Stratification of responders towards eculizumab using a structural epitope mapping strategy

www.nature.com/scientificreports OPEN received: 16 May 2016 accepted: 18 July 2016 Published: 11 August 2016 Stratification of responders towards eculizumab using a structural epitope mapping strategy Anna-Luisa Volk1,2, Francis Jingxin Hu1,2, Magnus M. Berglund3, Erik Nordling3, Patrik Strömberg3, Mathias Uhlen1,2,4,5 & Johan Rockberg1 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. The term precision medicine describes the idea of providing effective treatment based on a patient’s molecular make up. Recent advances in molecular diagnostic tools and handling of large data sets allow for the stratification of patients based on e.g. genetic or protein information and make it possible to provide tailored treatment for these sub-groups1. Here we describe how epitope information, exemplified by binding of the therapeutic antibody eculizumab on its target complement component 5 (C5), can be used to guide treatment. The complement system is an important part of the human innate immunity. It fulfils an important role in fighting bacterial infections and homeostasis and provides a link between innate and adaptive immune response2. Such powerful functions require a tight regulation to prevent the complement system from attacking the host’s cells. Paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS) are two disorders associated with a malfunctioning complement system. PNH is characterized by blood cells deficient of glycosylphosphatidylinositol (GPI)-anchored proteins, which protect cells from complement attack, caused by an acquired mutation in hematopoietic stem cells3–5. This consequently results in intravascular haemolysis, which in turn leads to anaemia and thrombosis4–6. Atypical HUS is a renal disease that is characterized by an unregulated activation of the alternative pathway of the complement resulting in thrombosis in kidney capillaries and renal failure7,8. The monoclonal antibody eculizumab (Soliris ) is the first FDA-approved therapeutic antibody for treatment of PNH2,4,5,9 and aHUS7,10. Eculizumab is a humanized monoclonal antibody targeting C5. The suggested mode of action is that eculizumab binding to C5 prevents the entry and subsequent cleavage of C5 by C5 convertase7,11. ® 1 KTH - Royal Institute of Technology, School of Biotechnology, Department of Proteomics and Nanobiotechnology, 106 91 Stockholm, Sweden. 2KTH Center for Applied Proteomics, School of Biotechnology, KTH - Royal Institute of Technology, Stockholm, Sweden. 3Swedish Orphan Biovitrum AB, 11276 Stockholm, Sweden. 4KTH - Royal Institute of Technology, Science for Life Laboratory, 17165 Stockholm, Sweden. 5Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2970 Hørsholm, Denmark. Correspondence and requests for materials should be addressed to J.R. (email: ) Scientific Reports | 6:31365 | DOI: 10.1038/srep31365 1 www.nature.com/scientificreports/ C5 is a central protein in the complement system cascade that is common to all three pathways. This makes it attractive as a therapeutic target. By inhibiting C5 cleavage the common downstream cascade can be disrupted independent of the activating pathway, i.e. no C5b-9 formation (membrane attack complex, MAC) and generation of the anaphylatoxin C5a occurs2,5. At the same time, the upstream pathways resulting in generation of C3b are kept intact and thus pathogen clearance via C3b-mediated opsonisation is not impaired4,5. Besides the approved treatment of PNH and aHUS, eculizumab has also proven effective in some cases for treatment of other conditions, like myasthenia gravis12,13, neuromyelitis optica14,15, membranoproliferative glomerulonephritis16,17, catastrophic antiphospholipid syndrome18–20 and prevention of rejection after renal transplants21–23. Despite the proven relevance of the drug, reports on non- or poorly responding patients have been published24–28. Among them a study on Japanese PNH patients which carried a mutation in the C5 gene, which changes the C5 protein so that it can no longer be bound by eculizumab24. This highlights the need for an exact determination of eculizumab’s epitope on C5. Such knowledge would enable the development of genotyping kits for the prediction of responsiveness of a patient to eculizumab treatment. Different efforts have been undertaken to identify the binding site of eculizumab on C511,29,30. In an earlier report to the Japanese Pharmaceutical and Food Safety Bureau29, eculizumab was reported to have a conformational epitope consisting of three distant amino acid stretches covering residues 822 to 826 (DVFLE), 879 to 883 (KSSKC), and 930 to 933 (VPEG). Later, Zuber et al.7 only refer to KSSKC as eculizumab’s epitope. In a recent publication30 based on transmission electron microscopy (TEM), eculizumab was found to interact with the MG7 domain with R885 and the KSSKC peptide being part of the binding site. While the present study was under review the crystal structure of C5 in complex with the eculizumab Fab fragment was solved11. While accurately determining the region of binding, the resolution of the co-crystal structure did not allow an unambiguous determination of intermolecular interactions at residue level. Here, we used an epitope mapping strategy based on deep mutational scanning of surface-displayed protein domains to explore in detail the structural epitope properties of eculizumab. While high-resolution X-ray crystallography of the antibody complex still represents the gold standard for epitope determination31 other epitope mapping methods have been developed, like peptide sc (...truncated)