A Novel Approach to Treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH): Both Hemolysis and C3 Deposition Are Blocked by a Monoclonal Antibody (mAb) Specific for the Alternative Pathway of Complement (APC) C3/C5 Convertase.

Abstract 157

In PNH, red blood cells (RBCs) lack key complement control proteins, CD55 and CD59 and are therefore sensitive to complement activation and intravascular hemolysis. These regulatory proteins function at two different steps in the complement cascade; CD55 (decay accelerating factor, DAF) controls the formation and stability of the APC C3 and C5 convertases, while CD59 (membrane inhibitor of reactive lysis, MIRL) blocks formation of the cytolytic membrane attack complex (MAC). The intravascular hemolysis of PNH can be inhibited in vivo by eculizumab, a humanized mAb that binds complement C5, thereby preventing formation of the MAC. However, PNH patients treated with eculizumab continue to manifest evidence of ongoing hemolysis as they remain anemic with an elevated reticulocyte count and low serum haptoglobin concentration, and approximately 50% of eculizumab-treated patients require transfusion. This observation is consistent with the hypothesis that, in patients treated with eculizumab, PNH RBCs undergo extravascular hemolysis as a consequence of C3 opsonization because eculizumab does not compensate for deficiency of DAF. Recent studies (Risitano et al., Blood, 2009) support this hypothesis as patients undergoing treatment with eculizumab were found to have a positive Coombs test for C3 but not IgG, and flow cytometry demonstrated C3 activation and degradation products bound to the PNH RBCs. This process appeared clinically relevant as transfusion requirement correlated with the percentage of C3 opsonized PNH RBCs. These observations suggest that blocking the APC C3/C5 convertase would be a better way to treat the hemolysis of PNH because this approach has the advantage of blocking both extravascular hemolysis by inhibiting C3 opsonization and preventing intravascular hemolysis by inhibiting MAC generation. We have developed a mAb 3E7 and its deimmunized chimeric humanized derivative H17 that specifically block the APC C3/C5 convertase by binding to a neoepitope expressed when complement C3 is activated. In vitro, 3E7/H17 prevents APC-mediated lysis of rabbit RBCs in human serum and blocks deposition of human C3 activation fragments on APC activator substrates such as zymosan (Mol Immunol, 2006; J Immunol, 2007). We now report that mAb H17/3E7 blocks lysis in acidified normal human serum (aNHS) (a process mediated by the APC) of RBCs from patients with PNH (n=5). Representative results for patients 1 and 2 are as follows: 60% and 40% of RBCs were lysed after a one hour incubation at 37°C; lysis was reduced to 10% and 6%, respectively, at 80 ug/ml of mAb H17, and to 1% lysis (both patients) at 170 ug/ml of mAb H17. We also showed that mAb H17/3E7 blocks deposition of C3 activation fragments on PNH RBCs. After lysis in aNHS, blood samples from PNH patients were probed with Al488 mAb 1H8, specific for C3b/iC3b/C3dg. Flow cytometry experiments revealed C3 fragment deposition on lysed cells corresponding to 30,500 molecules of equivalent soluble fluorochrome (MESF) compared to a background signal of 225 MESF on unlysed RBCs in the same sample. Addition of mAb H17 blocked C3 fragment deposition not only on the unlysed cells but also on the small number of recovered ghosts . Importantly, mAb H17/3E7 inhibits the APC specifically. PNH RBCs, opsonized with IgM in serum from a patient with chronic cold agglutinin disease, were lysed in NHS by the classical complement pathway, and this lysis was not inhibited by mAbH17/3E7. Together, these experiments demonstrate that both hemolysis and C3 opsonization of PNH RBCs can be inhibited by a novel mAb that specifically blocks the APC C3/C5 convertase while leaving intact the classical pathway of complement. These findings suggest an approach to therapy of PNH in which both intravascular and extravascular hemolysis can be inhibited while preserving the important immune functions of the classical pathway of complement.