C3d-targeted complement inhibitors to correct complement dysregulation in aHUS patients

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare and severe thrombotic microangiopathy caused by genetic or acquired abnormalities leading to activation of the complement alternative pathway on cell surfaces. This process leads to endothelial dysfunction and microvascular thrombosis. The introduction of anti-C5 antibodies has dramatically improved aHUS prognosis; however, these treatments require regular intravenous infusions and block systemic complement activity, exposing the patient to risk of infections. Recently complement inhibitors have been developed to selectively bind injury-associated target molecules, thereby concentrating the drug at specific cellular or tissue sites while preserving systemic complement function. This study evaluated the local complement inhibitory activity of new molecules that exploit the natural localization of C3d at complement activation sites on cells: namely the anti-C3d monoclonal antibody 3d8b conjugated with the first 10 or 17 short consensus repeats (SCRs) of complement receptor 1 (CR1_1-10 and CR1_1-17, respectively) or the first 5 SCRs of complement factor H (FH_1-5). To this purpose we tested their capability to block C3 deposition and C5b-9 formation on microvascular endothelial cells (HMEC-1) exposed to serum from patients with aHUS. We also assessed their ability to prevent loss of anti-thrombogenic properties in HMEC-1 pre-exposed to aHUS serum and then perfused with control blood. We demonstrate that anti-C3d-antibody conjugated with CR1_1-10, or CR1_1-17, or FH_1-5 effectively prevented aHUS serum-induced complement activation on HMEC-1, outperforming their non-targeted soluble counterparts. The efficacy of C3 convertase inhibition varied depending on the complement inhibitory component (CR1_1-17 > CR1_1-10 > FH_1-5). However, all the inhibitors successfully blocked C5 convertase activity and eliminated the pro-thrombogenic effects of aHUS patients' serum. These findings support the potential of tissue-targeted complement inhibition as a novel, non-systemic therapeutic strategy for aHUS and other diseases characterized by localized complement dysregulation.

Keywords: aHUS; complement inhibitors; complement system; endothelial cell (EC); thrombus formation.

Figure 1

C3 deposition induced on activated HMEC-1 by serum from aHUS patients carrying RVs in C3 (n=3), CFH (n=3) or CFI (n=3) genes. Data are expressed as area covered by C3 staining measured by anti-C3c mAb (mean ± SD). * p<0.05 vs NHS and corresponding +sCR1. RVs, rare variants; NHS, normal human serum.

Figure 2

C3 deposition induced on activated HMEC-1 by serum from aHUS patients (three with CFH RVs, three with CFI RVs, and three with C3 RVs) in the presence or absence of anti-C3d mAb conjugated with CR1_1-10 (1, 10, 100 nM) or the corresponding unconjugated CR1_1-10 (1, 10, 100 nM) (A); anti-C3d mAb conjugated with CR1_1-17 (1, 10, 100 nM) or the corresponding unconjugated CR1_1-17 (1, 10, 100 nM) (B); and anti-C3d mAb conjugated with FH_1-5 (10, 100, 1000 nM) or the corresponding unconjugated FH_1-5 (10, 100, 1000 nM) (C). Data are expressed as area covered by C3 staining (mean ± SD, nine independent experiments). Each experiment includes cells exposed to NHS, or serum from a specific aHUS patient, with or without complement inhibitors. *p<0.05 vs all groups; #p<0.05 vs equimolar CR1_1–10 or CR1_1-17; **p<0.05 vs aHUS. NHS: normal human serum.

Figure 3

Representative images of C3 staining (green) on activated HMEC-1 exposed to normal human serum (NHS) or serum from aHUS patients in the presence or absence of anti-C3d-CR1_1-10, anti-C3d-CR1_1-17, and anti-C3d-FH_1-5. Blue: DAPI. Scale bar: 20 µm.

Figure 4

C3 deposition induced on activated HMEC-1 by serum from aHUS patients in the presence or absence of C3d-targeted or non-targeted CR1_1-10 (A), C3d-targeted or non-targeted CR1_1-17 (B), and C3d-targeted or non-targeted FH_1-5 (C). Data are expressed as percentages of serum-induced C3 deposition in the presence of complement inhibitors, relative to aHUS serum alone, which is set at 100%. Upper panels: Patients carrying CFH RVs (n=3 independent experiments). Each experiment includes cells exposed to NHS, or serum from a specific aHUS patient with or without complement inhibitors. Colored points represent the percentage value for individual patients/experiments. #1: patient carrying p.R1210C RV in CFH gene. #2: patient carrying p.R78G RV in CFH gene. #3: patient carrying p.S1191L RV in CFH gene. Middle panels: Patients carrying C3 RVs (n=3 independent experiments). Each experiment includes cells exposed to NHS, or serum from a specific aHUS patient with or without complement inhibitors. Colored points represent the percentage value for individual patients/experiments. #4: patient carrying p.D61N RV in C3 gene. #5: patient carrying p.S1063R RV in C3 gene. #6: patient carrying p.T162R RV in C3 gene. Lower panels: Patients carrying CFI RVs (n=3 independent experiments). Each experiment includes cells exposed to NHS, or serum from a specific aHUS patient with or without complement inhibitors. Colored points represent the percentage value for individual patients/experiments. #7: patient carrying p.P50R RV in CFI gene. #8: patient carrying p.V412M RV in CFI gene. #9: patient carrying p.C67W RV in CFI gene. RVs, rare variants; NHS, normal human serum. Dashed line: 100% aHUS serum.

Figure 5

Effect of anti-C3d mAb conjugated with CR1_1–10 or CR1_1-17 (10 nM) and their corresponding non-targeted CR1_1-10, CR1_1-17 (10 nM) (A); or anti-C3d mAb conjugated with FH_1-5 (100 and 1000 nM) and its corresponding non-targeted FH_1-5 (100 and 1000 nM) (B), on C5b-9 formation on activated HMEC-1 cells exposed to serum from aHUS patients (one with RV in CFH, one with RV in CFI, and one with RV in C3). Data are expressed as area covered by C5b-9 staining (mean ± SD of three independent experiments). *p<0.05 vs all groups. NHS, normal human serum.

Figure 6

Representative images of C5b-9 staining (green) on activated HMEC-1 exposed to normal human serum (NHS) or serum from aHUS patients in the presence or absence of anti-C3d-CR1_1-10 (10 nM), anti-C3d-CR1_1-17 (10 nM), and anti-C3d-FH_1-5 (100 and 1000 nM). Blue: DAPI. Scale bar: 20 µm.

Figure 7

(A) Platelet adhesion and aggregation on activated HMEC-1 exposed to serum from aHUS patients (one with RV in CFH, one with RV in CFI, and one with RV in C3), in the presence or absence of anti-C3d mAb conjugated with CR1_1-17 (10 nM), or anti-C3d mAb conjugated with FH_1-5 (100 or 1000 nM), or sCR1 (150 µg/ml), or eculizumab (100 µg/ml), and then perfused with control whole blood. Data are expressed as area covered by platelet aggregates (mean ± SD). *p<0.05 vs all groups. (B) Representative images of experiments of platelet adhesion and aggregation (green staining) on HMEC-1 cells exposed to NHS, or aHUS serum with or without C3d-mAb-CR1_1-17 (10 nM) or C3d-mAb-FH_1-5 (1000 nM). NHS, normal human serum. Scale bar: 100 µm.