A C3-specific nanobody that blocks all three activation pathways in the human and murine complement system

Abstract

The complement system is a tightly controlled proteolytic cascade in the innate immune system, which tags intruding pathogens and dying host cells for clearance. An essential protein in this process is complement component C3. Uncontrolled complement activation has been implicated in several human diseases and disorders and has spurred the development of therapeutic approaches that modulate the complement system. Here, using purified proteins and several biochemical assays and surface plasmon resonance, we report that our nanobody, hC3Nb2, inhibits C3 deposition by all complement pathways. We observe that the hC3Nb2 nanobody binds human native C3 and its degradation products with low nanomolar affinity and does not interfere with the endogenous regulation of C3b deposition mediated by Factors H and I. Using negative stain EM analysis and functional assays, we demonstrate that hC3Nb2 inhibits the substrate–convertase interaction by binding to the MG3 and MG4 domains of C3 and C3b. Furthermore, we notice that hC3Nb2 is cross-reactive and inhibits the lectin and alternative pathway in murine serum. We conclude that hC3Nb2 is a potent, general, and versatile inhibitor of the human and murine complement cascades. Its cross-reactivity suggests that this nanobody may be valuable for analysis of complement activation within animal models of both acute and chronic diseases. The complement system is a tightly controlled proteolytic cascade in the innate immune system, which tags intruding pathogens and dying host cells for clearance. An essential protein in this process is complement component C3. Uncontrolled complement activation has been implicated in several human diseases and disorders and has spurred the development of therapeutic approaches that modulate the complement system. Here, using purified proteins and several biochemical assays and surface plasmon resonance, we report that our nanobody, hC3Nb2, inhibits C3 deposition by all complement pathways. We observe that the hC3Nb2 nanobody binds human native C3 and its degradation products with low nanomolar affinity and does not interfere with the endogenous regulation of C3b deposition mediated by Factors H and I. Using negative stain EM analysis and functional assays, we demonstrate that hC3Nb2 inhibits the substrate–convertase interaction by binding to the MG3 and MG4 domains of C3 and C3b. Furthermore, we notice that hC3Nb2 is cross-reactive and inhibits the lectin and alternative pathway in murine serum. We conclude that hC3Nb2 is a potent, general, and versatile inhibitor of the human and murine complement cascades. Its cross-reactivity suggests that this nanobody may be valuable for analysis of complement activation within animal models of both acute and chronic diseases. The complement system is a prominent part of our innate immune system and serves as a first line of defense through opsonization and lysis of intruding pathogens. Likewise, complement contributes to the clearance of dying host cells, which is crucial in homeostasis and minimizes the development of autoimmune diseases (1Merle N.S. Noe R. Halbwachs-Mecarelli L. Fremeaux-Bacchi V. Roumenina L.T. Complement system part II: role in immunity.Front. Immunol. 2015; 6 (26074922): 25710.3389/fimmu.2015.00257Crossref PubMed Scopus (419) Google Scholar). The system also plays a role in development and aids the sculpting of the early developing brain by promoting the pruning of excess synapses (2Stephan A.H. Barres B.A. Stevens B. The complement system: an unexpected role in synaptic pruning during development and disease.Annu. Rev. Neurosci. 2012; 35 (22715882): 369-38910.1146/annurev-neuro-061010-113810Crossref PubMed Scopus (544) Google Scholar). Complement is activated through three pathways, the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The complement cascade may be triggered upon binding of pattern-recognizing molecules (PRMs) to pathogen-associated molecular patterns and danger-associated molecular patterns presented on the activating surface. In the CP, the PRM C1q detects antibody-antigen complexes and multiple other patterns (3Kojouharova M. Reid K. Gadjeva M. New insights into the molecular mechanisms of classical complement activation.Mol. Immunol. 2010; 47 (20542571): 2154-216010.1016/j.molimm.2010.05.011Crossref PubMed Scopus (54) Google Scholar), whereas five different PRMs of the LP recognize specific carbohydrate structures or patterns of acetyl groups (4Kjaer T.R. Thiel S. Andersen G.R. Toward a structure-based comprehension of the lectin pathway of complement.Mol. Immunol. 2013; 56 (23810291): 222-23110.1016/j.molimm.2013.05.220Crossref PubMed Scopus (40) Google Scholar). Serine proteases associated with the PRMs cleave complement factor C4 (C4) and C2, leading to the appearance of the classical pathway C3 convertase C4b2a formed by C4b and the active serine protease C2a on the activator surface Thiel S. Andersen G.R. Complement and molecular for 2015; PubMed Scopus Google C3 convertase C3 into C3b and the The C3b on the activator with to the upon activation by to the active C3 The alternative pathway the of the and LP M. The of lectin complement pathway activation on alternative pathway Immunol. 47 PubMed Scopus Google M. L. M. The role of alternative pathway in classical pathway complement Immunol. PubMed Scopus Google Scholar), the may also through the of C3. Here, the of C3 which in C3 to a functional of C3b. for the with and the of a C3 B. The and regulation of the alternative complement C3 convertase Immunol. PubMed Scopus Google Scholar). complement activation to the C3b a surface on the the C3 to The appearance of which cleave into and the the of the the of the that and pathogens Complement and molecular mechanisms to therapeutic PubMed Scopus Google Scholar). host cells, the pathway is not activated C3b is to by with the of the factor H protein and complement The is of a C3 convertase with factor and the C3 convertase Thiel S. Andersen G.R. Complement and molecular for 2015; PubMed Scopus Google Scholar). In also as is the of the C3 and and by convertase and with for binding to C3b M. K. Thiel S. N.S. Fremeaux-Bacchi V. Andersen G.R. for and convertase in the human complement Immunol. PubMed Scopus Google Roumenina L. Thiel S. Fremeaux-Bacchi V. Andersen G.R. and into of complement alternative pathway PubMed Scopus Google Scholar). in associated with the development of a of the chronic diseases C3 and Fremeaux-Bacchi V. M. M. S. a Rev. PubMed Scopus Google S. S. of PubMed Scopus Google as as which to of M. complement in PubMed Scopus Google Scholar). In the chronic of the pathway to in for to lysis Rev. PubMed Scopus Google Scholar). Furthermore, complement activation has also been to multiple T.R. K. M. V. of the of complement component PubMed Scopus Google and early of S. S. S. Barres B.A. Stevens B. Complement and early in PubMed Scopus Google Scholar). In to chronic the complement system plays a role in the of acute as of complement Rev. PubMed Scopus Google Scholar). the of complement in a of different and been L. of complement Immunol. PubMed Scopus (40) Google Scholar). We the first nanobody complement that inhibits the in both human and murine R. Andersen Thiel S. N.S. Andersen G.R. complement factor C3 specific nanobody multiple in the alternative pathway of human and murine PubMed Scopus Google Scholar). the of the in on C3 by the C3 the of the in the of and diseases. Furthermore, degradation of which may be for in We to a that inhibits complement through of the negative regulation of C3b on host we hC3Nb2, which binds and a with low nanomolar The inhibits both the and C3 convertase in human with the degradation of C3b to and is also cross-reactive with C3 and is functional in murine serum. a of biochemical assays and negative stain EM we that hC3Nb2 its through of binding to C3 We a nanobody, for of the complement pathway in human diseases and murine models that may into a for therapeutic of We the of a of a with human C3b. this we human C3b. Using this we not the R. Andersen Thiel S. N.S. Andersen G.R. complement factor C3 specific nanobody multiple in the alternative pathway of human and murine PubMed Scopus Google Scholar), also the hC3Nb2 nanobody presented we the of hC3Nb2 in classical and lectin pathway assays in with or We the hC3Nb2 nanobody with our inhibitor and the of the by the C3 on the surface. In our we in to the the hC3Nb2 nanobody inhibits the C3 deposition upon activation of the classical pathway in excess with C3 the nanobody inhibits C3 deposition through the lectin that hC3Nb2 inhibits C3 by the C3 convertase through the and the LP In the nanobody has a on this We to the of the nanobody in an in which activation of the the and LP that the nanobody also inhibits the alternative pathway in both human and and the cross-reactivity of the nanobody, we the of the nanobody in the lectin pathway in murine to the lectin pathway in human hC3Nb2 the of the murine lectin assays that hC3Nb2 is a inhibitor of the complement system of C3 in all three pathways. we the of the nanobody to of which classical pathway with We the cells in human in the or of we the to to that the of hC3Nb2 to of the classical inhibits of C3b through the In with the that hC3Nb2 inhibits the classical deposition of the nanobody also the lysis of the that of C3 in the The nanobody the to a of in C3 hC3Nb2 by the inhibitor or its of lysis that an role in this that the the of in this we also the in in which is hC3Nb2 the in excess to and the lysis lysis that in to C3 hC3Nb2 is a inhibitor of the of the complement cascade the classical pathway to the The C3b deposition and assays in that a hC3Nb2 and the C3 is for of the as as the for binding and other for the of an We by surface plasmon Here, hC3Nb2 as a for binding to a in the of of and the as the The and for with hC3Nb2 by the binding to a binding The nanobody binds the three functional of C3 with in the low nanomolar The the three and on the of that the nanobody binds with affinity to a in and the C3 We the binding of hC3Nb2 to murine C3b. this we with hC3Nb2 and the to murine C3b to to the we the to a binding that hC3Nb2 binds murine C3b with affinity with binding to human we a with human C3b and the in a that the hC3Nb2 binds with affinity to murine and human C3b on the of for hC3Nb2 interaction with human C3 degradation we that the hC3Nb2 degradation products of murine C3. 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The nanobody inhibits the of the complement cascade by the substrate–convertase interaction of the C3 and binds both the C3 and C3b in the convertase with low nanomolar the nanobody does not interfere with endogenous regulation of C3b. We the nanobody for of the complement system in and murine in The hC3Nb2 nanobody as R. Andersen Thiel S. N.S. Andersen G.R. complement factor C3 specific nanobody multiple in the alternative pathway of human and murine PubMed Scopus Google Scholar). a with human C3b. purified and The into a a C3b by a with of C3b in for with to the to excess binding and in for by in by in The by for in The to a of with of C3b in by as R. Andersen Thiel S. N.S. 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