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

Abstract

• 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)

Iron oxide nanoparticles induce cytokine secretion in a complement-dependent manner in a human whole blood model

“…The C3 inhibitor compstatin analog Cp40 completely inhibited the cytokine response, whereas C5 inhibitor eculizumab only partly reduced the production of the inflammatory mediators, which is in agreement with previous findings. 26,27 While compstatin analog Cp40 inhibits both the formation of C3a and C5a, eculizumab only inhibited the formation of C5a, thus our data could be explained by a dual involvement of C3a and C5a for optimal cytokine response. In a study by Orning et al, 27 the differences between compstatin and eculizumab were also shown to be connected to the C3b/iC3b formation with subsequent cell-adhesion (by complement receptor CR3).…”

The renaissance of complement therapeutics

“…Although eculizumab prevents MAC formation and intravascular haemolysis, PNH erythrocytes continue to be opsonized by C3 fragments, which could lead to recognition by complement receptors on phagocytic cells and extravascular haemolysis via erythrophagocytosis, as shown in in vitro studies 62,73 . Insufficient dosing and/or could also potentially contribute to insufficient responses to eculizumab 62,74 . Indeed, an in vitro study showed that at high levels of complement activation, eculizumab alone was not sufficient to protect PNH erythrocytes from lysis; the addition of a second C5 inhibitor or an agent controlling upstream activation at the convertase level was required for full blockade 74 .…”

“…Insufficient dosing and/or could also potentially contribute to insufficient responses to eculizumab 62,74 . Indeed, an in vitro study showed that at high levels of complement activation, eculizumab alone was not sufficient to protect PNH erythrocytes from lysis; the addition of a second C5 inhibitor or an agent controlling upstream activation at the convertase level was required for full blockade 74 . The clinical availability of additional complement inhibitors for the treatment of PNH and other disorders is therefore an important goal.…”

“…This agent consists of the regulatory and surface-recognizing segments of FH connected by a peptide linker. Despite its reduced size in comparison to FH, the binding of mini-FH to opsonins is maintained or even improved, resulting in high efficacy in preclinical models of PNH 74,149 . Amyndas is also developing a peptide- based entity, compsorbin (5C6), that can be coated to biomaterials and cells, for example, the endothelium of transplanted organs, to recruit endogenous FH and therefore protect against complement attack 150,151 .…”

Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics

“…The underlying mechanisms which result in switching of the molecular arrangement and substrate specificities of C3 convertases to C5 convertases remain poorly understood. However, recent advances have been made in this area which have made use of high-affinity C5 targeted complement inhibitors (71, 72), and these new systems may better enable future mechanistic studies of cmp-5-based compounds. Thus, while important questions about the mechanism of cmp-5 inhibition remain to be addressed by future studies, it is feasible that cmp-5 could disrupt C5 convertase activity by binding this functionally critical region of C3b.…”