Comparative Analysis of Novel Complement-Targeted Inhibitors, MiniFH, and the Natural Regulators Factor H and Factor H–like Protein 1 Reveal Functional Determinants of Complement Regulation

Abstract

The serum proteins Factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product Factor H-like protein 1 (FHL-1; consisting of CCPs 1–7) are major regulators of the alternative pathway (AP) of complement activation. The engineered version of FH, miniFH, contains only the N- and C-terminal portions of FH linked by an optimized peptide and shows ~10-fold higher ex vivo potency. We explored the hypothesis that regulatory potency is enhanced by unmasking of a ligand-binding site in the C-terminal CCPs (19–20) that is cryptic in full-length native FH. Therefore we produced a FH variant lacking the central domains 10–15 (FHΔ10–15). To explore how avidity affects regulatory strength, we generated a duplicated version of miniFH, termed midiFH. We compared activities of FHΔ10–15 and midiFH to miniFH, FH and FHL-1. Relative to FH, FHΔ10–15 exhibited an altered binding profile toward C3 activation products and a 5-fold-enhanced complement regulation on PNH patient’s erythrocytes. Contrary to dogma, FHL-1 and FH exhibited equal regulatory activity, suggesting that the role of FHL-1 in AP regulation has been underestimated. Unexpectedly, a substantially increased avidity for complement opsonins, as seen in midiFH, did not potentiate the inhibitory potential on host cells. In conclusion, comparisons of engineered and native FH-based regulators have identified features that determine high AP regulatory activity on host cells. Unrestricted availability of FH CCPs 19–20 and an optimal spatial orientation between the N- and C-terminal FH regions are key.

Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation

“…This is consistent with previous data showing that the C3-opsonintargeting inhibitors mini-FH and TT30 efficiently control AP activation in several assays. 38,44,45,49,50 As observed for the double inhibition of C5 in NHS (above), addition of coversin or PAS-coversin to the patientderived, eculizumab-containing serum completely stopped the residual hemolysis if compared with eculizumab alone. We verified that the patient sera indeed contained an excess amount of eculizumab over C5 ( Figure 1F).…”

“…Immobilization of 800 response units (RUs) of C3b onto the carboxymethyldextran surface of a sensor chip via standard amine coupling enabled binding of mini-FH ( Figure 3A) as expected. 38,45 In contrast, no binding of C5 to amine coupled C3b was observed. However, when additional 900 RUs of C3b molecules were immobilized in a more physiological manner employing onchip assembled C3 convertases, C5 did efficiently adhere to such "physiologically" immobilized C3b in a concentration-dependent way ( Figure 3B).…”

“…Coupling of C3b to the sensor chip and binding analysis was performed as described previously 45 and detailed in supplemental Methods. The following analytes were applied as indicated: C5 alone, C5 with excess amounts of eculizumab or coversin, or a mix of eculizumab and coversin.…”

Dual modulating functions of thrombomodulin in the alternative complement pathway

“…C3 is composed of two polypeptide chains (α and β) linked by disulfide bonds (Figure 1). able to activate the alternative pathway (12,13). Under the condition in which C3 was partially cleaved to generate the α' chain by incubation of complement components of the alternative pathway (C3, factor B, and factor D), co-incubation of various concentrations of rTM with these complement components resulted in a decrease in the intensity of a band corresponding to the α chain (Figure 2, upper).…”

Protection of host cells by complement regulators

“…Published data indicate that the N‐ and C‐termini of FH cooperate in binding to C3b, since the affinity of FH for C3b (K D approximately 0.5–1 μM) is significantly higher than that of the N‐terminal (FH1–4, K D approximately 10–14 μM) or C‐terminal (FH19–20, K D approximately 5 μM) sites assayed in isolation . Remarkably, the affinity of FHL‐1 for C3b was recently determined to be 1.5 μM: just half that of FH . This result suggests that the N‐terminal binding site in FH for C3b actually extends beyond domains 1–4 and includes one or more of the domains 5–7.…”