Fig. 4.
LNP023 blocks complement activity in the serum of C3G patients and prevents lysis of human PNH erythrocytes. (A) Inhibition of C3 convertase activity in serum of C3G patients. Patient serum was incubated with normal human serum, and C3 degradation was detected by immunofixation electrophoresis and quantified. The AP is blocked in the presence of EDTA (maximum inhibition control) but not in the presence of Mg2+-EGTA, which blocks the classical and lectin pathways. LNP023 is shown in light blue at 1.1 µM and dark blue at 3.3 µM, and an FD inhibitor is shown in green. (B) Prevention of C3G patient serum-induced hemolysis of sheep erythrocytes. Erythrocytes were incubated with C3G patient serum and LNP023 (0.15 µM) or an FD inhibitor (0.15 µM). Hemolysis was measured by increase in optical density at 415 nm. Addition of EDTA and EGTA was used as negative and positive control, respectively. (C) Inhibition of nephritic factor-stabilized C3 convertase activity. Sheep erythrocytes coated with preformed C3 convertase were incubated with total IgGs from C3G patient sera in the absence or presence of LNP023 (0.15 µM) or an FD inhibitor (0.15 µM). C3 convertase stabilized at 15 min was calculated as % Z (Z15min/Z0min × 100). The assay was adjusted to yield one active C3 convertase per sheep erythrocyte (Z0min = 1; SI Appendix). (D) Inhibition of hemolysis of PNH erythrocytes. Blood from three patients was analyzed in 2–14 repeats, mean ± SEM. Inhibitors were used at 1 µM. Hemolysis of PNH erythrocytes was determined by FACS analysis. (E) Dose–response curves for inhibition of hemolysis of erythrocytes from PNH patients for LNP023, FD inhibitor, and anti-C5 antibody. Hemolysis was measured by FACS analysis. Curves are representatives of three patients measured in 2–14 repeats. For each curve, the average per person from all repeats was used. P values were calculated using one-way ANOVA followed by Dunnett’s multiple comparison test with a single pooled variance; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.