C5 Polymorphism in a Dutch Patient with Paroxysmal Nocturnal Hemoglobinuria (PNH) and No Asian Ancestry, Resistant to Eculizumab, but in Vitro Sensitive to Coversin
Authors: Langemeijer Saskia , Nishimura Jun-Ichi , Weston-Davies Wynne , Nunn Miles A , Kanakura Yuzuru , Mackie Ian J , Muus Petra 
Published: October 2019

Abstract
Eculizumab (Soliris®) is a humanized monoclonal antibody that targets complement factor C5and inhibits the production of C5a and formation of the terminal complement membrane attack complex. It is registered for the treatment of PNH and aHUS. Eculizumab results in significant reduction of hemolysis in PNH, improves symptoms and reduces the incidence of PNH related thrombosis . A poor response, defined as sustained high levels of LDH during treatment with eculizumab irrespective of improvement of clinical symptoms, has been reported in a subgroup of PNH patients (Nishimura et al, NEJM 2014;370:632-639). These patients had a genetic variant of C5 which occurs in approximately 3.5% of the Japanese and 1% of the Chinese Han populations and interferes with binding of eculizumab to C5. We describe the first patient with no known Asian ancestry with a poor response to eculizumab, who subsequently had a good in vitro response to protein rEV576 (syntheticOrnithodoros moubata complement C5 binding) or coversin. Coversin is a recombinant small protein derived from a tick salivary molecule which binds to C5 and, through steric hindrance, interferes with the access of C5 convertase to the active site and thus prevents cleavage to C5a and C5b in a similar fashion to eculizumab.
Our 30-year old male patient commenced treatment with Eculizumab because of PNH (granulocyte clone size: 90%), severe haemolysis (LDH 3-6x ULN, and peak value of 17xULN), transient renal failure, extreme fatigue and erectile dysfunction. He had no history of thrombosis and no underlying bone marrow disease. During eculizumab treatment (dosed 600 mg iv every 7 days, weeks 1-4 and 900 mg biweekly starting in week 5) he felt better, seemed less fatigued and experienced less erectile dysfunction. However, laboratory examination showed sustained elevated markers of hemolysis. Other causes of hemolysis were excluded. Underdosing of eculizumab was ruled out by demonstrating sustained high LDH levels at different time points in between subsequent eculizumab infusions and by measuring trough levels of eculizumab (>100ug/ml). In vitro terminal complement complex blockage by eculizumab through antibody-coated chicken red blood cell lysis was indicative of ongoing active hemolysis in our patient's serum. The presence of Human Anti-Drug Antibodies was excluded using an illuminescent MSD®assay. Treatment was discontinued when the patient experienced increased hemolysis (LDH 9x ULN) and macroscopic hemoglobinuria one day after receiving a dose of 900 mg eculizumab. As expected, discontinuation did not result in further increase of hemolysis parameters or clinical change.
To investigate whether a mutation of complement C5 might explain the eculizumab resistance in our patient, DNA analysis of the coding region of C5 was performed. This showed a single C5 heterozygous missense mutation, c.2653C>A, which predicts p.Arg885Ser. This new mutation was very similar to variants previously found in Japanese patients (c.2654G>A, which predicts p.Arg885His) and an Argentinian patient with Asian ancestry (c.2653C>T, which predicts p.Arg885Cys), indicating the importance of this amino acid in C5 recognition by eculizumab. The same mutation was demonstrated in the DNA of our patient's healthy father.
Since coversin binds to an epitope on C5 remote from the eculizumab binding site, we hypothesised that it might block C5 cleavage in our patient. Serum samples from our patient and 6 healthy controls were spiked with ascending doses of either eculizumab or coversin and complement activity was measured using a commercially available CH50 Equivalent ELISA (Quidel Corporation ®). In agreement with our in vivo observation eculizumab was incapable of inhibiting CH50 activity in the patient's serum beyond approximately 75%, even at concentrations of 100ug/ml. In contrast coversin, even in concentrations of 10 ug/ml inhibited complement activity completely, both in serum of our patient and serum of healthy controls. We conclude that coversin may prove a useful alternative to eculizumab for patients with resistance due to C5 polymorphisms.
Figure 1. Change in serum complement C5 activity in response to ascending doses of coversin (Cov) and eculizumab (Ecu). R2 = patient sample, NC3 = normal control Figure 1. Change in serum complement C5 activity in response to ascending doses of coversin (Cov) and eculizumab (Ecu). R2 = patient sample, NC3 = normal control

Disclosures
Nishimura: Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Weston-Davies:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Nunn:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mackie:Volution Immuno Pharmaceuticals (Uk) Ltd: Research Funding. Muus:Alexion Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.