F0201

The ability of CPV-104 and sd-FH to inhibit C5b-9 formation on endothelial cells was assessed as previously described (59 (link)). Briefly, HMEC-1 cells were activated with 10 µM ADP, then incubated with either pooled NHS or primary aHUS serum (patients aHUS3–5, diluted 1:2 in HBSS + 0.5% BSA) in the presence or absence of CPV-104 (500 µg/ml), sd-FH (500 µg/ml) or pegcetacoplan (1 mg/ml) for 4 h at 37°C. The cells were then stained with an anti-human C5b-9 antibody (Calbiochem, 204903, 1mg/ml, 1h at room temperature, 1/200 final dilution) followed by a fluorochrome-conjugated secondary antibody (Jackson Immuno Research Laboratories, 1.5 mg/ml, 111-095-144, 1h at room temperature, 1/50 final dilution). Fifteen fields per sample were acquired and the areas with fluorescent staining were evaluated using the built-in automatic edge detection function of Image J. The highest and lowest values were discarded, and the mean was calculated on the remaining 13 fields.
For the examination of C3 deposits, HMEC-1 cells prepared as described above were incubated with either NHS or aHUS serum containing FH autoantibodies (patient aHUS2) for 4 h at 37°C. The cells were fixed in 4% paraformaldehyde (PFA) and stained with a rabbit anti-human C3c FITC-labeled antibody (F0201, 2.9 mg/ml, Dako, 1:300) to detect C3 and iC3b and DAPI to counterstain the nuclei.

For the experiments we used the following in-house produced antibodies (Abs): mouse anti-ficolin-2 mAb FCN219 (6 (link)) and mouse anti-ficolin-1 mAb cross-reacting with ficolin-2 (7 (link)). Moreover, we applied the following commercial Abs: mouse anti-MBL mAb (HYB 131-1, Bioporto Diagnotics, Gentofte, Denmark), rabbit anti-C1q pAb (A0136, Dako, Glostrup, Denmark), rabbit anti-IgM and anti-IgG pAbs (0425 and 0423, Dako), rabbit anti-C4c and -C3c pAbs (0369 and F0201, Dako), and mouse anti-TCC mAb clone aE11 (011-01, AntibodyChain, Utrecht, Netherlands). The isotype controls included were: mouse IgG1κ and IgG2α isotype controls (557273 and 555571, BD Biosciences, Albertslund, Denmark) and rabbit IgG isotype control (10500C, Invitrogen, Naerum, Denmark).

The ability of CPV-104 and sd-FH to prevent C3 deposition on the surface of PNH erythrocytes was tested as previously described (61 (link)). Briefly, EDTA-treated whole blood was centrifuged for 5 min at 400 g and the erythrocytes were reconstituted in an equivalent amount of saline for three wash cycles. We then mixed 2 µl of erythrocytes with PBS alone or with 0.5 µM eculizumab combined with increasing concentrations of CPV-104 (0, 0.2, 0.5, 1, 2 and 5 µM) or 12 µM pegcetacoplan in a total volume of 10 µl. We then added 30 µl of ABO-matched, acid-activated (0.1 M HCl, diluted 1:10) NHS supplemented with 2 mM MgCl₂ and incubated the cells for 24 h at 37°C. The cells were washed with PBS containing 2 mM EDTA and stained with antibodies against CD59 (376004, anti-human CD59 PE; 50 µg/ml, Biolegend, USA, 1:200) and C3c to detect C3 and iC3b (F0201, 2.9 mg/ml, Dako, USA, 1:25) before analysis by flow cytometry.