What Was Old Is New Again

The chronic intravascular hemolysis that is the hallmark clinical manifestation of paroxysmal nocturnal hemoglobinuria (PNH) is mediated by the alternative pathway of complement (APC) (Figure 1). Because the antibody-independent APC is primed for attack at all times, elaborate mechanisms for self-recognition and protection of the host against complement-mediated injury have evolved. Both fluid-phase and membrane-bound proteins are involved in these protective and self-recognition processes. Normal human erythrocytes are protected against APC-mediated destruction primarily by decay accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL, CD59). These proteins act at different steps in the complement cascade (Figure 1). CD55 regulates the formation and stability of the C3 and C5 convertases (Figure 1, inside green boxes), while CD59 blocks the formation of the membrane attack complex (MAC) (Figure 1, inside blue box). Deficiency of both CD55 (brown ovals) and CD59 (gold ovals) on the affected erythrocytes is the pathophysiologic basis of the Coombs’-negative, intravascular hemolysis that defines PNH (Figure 2).

The complement-mediated intravascular hemolysis of PNH can be inhibited by blocking formation of the MAC. The MAC consists of complement components C5b, C6, C7, C8, and multiple molecules of C9. Eculizumab is a humanized monoclonal antibody that binds to complement C5, preventing its activation to C5b and thereby inhibiting MAC formation (Figure 1). In 2007, eculizumab was approved by both the FDA and the European Union Commission for treatment of the hemolysis of PNH. Treatment with eculizumab reduces transfusion requirements, ameliorates the anemia of PNH, and improves quality of life by attenuating the debilitating constitutional symptoms (fatigue, lethargy, asthenia) associated with chronic complement-mediated intravascular hemolysis. Following treatment, serum lactate dehydrogenase concentration (LDH), a surrogate marker for intravascular hemolysis (Figure 2), returns to normal, but mild to moderate anemia, hyperbilirubinemia and reticulocytosis often persist.¹  This could be the result of ongoing extravascular hemolysis of PNH erythrocytes as a consequence of C3 opsonization as eculizumab does not block the activity of the APC C3 convertase (Figure 1).

Support for this hypothesis is provided by the recent studies of Antonio Risitano and colleagues from Italy. By using two-color flow cytometric analysis, the investigators showed that, in patients treated with eculizumab, a portion of the PNH erythrocytes (i.e., the CD59-deficient population) had C3 bound. The studies of Risitano et al. also confirmed the Coombs’-negative designation of PNH, as no C3 was found bound to PNH erythrocytes prior to initiation of treatment with eculizumab, consistent with a model in which, in the absence of eculizumab, PNH erythrocytes upon which complement has been activated are destroyed directly as a consequence of MAC-mediated cytolysis (Figure 2). Post hoc analysis of their data suggested that the percentage of C3+ erythrocytes negatively influenced response (based on steady-state hemoglobin concentration, transfusion requirement, and LDH concentration) in patients treated with eculizumab, but considerable overlap in the percentage of C3+ erythrocytes among the designated treatment groups was observed.

So, is there a better way to treat PNH? Why not block C3 and thereby inhibit not only MAC-mediated lysis, but also C3 opsonization? The major problems with this therapeutic approach are the high concentration of C3 in plasma and, more importantly, the central role that C3 plays in both the classical and the lectin pathways of complement as well as the APC. Blocking all three complement pathways would almost certainly result in unacceptable toxicity as congenital deficiency of C3 is associated with recurrent bacterial infection and early mortality. Still, it may be possible to target only the APC by blocking factor B (Figure 1) or by developing antibodies that inhibit APC C3 convertase formation without affecting C3 function within the classical and lectin pathways.