TT30, a Novel Human Complement Inhibitor in Development for Paroxysmal Nocturnal Hemoglobinuria and Other Hemolytic Disorders, Demonstrates Red Blood Cell Surface Targeting and Retention In a Model of Complement Alternative Pathway-Mediated Hemolysis (2010)

Abstract Abstract 638 Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases, especially genetically linked hemolytic disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and thrombotic microangiopathy (TMA) disorders such as atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura. The complement system can be activated through three unique pathways (classical, lectin/mannose and alternative). In PNH, the lack of CD55 on RBC allows CAP-initiated complement C3 activation by C3 convertases, while the lack of CD59 allows C5 activation by C5 convertase to proceed to formation of the membrane attack complex (MAC; C5b-9), resulting in intravascular hemolysis (IVH). Treatment of patients with the anti-C5 monoclonal antibody (mAb) eculizumab abrogates IVH; however, because eculizumab does not inhibit CAP activity prior to C5, covalently bound C3 fragments (C3frag) and both C3 and C5 convertases continue to accumulate on PNH red blood cells (RBCs). Clearance of PNH RBCs that are C3frag-coated by complement receptors within the reticuloendothelial system (RES) is the putative cause of continued extravascular hemolysis (EVH) in patients who receive eculizumab. Continued anemia and transfusion requirements are found in a substantial proportion of eculizumab-treated patients, and correlate with PNH RBC-bound C3frag. High levels of C5 convertases on the same cells may also contribute to intermittent escape from eculizumab control of IVH due to pharmacodynamic breakthrough. To selectively modulate CAP activity on PNH RBC and replace the CD55-mediated control of CAP activation, we developed TT30, a novel therapeutic fusion protein linking the C3frag-binding domain of human complement receptor type 2 (CR2/CD21) with the CAP inhibitory domain of human factor H (fH). TT30 delivers cell surface-targeted (via CR2) inhibition of CAP activity (via fH) and blocks the ex vivo hemolysis of PNH RBCs, while at the same time retaining the normal ability of the complement system to efficiently activate C3 through the classical and lectin pathways. We studied the mechanism of TT30 prevention of hemolysis by control of CAP activity in human serum using: 1) an in vitro model of CAP-mediated hemolysis in which rabbit RBCs are exposed to normal human serum under conditions promoting CAP activation (Mg++/EGTA) and the extent of hemolysis is quantified by measuring hemoglobin release; 2) flow cytometric phenotyping of C3frag accumulation on rabbit RBCs exposed to normal or C5-deficient human serum using mAbs specific for human iC3b (A710, Quidel) or C3d (A702, Quidel); 3) flow cytometric demonstration of TT30 binding to C3frag+ rabbit RBCs with a noncompeting mAbs against CR2 (HB5, Taligen) or fH (A255, Quidel); and 4) an in vitro model of CAP-mediated MAC formation in which human serum is exposed to an LPS-coated surface in the presence of Mg++/EGTA and CAP activation through to the MAC is quantified by detection of a neoantigen in poly-C9 by ELISA. The results demonstrate that TT30 efficiently inhibits CAP-mediated MAC formation (IC50 of 3.2 ug/ml) and hemolysis (IC50 of 50.1 ug/ml) and that both of these activities are dependent upon targeting to C3frag+ surfaces by CR2, as evidenced by complete reversal of TT30 inhibitory activity in the presence of a 2-fold molar excess of a competing anti-CR2 mAb (1048, Taligen). Rabbit RBCs were shown to become coated with C3frag in the presence of normal and C5-deficient serum and to undergo lysis with normal serum. TT30 was readily demonstrated to be bound to C3frag+ RBCs during prevention of hemolysis and to remain detectable on RBCs for at least 24 hours. The amount of bound TT30 was proportional to the accumulation of C3frags. Collectively, these results demonstrate that TT30 displays targeted control of cell surface CAP activation, with both effective and prolonged blockade of MAC formation, and dose-dependent inhibition of hemolysis. Therefore, the CAP-specific novel therapeutic TT30 has potential utility for the treatment of human complement-mediated diseases, such as PNH and aHUS, in which modulation of CAP activation is predicted to be clinically beneficial. Disclosures: Fridkis-Hareli: Taligen Therapeutics: Employment. Storek:Taligen Therapeutics: Employment. Risitano:Taligen Therapeutics: Consultancy, Research Funding. Lundberg:Taligen Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees. Horvath:Taligen Therapeutics: Employment. Holers:Taligen Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.