Using an in vitro xenoantibody-mediated complement-dependent cytotoxicity model to evaluate the complement inhibitory activity of the peptidic C3 inhibitor Cp40

Abstract

Simple and reliable methods for evaluating the inhibitory effects of drug candidates on complement activation are essential for preclinical development. Here, using an immortalized porcine aortic endothelial cell line (iPEC) as target, we evaluated the feasibility and effectiveness of an in vitro xenoantibody-mediated complement-dependent cytotoxicity (CDC) model for evaluating the complement inhibitory activity of Cp40, a potent analog of the peptidic C3 inhibitor compstatin. The binding of human xenoantibodies to iPECs led to serum dilution-dependent cell death. Pretreatment of the human serum with Cp40 almost completely inhibited the deposition of C3 fragments and C5b-9 on the cells, resulting in a dose-dependent inhibition of CDC against the iPECs. Using the same method to compare the effects of Cp40 on complement activation in humans, rhesus and cynomolgus monkeys, we found that the inhibitory patterns were similar overall. Thus, the in vitro xenoantibody-mediated CDC assay may have considerable potential for future clinical use.

Keywords: Complement inhibitor; Cp40; Nonhuman primate; SV40-immortalized porcine aortic endothelial cell.

Fig. 1

An in vitro human xenoantibody-mediated complement-dependent cytotoxic model. (A) FACS analysis for α-Gal expression on iPECs with FITC-GS-IB4 staining. Cells incubated with PBS alone were used as a negative control. (B) After incubation with 20% HINHS, the binding of human IgG and IgM on iPECs was analyzed by flow cytometry. The geometric mean fluorescence intensity (Gmean) was used to evaluate the degree of xenoantibody binding to iPECs. Cells incubated with secondary antibody alone served as negative controls. (C) Deposition of C3c- and C4c-containing complement opsonins and C5b-9 on iPECs after incubation with 20% NHS was detected by flow cytometry and evaluated by Gmean. Negative controls were cells incubated with 20% HINHS. (D) Cell death was analyzed by flow cytometry after the incubation of iPECs with sequentially diluted NHS. Representative FACS analyses for the gating of iPECs (a) and PI-positive iPECs (b, 1:5 diluted NHS) are shown. The percentages of iPEC lysis mediated by sequentially diluted NHS (1:2.5 to 1:80) are shown as a line graph (c). All results shown are representative of three independent experiments.

Fig. 2

No impact of Cp40 on the binding of human xenoreactive antibodies to iPECs. Binding of human xenoreactive antibodies (IgG/IgM) to iPECs was measured by FACS (A and B) and immunofluorescent staining (C, ×100) after the incubation of iPECs with either 20% HINHS (N) or Cp40-pretreated 20% HINHS (Cp40). iPECs incubated with secondary antibody alone served as negative controls (Control). For FACS analysis, the Gmean was used to evaluate the degree of xenoantibody binding to iPECs. (A) FACS results shown are representative of three independent experiments. (B) The extent of human IgG and IgM binding to iPECs is shown in the bar graphs. Data shown are means ± SEM (NS, P > 0.05 vs. N group; n = 3 per group). (C) Images are representative of three independent experiments.

Fig. 3

Cp40 significantly inhibits the cleavage of complement C3 and the formation of C5b-9. Deposition of C3c- and C4c-containing complement opsonins and C5b-9 on iPECs was detected by FACS (A and B) and immunofluorescent staining (C, ×100) after the incubation of iPECs with either 20% NHS (N) or Cp40-pretreated 20% NHS (Cp40). Cells incubated with HINHS were used as negative controls. For FACS analysis, the Gmean was used to evaluate the degree of complement deposition. (A) FACS results shown are representative of three independent experiments. (B) The degree of deposition of C3c- and C4c-containing opsonins, and C5b-9 on iPECs is shown in the bar graphs. Data shown are means ± SEM (*P < 0.05, **P < 0.01 vs. the N group; n = 3 per group). (C) Images are representative of three independent experiments.

Fig. 4

Cp40 inhibits human serum-mediated CDC in a dose-dependent manner. PI-positive dead cells were detected by FACS after the incubation of iPECs with 20% NHS pretreated with various doses of Cp40 (final concentrations from 1.25 to 1280 μg/ml). iPECs incubated with 20% HINHS were used as negative controls. The percentage of the total cells that were PI-positive was used to evaluate the degree of cell death. (A) Representative FACS results for negative controls and Cp40 pretreatment at 0, 1.25, 10, 20, and 1280 μg/ml. (B) The percentages of iPEC lysis mediated by 20% NHS that had been pretreated with various doses of Cp40 are shown as a line graph. Data shown are means ± SEM (n = 3).

Fig. 5

Effect of Cp40 on NHP serum-mediated CDC. PI-positive dead cells were detected by FACS after incubation of iPECs with 20% rhesus (A) or cynomolgus monkey (B) serum that had been pretreated with various doses of Cp40 (final concentrations from 1.25 to 1280 μg/ml). iPECs incubated with 20% heat-inactivated normal monkey serum (HINMS) were used as negative controls. Data shown are means ± SEM (n = 3).

Fig. 6

Comparison of the complement inhibitory activity of Cp40 in human and NHP (rhesus and cynomolgus monkey) sera. The percentage of inhibition was calculated according to CDC results to compare the complement inhibitory activity of Cp40 on human and NHP sera. All data are expressed as means ± SEM (*P < 0.05, **P < 0.01 vs. both rhesus and cynomolgus monkeys; n = 3).