of mice with 14E1 markedly ameliorated vaso-occlusion and reduced both C3b and C5b-9 deposition on RBCs, and hemolysis biomarkers. Image:Summary/Conclusion: These data strengthen the hypothesis that in SCD, cell-free heme is a potent trigger of complement AP activation, which can be blocked by targeting properdin. Investigating the efficacy and safety of anti-properdin ALXN1820 in patients with SCD is warranted as a novel approach to treatment of acute and chronic SCD complications.