A potent complement factor C3-specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

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.

Keywords: alternative pathway; antibody; complement; convertase; inhibitor; innate immunity; structural biology.

Figure 1.

The alternative pathway and negative stain EM analysis. A, the AP can be initiated by tick-over generated C3(H₂O) or from C3b generated within the classical or lectin activation pathways. Both C3b or C3(H₂O) can form the proconvertase with FB. C3b can be degraded to iC3b by FI assisted by a cofactor. The (P) signifies that the AP convertases may bind and be stabilized by properdin. B, negative stain EM 2D classes of the complex between hC3Nb1 and C3b. The TE domain adopts multiple alternative positions as compared with the C3b MG1–8 domains. Scale bar, 100 Å. C, 3D reconstruction of negative stain EM particles of the C3b–hC3Nb1 complex with the crystal structure of C3b (PDB entry 5FO7) fitted to the envelope. Excess density corresponding to the Nb is marked by asterisks.

Figure 2.

The crystal structure of the C3b–hC3Nb1 complex. A, cartoon representation of the complex showing the Nb located close to the MG7 domain and the Nt-α′ region. B, omit 2mF_o − DF_c electron density contoured at 1σ around the CDR3 loop of hC3Nb1 and Arg⁸⁵⁵ of C3b. C, overview of the interaction surface between C3b and hC3Nb1 involving CDR2 and CDR3 from the Nb and MG7 and Nt-α′ of C3b. D–G, details of the interaction between hC3Nb1 and C3b. Salt bridges and hydrogen bonds are indicated by black dotted lines.

Figure 3.

The hC3Nb1 is a potent inhibitor of the alternative pathway. A, assay in human serum on a zymosan-coated AP activating surface. The horizontal axis gives the concentration of nanobodies present in the serum. The vertical axis shows the level of C3 fragment deposition with 100% deposition defined as the signal from serum without added nanobody. The dotted line indicates the final molar concentration of C3 in the assay based on a concentration of 5.3 μm in undiluted plasma (61). B, multiple sequence alignment of the hC3Nb1 epitope from human C3 and four animals of relevance for animal models for which residues marked with gray shading differ from the human sequence. Colored triangles indicate residues involved in interactions with hC3Nb1. C, assay in mouse serum on a zymosan-coated alternative pathway activating surface depicted as in A. The dotted line represents the final molar concentration of C3 in the assay assuming 4.2 μm C3 in 100% serum from C57BL6 male mice (62).

Figure 4.

hC3Nb1 inhibits convertase assembly and FH-mediated FI cleavage of C3b caused by spatial overlap with FH and FB. A, superposition of the C3b–hC3Nb1 and the C3bB (PDB entry 2XWB) structures shows that the N122 linked glycan of FB overlaps significantly with the Nb. B–E, SEC analysis of the AP proconvertase with and without hC3Nb1. The Nb inhibits the formation of the AP proconvertase only when the Asn¹²²–linked glycan is present on FB. F, superposition of the C3b–hC3Nb1 structure onto the structure of C3b bound to FH CCP1–4 (PDB entry 2WII) reveals that the binding sites of FH CCP1 and the Nb overlap substantially. G, SDS-PAGE analysis of a FH-assisted FI cleavage assay showing that hC3Nb1 completely inhibits iC3b formation.

Figure 5.

SPR analysis of the interaction of hC3Nb1 with C3b and C3. A and B, representative SPR concentration series for C3b (A) or C3 (B) are displayed. The measured (gray) and fitted (red) SPR curves are shown for 50, 20, 10, 5, 2, 1, 0.25, and 0.1 nm for C3b or 20, 10, 5, 2.5, 1.25, 0.61, 0.32 and 0.15 nm for C3. The k_a, k_d, and K_D values are given as their average fitted from three independent concentration series ± the standard deviation. C, competition SPR assay with 50 nm C3b preincubated with 500 nm of different hC3Nb1 mutants. D, competition SPR assay with 50 nm C3b preincubated with 500 nm FB or FH. In C the curve for hC3Nb1(W102A) is hidden below the C3b curve. RU, response units.

Figure 6.

The hC3Nb1 nanobody also inhibits C3 cleavage at the substrate level. A, C3 cleavage assay with the CVFBb convertase revealing that also for this convertase not containing C3b, hC3Nb1 is an inhibitor of C3 cleavage. B, C5 cleavage assay with the CVFBb convertase demonstrating that hC3Nb1 has no effect on C5 cleavage. The bands around at and below the 200-kDa marker represents C5b not completely reduced/denatured. C, assay in 1% human serum on a mannan-coated LP activating surface. The horizontal axis gives the concentration of nanobodies added to the serum. The vertical axis shows the level of C3 fragment deposition with 100% deposition defined as the signal from serum without added nanobody. The dotted line indicates the final molar concentration of C3 in the assay based on a concentration of 5.3 μm in undiluted plasma.