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Eculizumab-C5 complexes express a C5a neoepitope in vivo: Consequences for interpretation of patient complement analyses.
Mol Immunol. 2017 09; 89:111-114.MI

Abstract

The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab.

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Authors+Show Affiliations

Nilsson PH
Department of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway; K.G. Jebsen Inflammatory Research Center, University of Oslo, Oslo, Norway; Linnaeus Centre for Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden.
Thomas AM
Department of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway.
Bergseth G
Research Laboratory, Nordland Hospital, Bodø, Norway.
Gustavsen A
Department of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway.
Volokhina EB
Department of Pediatric Nephrology (830), Radboud University Medical Center, Nijmegen, The Netherlands; Department of Laboratory Medicine (830), Radboud University Medical Center, Nijmegen, The Netherlands.
van den Heuvel LP
Department of Pediatric Nephrology (830), Radboud University Medical Center, Nijmegen, The Netherlands; Department of Laboratory Medicine (830), Radboud University Medical Center, Nijmegen, The Netherlands; Department of Paediatric Nephrology & Growth and Regeneration, University Hospitals Leuven, Leuven, Belgium.
Barratt-Due A
Department of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway.
Mollnes TE
Department of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway; K.G. Jebsen Inflammatory Research Center, University of Oslo, Oslo, Norway; Research Laboratory, Nordland Hospital, Bodø, Norway; Faculty of Health Sciences, K.G. Jebsen TREC, University of Tromsø, Tromsø, Norway; Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway. Electronic address: t.e.mollnes@gmail.com.

MeSH

Antibodies, Monoclonal, HumanizedAtypical Hemolytic Uremic SyndromeChromatography, GelComplement ActivationComplement C5Complement C5aComplement C5bComplement Inactivating AgentsEnzyme-Linked Immunosorbent AssayEpitopesHemoglobinuria, ParoxysmalHumansHydrogen-Ion ConcentrationImmunoglobulin GOutcome Assessment, Health CareProtein Binding

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28610663

Citation

Nilsson, Per H., et al. "Eculizumab-C5 Complexes Express a C5a Neoepitope in Vivo: Consequences for Interpretation of Patient Complement Analyses." Molecular Immunology, vol. 89, 2017, pp. 111-114.
Nilsson PH, Thomas AM, Bergseth G, et al. Eculizumab-C5 complexes express a C5a neoepitope in vivo: Consequences for interpretation of patient complement analyses. Mol Immunol. 2017;89:111-114.
Nilsson, P. H., Thomas, A. M., Bergseth, G., Gustavsen, A., Volokhina, E. B., van den Heuvel, L. P., Barratt-Due, A., & Mollnes, T. E. (2017). Eculizumab-C5 complexes express a C5a neoepitope in vivo: Consequences for interpretation of patient complement analyses. Molecular Immunology, 89, 111-114. https://doi.org/10.1016/j.molimm.2017.05.021
Nilsson PH, et al. Eculizumab-C5 Complexes Express a C5a Neoepitope in Vivo: Consequences for Interpretation of Patient Complement Analyses. Mol Immunol. 2017;89:111-114. PubMed PMID: 28610663.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Eculizumab-C5 complexes express a C5a neoepitope in vivo: Consequences for interpretation of patient complement analyses. AU - Nilsson,Per H, AU - Thomas,Anub Mathew, AU - Bergseth,Grethe, AU - Gustavsen,Alice, AU - Volokhina,Elena B, AU - van den Heuvel,Lambertus P, AU - Barratt-Due,Andreas, AU - Mollnes,Tom E, Y1 - 2017/06/10/ PY - 2017/05/05/received PY - 2017/05/24/revised PY - 2017/05/25/accepted PY - 2017/6/15/pubmed PY - 2017/10/24/medline PY - 2017/6/15/entrez KW - C5 KW - C5a KW - Complement KW - Eculizumab KW - Neoepitope SP - 111 EP - 114 JF - Molecular immunology JO - Mol Immunol VL - 89 N2 - The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab. SN - 1872-9142 UR - https://www.unboundmedicine.com/medline/citation/28610663/Eculizumab_C5_complexes_express_a_C5a_neoepitope_in_vivo:_Consequences_for_interpretation_of_patient_complement_analyses_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0161-5890(17)30155-4 DB - PRIME DP - Unbound Medicine ER -