The complement alternative pathway in paroxysmal nocturnal hemoglobinuria: From a pathogenic mechanism to a therapeutic target

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal, not malignant, hematological disease characterized by intravascular hemolysis, thrombophilia and bone marrow failure. While this latter presentation is due to a T-cell mediated auto-immune disorder resembling acquired aplastic anemia, the first two clinical presentations are largely driven by the complement pathway. Indeed, PNH is characterized by a broad impairment of complement regulation on affected cells, which is due to the lack of the complement regulators CD55 and CD59. The deficiency of these two proteins from PNH blood cells is due to the somatic mutation in the phosphatidylinositol N-acetylglucosaminyltransferase subunit A gene causing the disease, which impairs the surface expression of all proteins linked via the glycosylphosphatidylinositol anchor. The lack of the complement regulators CD55 and CD59 on PNH erythrocytes accounts for the hallmark of PNH, which is the chronic, complement-mediated intravascular hemolysis. This hemolysis results from the impaired regulation of the alternative pathway upstream in the complement cascade, as well as of the downstream terminal pathway. PNH represented the first indication for the development of anti-complement agents, and the therapeutic interception of the complement cascade at the level of C5 led to remarkable changes in the natural history of the disease. Nevertheless, the clinical use of an inhibitor of the terminal pathway highlighted the broader derangement of complement regulation in PNH, shedding light on the pivotal role of the complement alternative pathway. Here we review the current understanding of the role of the alternative pathway in PNH, including the emergence of C3-mediated extravascular hemolysis in PNH patients on anti-C5 therapies. These observations provide the rationale for the development of novel complement inhibitors for the treatment of PNH. Recent preclinical and clinical data on proximal complement inhibitors intercepting the alternative pathway with the aim of improving the treatment of PNH are discussed, together with their clinical implications which are animating a lively debate in the scientific community.

Keywords: C3; FB; FD; alternative pathway; extravascular hemolysis; intravascular hemolysis; paroxysmal nocturnal hemoglobinuria.

FIGURE 1

Complement alternative pathway in PNH. (A), Untreated PNH. C3:H₂O generated by spontaneous hydrolysis of C3 (the so‐called “C3 tick‐over”) continuously initiates the complement cascade through its alternative pathway in the fluid phase. Due to the lack of CD55, PNH erythrocytes are unable to regulate complement activation on their surface, and C3bBb C3 convertase can be generated from C3 tick‐over and factor B cleavage operated by factor D. These C3 convertases generate further C3b, eventually self‐transforming into the C3bBbC3b C5 convertases, which cleave C5 into C5a and C5b. This latter may start the formation of the membrane attack complex with C6, C7, C8 and C9, eventually leading to intravascular hemolysis. (B), PNH on C5‐inhibitors. Terminal complement inhibitors (ie, anti‐C5 agents) prevent the cleavage of C5 into C5a and C5b, thereby disabling the formation of the membrane attack complex (MAC) and inhibiting intravascular lysis of PNH erythrocytes. Nevertheless, early steps of complement activation and upstream C5 cleavage remain uncontrolled, leading to opsonization of PNH erythrocytes with C3 fragments. C3‐opsonized erythrocytes can be recognized by C3‐specific receptors, expressed on professional macrophages in the liver and in the spleen, eventually resulting in extravascular hemolysis. (C), PNH on alternative pathway inhibitors. The alternative pathway may be intercepted at the level of different key components, such as FD, FB, C3 and C3 convertases. Inhibitors of any of these target, if the inhibition is pharmacologically sustained, disable the complement cascade in its early phases (these agents are also known as proximal complement inhibitors), preventing the generation of C3(H₂O) Bb in the fluid phase, as well as disabling surface amplification activity acting on surface‐bound C3bBb. On PNH erythrocytes, this combined effect results in inhibition of the MAC‐mediated intravascular hemolysis, and in the prevention of C3 opsonization (and thus of extravascular hemolysis), eventually leading to normal life‐span of affected erythrocytes

FIGURE 2

Dose–response curve of different alternative pathway inhibitors. (A), TT30. Effect of TT30 on lysis of PNH erythrocytes in vitro. (B), Mini‐FH. Effect of mini‐FH on lysis of PNH erythrocytes in vitro. (C), Compstatin. Effect of the compstatin analogue Cp40 on lysis of PNH erythrocytes in vitro. (D), FD inhibitor. Effect of the FD inhibitor “compound 7” on lysis of PNH erythrocytes in vitro. (E), FB inhibitor. Effect of the FB inhibitor LNP023/iptacopan on lysis of PNH erythrocytes in vitro. (F), Proximal inhibitor combined with anti‐C5. Effect of combination treatment including the proximal inhibitor TT30 and the anti‐C5 eculizumab (at fixed dose, 3.3 μM); effect on C3 opsonization is shown in addition to effect on lysis. In presence of anti‐C5, the contribution of the proximal inhibitors is better seen as inhibition of C3 opsonization, which becomes evident at doses overlapping to those of the dose–response in absence of anti‐C5 (see panel A of this figure). At the same concentration, the additive (and not synergic) effect on lysis also appears, leading to full inhibition of lysis (while at suboptimal concentration the effect on lysis was entirely driven by the anti‐C5)