Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

Abstract

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

Cryo-EM analysis of complement C3 reveals a reversible major opening of the macroglobulin ring

“…Here we used the nanobody hC3Nb1 which binds with low nanomolar affinity to C3b, and sterically inhibits progression of the alternative pathway, but does not alter the conformation of C3b 20 . Although originally found to bind to C3, we recently described that hC3Nb1 does not bind native C3 20,21 . Inspired by the crystal packing for a complex between the hC3Nb1 nanobody and C3b 20 , we mutated Asn108 in the nanobody to cysteine.…”

“…We therefore measured the rate of C3MA formation in the presence and absence of the MG3/MG4-interface-binding nanobody, hC3Nb2 25 , and followed the transformation using ion exchange chromatography. In an orthogonal approach, we took advantage of the tight interaction between C3MA and the EWEµH fusion protein 21 to follow the formation of the C3MA by mass photometry in the absence or presence of hC3Nb2. The EWEµH encompasses a modified version of hC3Nb1 called EWE fused with a linker to the FH fragment CCP2-4.…”

“…Peak fractions were analyzed on SDS-PAGE. The hC3Nb2 nanobody and the EWEµH fusion proteins were prepared as described 21,25 . C3ΔTE was expressed from a modified C3 expression plasmid where the residues 990-1287 were deleted and residues 989 and 1286 connected.…”

Structure determination of an unstable macromolecular complex enabled by nanobody‐peptide bridging

“…Human complement C3 was prepared in small scale essentially as described for rat C3 33 . Alternatively, large‐scale preparation of C3, C3b, and biotinylated C3b was conducted as described in Reference 7.…”

“…Human complement C3 was prepared in small scale essentially as described for rat C3. 33 Alternatively, large‐scale preparation of C3, C3b, and biotinylated C3b was conducted as described in Reference 7 . Recombinant FB and BC2T‐FB carrying the stabilizing mutation D279G were prepared as described.…”