Inadequate control of the complement system is the underlying or aggravating factor in many human diseases. While treatment options that specifically target the alternative pathway (AP) of complement activation are considered highly desirable, no such option is available in the clinic. Here we present a successful example of protein engineering, guided by structural insight on the complement regulator factor H (FH), yielding a novel complement-targeted therapeutic (mini-FH) with clinical potential. Despite a 70% reduction in size, mini-FH retained and in some respects exceeded the regulatory activity and cell surface-recognition properties of its parent protein FH, including the recently described recognition of sites of oxidative stress. Importantly, the chosen design extended the functional spectrum of the inhibitor, as mini-FH showed increased binding to the surface-bound opsonins iC3b and C3dg when compared to FH. Thus, mini-FH is equipped with a unique and clinically valuable triple-targeting profile towards diseased host cells, through its binding to sites of ongoing complement activation, markers of oxidative damage, and host surface-specific polyanions. When assessed in a clinically relevant AP-mediated disease model of paroxysmal nocturnal hemoglobinuria, mini-FH largely outperformed factor H and indicated advantages over clinically evaluated AP inhibitors. Thus, the rational engineering of a streamlined FH construct not only provided insight into the function of a key complement regulator but also yielded a novel inhibitor that combines a triple targeting approach with high AP-specific inhibitory activity (IC50 ~ 40 nM), which may pave the way towards new options for the treatment of complement-mediated diseases.

• Peptidic C3 inhibitors of the compstatin family (Cp40) efficiently prevent hemolysis and opsonization of PNH erythrocytes in vitro.• Pharmacokinetic studies show that sustained therapeutic concentrations can be achieved with both Cp40 and its PEGylated derivative, PEG-Cp40.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 IC 50 ∼4 mM and full inhibition at 6 mM. 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. (Blood. 2014;123(13):2094-2101 IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a complex hematologic disorder characterized by the expansion of hematopoietic cells deficient in glycophosphatidylinositol-anchored surface proteins, including the complement regulators CD55 and CD59. 1 Affected erythrocytes suffer from uncontrolled complement activation on their surface, and subsequent membrane attack complex (MAC)-mediated intravascular hemolysis.2 The therapeutic anti-C5 antibody eculizumab (Soliris, Alexion) has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in a majority of PNH patients.3,4 Yet, persistent C3 activation occurring during eculizumab treatment may lead to progressive deposition of C3 fragments on affected erythrocytes and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematologic benefit of anti-C5 treatment. 5,6 Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current complement-targeted treatment. 7,8 Indeed, it has been recently documented that protein inhibitors of the alternative pathway (AP) of complement activation, such as the CD21/factor H (FH) fusion protein TT30 (Alexion) or the engin...

Regulators of complement activation (RCA) inhibit complement‐induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i–iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b‐binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease‐related mutations and immune evasion.

Key Points The complement opsonin C3dg, which is found on PNH erythrocytes of patients under anti-C5 therapy, can bind to complement receptor 3 (CR3). Interaction of C3dg with CR3 on activated monocytes induces erythrophagocytosis, thereby corroborating a model of extravascular hemolysis.

Background:Staphylococcus aureus has evolved a web of mechanisms to disrupt the human complement system. Results: We report the structures of two staphylococcal complement inhibitor proteins, SCIN-B and SCIN-D. Conclusion:We have identified differences in C3b recognition within active SCIN proteins and suggest a physical basis for lack of C3b binding by SCIN-D. Significance: This analysis may inform future design of complement-targeted therapeutics.