Peptide inhibitors of C3 activation as a novel strategy of complement inhibition for the treatment of paroxysmal nocturnal hemoglobinuria

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis due to the lack of CD55 and CD59 on affected erythrocytes. The anti-C5 antibody eculizumab has proven clinically effective, but uncontrolled C3 activation due to CD55 absence may result in opsonization of erythrocytes, possibly leading to clinically meaningful extravascular hemolysis. We investigated the effect of the peptidic C3 inhibitor, compstatin Cp40, and its long-acting form (polyethylene glycol [PEG]-Cp40) on hemolysis and opsonization of PNH erythrocytes in an established in vitro system. Both compounds demonstrated dose-dependent inhibition of hemolysis with IC50 ∼4 µM and full inhibition at 6 µM. Protective levels of either Cp40 or PEG-Cp40 also efficiently prevented deposition of C3 fragments on PNH erythrocytes. We further explored the potential of both inhibitors for systemic administration and performed pharmacokinetic evaluation in nonhuman primates. A single intravenous injection of PEG-Cp40 resulted in a prolonged elimination half-life of >5 days but may potentially affect the plasma levels of C3. Despite faster elimination kinetics, saturating inhibitor concentration could be reached with unmodified Cp40 through repetitive subcutaneous administration. In conclusion, peptide inhibitors of C3 activation effectively prevent hemolysis and C3 opsonization of PNH erythrocytes, and are excellent, and potentially cost-effective, candidates for further clinical investigation.

Figures

Figure 1

Preparation and characterization of compstatin Cp40 and long-acting derivatives thereof. (A) Schematic representation of the PEGylation strategy: PEG-Cp40 was prepared by adding a reactive two-arm branched PEG moiety of 40 kDa to the unprotected amino terminus of Cp40. In the case of Ac-Cp40-K-PEG, the N terminus was acetylated, a C-terminal lysine residue was added to the peptide during synthesis, and the PEG reagent was reacted with the lysine side chain. The dipeptide fragment used for the mass spectrometric quantification of PEG-Cp40 in plasma is indicated in red. (B) Evaluation of the complement inhibitory activity of Cp40 and its PEGylated derivatives using a complement activation ELISA. The clinically developed analog 4(1MeW) is shown as a control (for more information on individual compstatin analogs, see also supplemental Figure 1). The panel shows a representative plot out of 3 separate experiments.

Figure 2

Effect of C3 inhibitors on hemolysis and C3 fragment deposition of PNH erythrocytes. (A) Dose-response curves from the in vitro hemolysis assay with Cp40 (red) and its long-acting derivatives PEG-Cp40 (blue) and Ac-Cp40-K-PEG (green). Lysis of PNH erythrocytes (y-axis) is expressed as a relative percentage of the lysis observed without any inhibitor (in each experiment, 100% represents the lysis observed in AcNHS) relative to the concentration of the C3 inhibitors (x-axis). Curves represent the mean of 10 experiments performed on samples obtained from 2 PNH patients; error bars represent standard deviations. (B) Flow cytometry assessment of C3 fragment deposition on the surface of erythrocytes from untreated PNH patients. Dot plots show intact erythrocytes as gated by physical parameters; CD59 (59-PE monoclonal Ab; y-axis) vs C3 fluorescein isothiocyanate (Ab14396 polyclonal Ab; x-axis). Scheme of erythrocyte populations and potential pattern of C3-fragment deposition on normal, type II, and type III PNH erythrocytes as observed in the fluorescence-activated cell sorter plots (top); unmanipulated fresh erythrocytes (negative control) and erythrocytes incubated in the presence of eculizumab (acidified ABO-matched serum from a patient on eculizumab, positive control for C3 deposition) (middle, from left to right); and erythrocyte pellets after incubation in acidified ABO-matched NHS in the absence of inhibitors (positive control for hemolysis) and in the presence of blocking concentrations of Cp40 and PEG-Cp40. Plots show representatives out of the 10 experiments (bottom, from left to right). (C) Schematic representation of activity spectrum of Cp40 in comparison with anti-C5 therapy. Eculizumab only blocks MAC formation and thus intravascular lysis, thereby enabling possible C3 opsonization of surviving erythrocytes and subsequent extravascular hemolysis. C3 inhibitors such as Cp40 prevent C3 activation upstream, thereby preventing both intravascular lysis and possible C3-mediated extravascular lysis via CR.

Figure 3

Pharmacokinetic evaluation of PEG-Cp40 in NHPs. (A) Dose scheme for PEG-Cp40 administration: a single dose of 200 mg PEG-Cp40 was injected intravenously into 2 cynomolgus monkeys at time 0 (green arrow) and blood samples were drawn at various time points (red arrows). Given the size difference between Cp40 (1.7 kDa) and PEG-Cp40 (∼40 kDa), the selected dose corresponds to ∼2 mg of active peptide per kg. (B) Monitoring of PEG-Cp40 plasma concentrations as determined by ultra performance liquid chromatography-high definition mass spectrometry (after fragmentation using subtilisin-A and SPE). Baseline levels of plasma C3 (measured by ELISA in T0 sample) are depicted as dotted lines for each animal. Note that the 1 and 2 hour postinjection blood draws were not included in this analysis. (C) Change of plasma C3 levels during the treatment with PEG-Cp40; western blot analysis after 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (reducing conditions) was performed with plasma samples collected at different time points and a polyclonal C3 antibody was used for detection (top). Although the antibody recognized both chains of C3, the reactivity with the α-chain was generally higher. Plasma levels of an unrelated protein (transferrin) were used as an internal control on the same membrane after stripping and reprobing with a transferrin antibody (bottom). Panel shows a representative blot from at least 2 independent analyses of samples from 2 animals each.

Figure 4

Subcutaneous administration of Cp40 in NHPs. (A) Pharmacokinetic profile of Cp40 in plasma samples collected 2, 5, and 30 minutes and 1, 2, 4, 6, and 24 hours after a single subcutaneous injection (2 mg/kg) in cynomolgus monkeys. Baseline levels of plasma C3 (measured by ELISA) are depicted as dotted lines for each animal. (B) Dose scheme for optimized Cp40 administration study: 4 doses of Cp40 (1 mg/kg each) were injected subcutaneously into 2 cynomolgus monkeys at a time interval of 12 hours (green arrows) and blood samples were collected 30 minutes, and 4, 8, and 12 hours after each injection, with an additional collection 72 hours after the start of the experiment (red arrows). (C) Monitoring of Cp40 plasma concentrations as determined by ultra performance liquid chromatography-high definition mass spectrometry (after SPE). Baseline levels of plasma C3 (measured by ELISA in T0 sample) are depicted as dotted lines for each animal. The dependence of the Cp40 concentration profiles on the baseline C3 level of each animal is expected to be caused by target-dependent elimination kinetics, as suggested in previous studies. (D) Plasma levels of C3 (top) and transferrin (internal standard; bottom) during the treatment with Cp40 as determined by western blot analysis (in analogy to Figure 3C). Panel shows a representative blot from at least 2 independent analyses of samples from 2 animals each.