Stratification of responders towards eculizumab using a structural epitope mapping strategy.

Anna-Luisa Volk,Johan Rockberg,Francis Jingxin Hu,Magnus M Berglund,Mathias Uhlen,Patrik Strömberg,Erik Nordling

doi:10.1038/srep31365

Anna-Luisa Volk, Johan Rockberg + Show 5 more

Open Access

https://doi.org/10.1038/srep31365

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Abstract

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.

Highlights

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome
Paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome are two disorders associated with a malfunctioning complement system
C5 is a central protein in the complement system cascade that is common to all three pathways

Summary

Introduction

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. It fulfils an important role in fighting bacterial infections and homeostasis and provides a link between innate and adaptive immune response[2] Such powerful functions require a tight regulation to prevent the complement system from attacking the host’s cells. Besides the approved treatment of PNH and aHUS, eculizumab has proven effective in some cases for treatment of other conditions, like myasthenia gravis[12,13], neuromyelitis optica[14,15], membranoproliferative glomerulonephritis[16,17], catastrophic antiphospholipid syndrome[18,19,20] and prevention of rejection after renal transplants[21,22,23]

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