The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment opsonization

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complementmediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membranetargeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membranetargeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients. (Blood. 2012;119(26):6307-6316) IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a blood disorder clinically characterized by intravascular hemolysis, thrombophilia, and bone marrow failure. 1-3 A unique feature of PNH is the presence of clonal populations of blood cells that are defective in glycosylphosphatidylinositol (GPI)-anchor biosynthesis, 4 because they derive from stem cells harboring an acquired somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene. 5,6 GPIlinked surface proteins include CD55 (also known as decayaccelerating factor [DAF]) 7,8 and CD59 (or membrane inhibitor of reactive lysis [MIRL]), 9,10 2 major complement regulators on the cell surface. Because of the lack of these 2 regulators, PNH erythrocytes (red blood cells [RBCs]) are exquisitely vulnerable to complement activation. Indeed, the main mechanism of hemolysis in PNH is the intravascular destruction of CD59 deficient RBCs by the membrane attack complex (MAC; supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). 11 MAC formation in patients can be abrupt and massive when complement is triggered by specific conditions, as with an infection, explaining the paroxysms of hemoglobinuria that have given PNH its name. However, at a lower rate, hemolysis in PNH is continuous, and it is accounted for by the so-called tickover of the complement alternative pathway (CAP). Tickover occurs through spontaneous hydrolysis of C3, binding of factor B to this form of C3, and subsequent formation of a C3 cleavage and activating multiprotein complex designated a C3 convertase (supplemental Figure 1). 12,1...

An Engineered Construct Combining Complement Regulatory and Surface-Recognition Domains Represents a Minimal-Size Functional Factor H

“…FH plays a role in the protection of PNH erythrocytes, which are characterized by strongly reduced numbers of the membrane-anchored complement regulatory proteins CD55 and CD59 due to a defect in the glycosylphosphatidylinositol anchor (67). A recent report showed that targeting CCPs 1-5 of FH to sites of complement activation at the surface of PNH erythrocytes by using a chimeric molecule confers protection of these erythrocytes from C3 fragment deposition and lysis (68).…”

Polymorphism of the complement receptor 1 gene correlates with the hematologic response to eculizumab in patients with paroxysmal nocturnal hemoglobinuria

“…The RBC were then incubated with sera containing eculizumab as previously described. 13,14 Briefly, a 2% suspension of RBC from PNH patients was incubated at 37°C with a pool of ABO-compatible sera from patients on eculizumab, and the complement alternative pathway was activated by mild acidification (HCl 0.016 M). At serial time points (15,30, 60, and 120 min) after complement activation the fraction of GPI-negative RBC with newly bound C3 fragments was measured by flow cytometry (AccuriC6, Becton Dickinson, NJ, USA) after staining with anti-CD59 Alexa647 (Mem43, Serotec, UK) and anti-C3d-neoantigen (A250, Quidel, CA, USA); secondary staining was performed with phycoerythrin-labeled polyclonal rabbit-anti-mouse antibodies (Dako Cytomation, Denmark).…”

“…29,30 In addition, CR1 plays a role in the clearance of immune complexes and in phagocytosis. [31][32][33] Thus, the lower levels of CR1 expression on the red cell surface associated with the H/L and L/L genotypes 22 are expected to result in increased complement activation and C3 binding on PNH red cells.In order to test this hypothesis we investigated the kinetics of C3 binding to GPI-negative red cells in vitro 13,14 from patients with the three CR1 HindIII genotypes. When GPI-negative red cells were exposed to activated complement in the presence of eculizumab, we promptly detected C3-positive PNH red cells, the percentage of which increased with time.…”

Complement inhibitors to treat IgM-mediated autoimmune hemolysis

“…53 Risitano and co-workers reported TT30 to efficiently inhibit C3 deposition on and hemolysis of PNH RBCs. 55 Therefore, TT30 might be a suitable therapeutic, not only to inhibit intravascular hemolysis, but also extravascular hemolysis in PNH patients (Figure 2). In analogy to H17/3E7, TT30 is not effective in the inhibition of classical pathway activation.…”