• Strong complement activation overrides the terminal pathway inhibition by the anti-C5 antibody eculizumab.• The more powerful complement is activated, the less effective is terminal pathway inhibition by diverse anti-C5 agents.Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drug's therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general. (Blood. 2017;129(8):970-980)

The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.

Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naïve macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1β, IL-1ra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-I-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection.

Background: IFN␤ constitutes an approved drug to treat multiple sclerosis (MS), but it has limited efficacy. Results: A modified human IFN variant, which exhibits both superagonist properties and 10-fold increased lifespan, outperforms IFN␤ in an animal MS model. Conclusion:This drug candidate has potential to supersede IFN␤ for the treatment of MS. Significance: Protein engineering allows development of more effective drugs to treat autoimmune diseases.