Abstract
The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance, and inhibition of the complement alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds to multiple functional states of C3 with subnanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable with that of C3, and hC3Nb1 is shown to prevent proconvertase assembly, as well as binding of the C3 substrate to C3 convertases. Our crystal structure of the C3b-hC3Nb1 complex and functional experiments demonstrate that proconvertase formation is blocked by steric hindrance between the nanobody and an Asn-linked glycan on complement factor B. In addition, hC3Nb1 is shown to prevent factor H binding to C3b, rationalizing its inhibition of factor I activity. Our results identify hC3Nb1 as a versatile, inexpensive, and powerful inhibitor of the alternative pathway in both human and murine in vitro model systems of complement activation.
Highlights
The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance, and inhibition of the complement alternative pathway is a subject of intense research
To study the interaction between C3b and this Nb, we isolated the complex by size-exclusion chromatography (SEC), and aliquots taken from the early part of the SEC peak were analyzed by single particle negative stain EM (Fig. 1B)
Using the MG domains of C3b filtered to a resolution of 80 Å as a reference model, we obtained a 3D reconstruction with an approximate resolution of 25 Å in which the MG domains, the C345c domain, the CUB, and the associated thioester (TE) domains of C3b were recognized (Fig. 1C)
Summary
A potent complement factor C3–specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement. The C5-specific mAb eculizumab inhibits C5 cleavage and thereby functions as an overall inhibitor of the terminal pathway This antibody has successfully been used for 10 years to treat patients suffering from the two rare diseases: atypical hemolytic uremia and paroxysmal nocturnal hemoglobinuria, proving that systemic and chronic inhibition of the complement terminal pathway can be done without severe adverse effects [9, 10]. This has sparked substantial interest in developing putative complement modulating drugs targeting the upstream classical, lectin, and alternative pathways [8, 11]. Our results, combined with the ease of producing and modifying Nbs, identifies hC3Nb1 as a versatile and powerful inhibitor of the alternative pathway that demonstrates great promise as a unique tool for studying complement C3
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