Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of "miniFH" and the clinically developed "FH-CR2" fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH.

Keywords: Alternative pathway; Complement targeted inhibition; Paroxysmal nocturnal hemoglobinuria; miniFH.

Figure 1. C3-opsonin targeted complement inhibitors: design and production

(A) Schematic representation of the molecular composition of inhibitors enrolled in this study. Numbering based on encoded protein sequences including signal sequence according to UniProt (accession number in parenthesis). Each oval represents a CCP domain (domain numbers are indicated). Native N- and C-terminal residues are denoted in one letter code; non-native linker sequences and purification tags are indicated by box. Key functional properties of CCP domains are highlighted at the bottom. In terms of regulating the AP (via C3b) FH-CR2 and miniFH proteins all utilize N-terminal portions of the natural regulator Factor H (FH). In regards to targeting to C3-inactivation products, FH-CR2 relies on an N-terminal portion of the complement receptor 2 (CR2), while the miniFH approach utilizes C-terminal portions of FH, which are cryptic and not fully available in natural FH. MiniFH-1 is polymorphic for isoleucine at position 62. (B) SDS-PAGE gel analysis of C3-targeted inhibitors produced for this study under reducing and non-reducing conditions (2μg each lane).

Figure 2. Regulatory activity of C3-opsonin targeted complement inhibitors on LPS and PNH erythrocytes

(A) Inhibition of LPS-driven AP-activation. Serum spiked with C3-opsonin targeted inhibitors or FH was exposed to LPS coated on an ELISA plate (25% final serum conc.). Deposition of C3b/iC3b was detected as a measure for complement activation (Average of 2 independent assays with SD is shown). (B) Protection of PNH cells from complement AP mediated lysis. PNH erythrocytes were incubated for 24h in acidified human serum spiked with complement inhibitors (75% final serum conc.). The lysis of PNH II & III erythrocytes was measured by flow cytometry and normalized to lysis of PNH II & III erythrocytes observed in acidified serum in absence of spiked inhibitors (Average of 4 independent assays with SD).

Figure 3. Protection of rabbit erythrocytes from complement AP mediated lysis

Rabbit erythrocytes were incubated for 30 min in serum (from healthy donors) spiked with complement inhibitors (75% final serum conc.). The lysis of rabbit erythrocytes was measured by hemoglobin release and normalized to lysis observed in water (Average of 3 or 2 independent assays with SD is shown for miniFH-1 and FH-CR2, respectively).

Figure 4. Complement mediated killing of E. coli in NHS

E. coli cells containing a selective marker were incubated for 1h in NHS spiked with complement inhibitors or PBS. Colony forming units (CFU) were evaluated by colony counting after plating the reactions on selective medium. Eculizumab was used at 0.3 μM, which nearly corresponds to the known human C5 plasma conc. of 0.38μM (HIS: heat inactivated serum; average of 3 assays with SD is shown, except for eculizumab which was assayed only twice). Selected points were analyzed for significant changes in CFU (non-parametric one-way analysis of variance with a significance level of p < 0.05; GraphPad Prism). The number of CFU of the serum control and the two selected concertation points of the targeted inhibitors that fully protected PNH erythrocyte hemolysis (0.2 μM miniFH-1 (green arrow); 0.8 μM FH-CR2 (black arrow)) were significantly smaller (*) than the number of CFU for heat-inactivated serum. No statistical significant (n.s.) change was noted between the serum control and the concentration points of miniFH-1 and FH-CR2.