“Red Cell Complement Loading In PNH Patients On Eculizumab Is Associated With a C3 Polymorphism Which Influences C3 Function, Predicts For Increased Extravascular Hemolysis and Provides a Rationale For C3 Inhibition.”
Authors: Kaudlay Praveen , Hua Haiying , He Guansheng , Newton Darren J , Varghese Abraham M , Munir Talha , Hill Anita , Kelly Richard J , Richards Stephen John , Hillmen Peter 
Published: October 2019

Abstract

Introduction
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired bone marrow disorder characterised by intravascular hemolysis and hemoglobinuria, potentially life-threatening thrombosis and an association with aplastic anemia. Most of the clinical features and complications of PNH are due to the unopposed activity of complement due to the absence of CD59 and CD55, two key regulators of complement. Eculizumab prevents the cleavage of C5 complement thereby preventing terminal complement activity and protecting PNH cells from lysis. The inhibition of C5 preserves the early part of complement pathway and leads to the build up of C3 on the PNH red cells, perhaps in part due to their lack of CD55. The majority of PNH patients receiving eculizumab have evidence of extravascular haemolysis that can be clinically significant, including with anemia, hyperbilirubinemia and in some a continued requirement for transfusions. This extravascular hemolysis in thought to be due to the C3 loading of PNH red cells.

Methods
We report the C3-loading of the PNH red cells from 119 patients treated with eculizumab and correlate this with hemoglobin, LDH, bilirubin, reticulocytes and transfusions. We have studied genetic polymorphisms that affect both C3 and FCγR. We have genotyped 46 eculizumab patients for a functional mutation in the C3 gene (rs2230199). The two alleles of this gene can be distinguished by the presence or absence of a HindIII restriction site that distinguishes the electophoretically slow (arg80) from the electrophoretically fast (gly80) allotype. The fast (C3F) allotype allele of this snp is associated with a range of disorders including age-related macular degeneration, IgA nephropathy, systemic vasculitis and partial lipodystrophy. APL-1 is a small cyclic peptide that binds to and inhibits the activation of complement C3. APL-2 is a large conjugate of APL-1 with enhanced bioactivity and a long systemic half-life. APL-1 and APL-2 molecules as well as other complement inhibitors were studied for lysis of red cells and C3 loading in vitro in a modified Ham test in which flow cytometry was used to identify non-lysed cells.

Results
Out of the 119 Eculizumab treated patients, 55 (46.2%) required at least one transfusion on treatment. 110 patients had C3 detectable by flow cytometry on their PNH red cells (mean of 19.8%; range: <0.1 to 64.6%). C3-loading was not seen on the normal red cells from the same patients on treatment nor on the PNH red cells in patients not receiving eculizumab. The mean LDH (735IU/l) and reticulocyte count (193 x 109/l) were not statistically significantly different for the transfused group compared to the non-transfused group (518 and 163 respectively). Mean PNH C3 and RBC C3 did not differ stastistically between the transfused and non-transfused groups (26.20 Vs 24.78 PNH C3;15.96 vs 15.09 RBC C3 respectively).
We studied one functional polymorphism in the Fcγ receptor but this showed no correlation with haemolytic parameters. Conversely, for the C3 polymorphism eculizumab-treated patients homozygous for the slow (C3S) allele had a significantly higher degree of C3 loading on PNH red blood cells with C3S/C3S having a mean of 33.7% C3 loaded PNH red cells (n=26), C3S/C3F 19.0% (n=19) and C3F/C3F 12.8% (n=3)(all P<0.01). Homozygote C3S also had increased reticulocytes (P<0.01) and bilirubin (P<0.01). The C3S allele has previously been shown to be more efficient in a haemolysis assay using sheep erythrocytes. This polymorphism appears to explain much of the variation in C3 loading between different patients with PNH.
In vitro experiments show that inhibitors of C5, such as eculizumab, protect the PNH red cells from lysis but lead to a very rapid deposition of C3 on the PNH red cells. However both APL1 and APL2 demonstrated similar protection of PNH red cell lysis with virtually no C3 loading on PNH red cells.

Conclusion
A significant proportion of patients on eculizumab experience extravascular haemolysis with the majority of patients developing C3 loading. We show for the first time that a functional polymorphism in the C3 gene is associated parameters of hemolysis. The S(low) allele alters the function of C3 protein and appears to be associated with extravascular haemolysis in patients with PNH. The C3 inhibitors, APL-1 and APL-2, protect PNH red cells and prevent C3 loading in vitro and if safe to be given chronically would be expected to reduce extravascular hemolysis significantly.

Disclosures:
Hill: Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kelly:Alexion: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Hillmen:Alexion: Honoraria, Membership on an entity’s Board of Directors or advisory committees.