Abstract
Background: Eculizumab blocks the lytic complement pathway by inhibiting C5 and has become the standard of care for certain complement-mediated diseases. Previously, we have shown that strong complement activation in vitro overrides the C5 inhibition by Eculizumab, which accounts for residual terminal pathway activity.Results: Here we show that the levels of residual hemolysis in ex vivo assays differ markedly (up to 3.4-fold) across sera collected from different paroxysmal nocturnal hemoglobinuria (PNH) patients on Eculizumab treatment. This large variability of residual activity was also found in sera of healthy donors, thus cross-validating the findings in patients. While PNH patients with residual lytic activities of 11–30% exhibited hemolysis levels around the upper limit of normal (i.e., plasma LDH of ~250 u/L), as expected for PNH patients on Eculizumab therapy, we found sustained and markedly increased LDH levels of around 400 u/L for the patient with the highest residual activity of 37%. Furthermore, the clinical history of nine out of 14 PNH patients showed intravascular breakthrough hemolysis at the time of documented infections despite ample amounts of administered Eculizumab and/or experimentally determined excess over C5.Conclusion: The occurrence of extraordinary high levels of residual terminal pathway activity in PNH patients receiving Eculizumab is rare, but can impair the suppression of hemolysis. The commonly observed low levels of residual terminal pathway activity seen for most PNH patients can exacerbate during severe infections and, thus, can cause pharmacodynamic breakthrough hemolysis in PNH patients treated with Eculizumab.
Highlights
Chronic intravascular hemolysis is the clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) and occurs as a consequence of an un- or under-regulated complement alternative pathway (AP) [1, 2]
We employed a rabbit erythrocyte lysis assay with human serum that strongly activates complement to probe if sera obtained from different PNH patients on Eculizumab treatment exhibit variable levels of residual hemolytic activity
As described before, addition of the orthogonal C5 inhibitor Coversin or the engineered alternative pathway (AP) inhibitor miniFH to the patient sera led to C5 double inhibition or proximal complement pathway inhibition, respectively, and resulted in complete inhibition of C5 activity (Figure 1A) [19]
Summary
Chronic intravascular hemolysis is the clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) and occurs as a consequence of an un- or under-regulated complement alternative pathway (AP) [1, 2]. Treatment with Eculizumab, a monoclonal antibody that targets the human complement component C5 and blocks the terminal and lytic complement pathway, has dramatically improved the clinical outcome and high mortality of PNH patients [3,4,5,6,7,8] [reviewed in Brodsky [9], Parker [10], Hill et al [11], and Brodsky [12]]. Different mechanisms can cause this incomplete clinical response, for example marrow failure or switch to extravascular hemolysis, i.e., removal of C3opsonised PNH erythrocytes via the reticuloendothelial system (C3-opsonisation can still occur under therapy since Eculizumab only blocks the terminal complement pathway) [14, 16,17,18]. We have shown that strong complement activation in vitro overrides the C5 inhibition by Eculizumab, which accounts for residual terminal pathway activity
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