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Cited by 15Complement system in diseasesMonoclonal and Polyclonal Antibodies ResearchBiomarkers in Disease Mechanisms

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Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies

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Struijf, Eva M., De la O Becerra, Karla I., Ruyken, Maartje, de Haas, Carla J.C., van Oosterom, Fleur, Siere, Danique Y., van Keulen, Joanne E., Heesterbeek, Dani A.C., Dolk, Edward, Heukers, Raimond, Bardoel, Bart W., Gros, Piet, Rooijakkers, Suzan H.M.·2023·Journal of Biological Chemistry
DOI 10.1016/j.jbc.2023.104956PMC PMC10374974

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Abstract

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.

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Metadata

Authors
Struijf, E. M., De la O Becerra, K. I., Ruyken, M., de Haas, C. J. C., van Oosterom, F., Siere, D. Y., van Keulen, J. E., Heesterbeek, D. A. C., Dolk, E., Heukers, R., Bardoel, B. W., Gros, P., & Rooijakkers, S. H. M.
Affiliations
Utrecht University (NL)
Corresponding author
Rooijakkers, Suzan H.M.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2023-08
Volume
299
Issue
8
Pages
104956
ISSN
0021-9258 (ISSN-L)
DOI
10.1016/j.jbc.2023.104956
PMC
PMC10374974
OpenAlex
W4381996895
Cited by
15
References
3
Citations by year
2026: 6 · 2025: 7 · 2024: 2
FWCI
1.64
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
Funders
  • Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWA.ID.17.036)
  • Horizon 2020 (101001937)
  • Horizon 2020 Framework Programme
  • ERC
  • Consejo Nacional de Ciencia y Tecnología (CVU 604718)
  • European Research Council
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Complement system in diseases
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Immunology and Microbiology › Immunology › Biomarkers in Disease Mechanisms
Keywords
complement component C5, nanobodies, C5a, membrane attack complex, eculizumab, epitope mapping, cryo-EM structure, complement system
MeSH terms
Antibodies, Monoclonal, Complement System Proteins, Complement Activation, Complement C5, Humans, Complement Membrane Attack Complex, Single-Domain Antibodies

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