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

The serum proteins factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product FH-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 an 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 a paroxysmal nocturnal hemoglobinuria 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.