Identification of a C3b-specific nanobody that does not bind C3 and blocks alternative pathway convertases
- 1University Medical Center Utrecht
Abstract
The human complement system is a protein network in blood and other body fluids that fights invading pathogens and is involved in maintaining homeostasis. A central step in the complement cascade is the conversion of complement protein C3 to C3b by convertase enzymes. Upon cleavage of C3, the nascent C3b molecule undergoes a large conformational change which exposes new epitopes. Molecules that discriminate between C3b and C3 could provide a powerful tool to selectively bind surface-bound complement activation products while leaving the circulating precursors untouched. In this study, we developed C3b-specific nanobodies by generating phage libraries from llamas immunized with purified and surface-bound C3b. We describe UNbC3b-1 as a high-affinity binder that recognizes soluble and surface-bound C3b molecules but does not bind to C3 in its native state. A 4.2 [A] structure of the UNbC3b-1:C3b complex generated by cryogenic electron microscopy shows that UNbC3b-1 binds C3b at the interface of MG3, MG4, and MG6, overlapping with the complement receptor immunoglobulin (CRIg) binding site. Functionally, we show that UNbC3b-1 inhibits complement activity in the alternative pathway (AP), likely by preventing association between substrate C3 and the AP C3 convertase (C3bBb). Altogether, nanobody UNbC3b-1 is a valuable tool to specifically bind C3b molecules and to selectively inhibit the AP convertases of the complement system.
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BibTeX
@misc{struijf2026identification,
title = {{Identification of a C3b-specific nanobody that does not bind C3 and blocks alternative pathway convertases}},
author = {E. M. Struijf and B. W. Bardoel and J. E. van Keulen and M. Ruyken and D. Y. Siere and D. J. Dijkstra and G. Dekkers and P. Gros and R. Heukers and D. A. C. Heesterbeek and S. H. M. Rooijakkers and J. S. Depelteau},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.30.748118},
url = {https://www.biorxiv.org/content/10.64898/2026.08.30.748118v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Identification of a C3b-specific nanobody that does not bind C3 and blocks alternative pathway convertases
AU - E. M. Struijf
AU - B. W. Bardoel
AU - J. E. van Keulen
AU - M. Ruyken
AU - D. Y. Siere
AU - D. J. Dijkstra
AU - G. Dekkers
AU - P. Gros
AU - R. Heukers
AU - D. A. C. Heesterbeek
AU - S. H. M. Rooijakkers
AU - J. S. Depelteau
PY - 2026
DA - 2026/08/30
PB - bioRxiv
DO - 10.64898/2026.08.30.748118
UR - https://www.biorxiv.org/content/10.64898/2026.08.30.748118v1
AB - The human complement system is a protein network in blood and other body fluids that fights invading pathogens and is involved in maintaining homeostasis. A central step in the complement cascade is the conversion of complement protein C3 to C3b by convertase enzymes. Upon cleavage of C3, the nascent C3b molecule undergoes a large conformational change which exposes new epitopes. Molecules that discriminate between C3b and C3 could provide a powerful tool to selectively bind surface-bound complement activation products while leaving the circulating precursors untouched. In this study, we developed C3b-specific nanobodies by generating phage libraries from llamas immunized with purified and surface-bound C3b. We describe UNbC3b-1 as a high-affinity binder that recognizes soluble and surface-bound C3b molecules but does not bind to C3 in its native state. A 4.2 [A] structure of the UNbC3b-1:C3b complex generated by cryogenic electron microscopy shows that UNbC3b-1 binds C3b at the interface of MG3, MG4, and MG6, overlapping with the complement receptor immunoglobulin (CRIg) binding site. Functionally, we show that UNbC3b-1 inhibits complement activity in the alternative pathway (AP), likely by preventing association between substrate C3 and the AP C3 convertase (C3bBb). Altogether, nanobody UNbC3b-1 is a valuable tool to specifically bind C3b molecules and to selectively inhibit the AP convertases of the complement system.
ER -