An Ultrahigh-Affinity Complement C4b-Specific Nanobody Inhibits in Vivo
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Published August 7, 2020, doi:10.4049/jimmunol.2000528 The Journal of Immunology An Ultrahigh-Affinity Complement C4b-Specific Nanobody Inhibits In Vivo Assembly of the Classical Pathway Proconvertase Alessandra Zarantonello,* Jessy Presumey,† Le´a Simoni,† Esra Yalcin,† Rachel Fox,‡ Annette Hansen,x Heidi Gytz Olesen,* Steffen Thiel,x Matthew B. Johnson,‡,{ Beth Stevens,‡,{,‖,# Nick Stub Laursen,* Michael C. Carroll,†,** and Gregers R. Andersen* The classical and lectin pathways of the complement system are important for the elimination of pathogens and apoptotic cells and stimulation of the adaptive immune system. Upon activation of these pathways, complement component C4 is proteolytically cleaved, and the major product C4b is deposited on the activator, enabling assembly of a C3 convertase and downstream alternative pathway amplification. Although excessive activation of the lectin and classical pathways contributes to multiple autoimmune and inflammatory diseases and overexpression of a C4 isoform has recently been linked to schizophrenia, a C4 inhibitor and structural characterization of the convertase formed by C4b is lacking. In this study, we present the nanobody hC4Nb8 that binds with picomolar affinity to human C4b and potently inhibits in vitro complement C3 deposition through the classical and lectin pathways in human serum and in mouse serum. The crystal structure of the C4b:hC4Nb8 complex and a three- dimensional reconstruction of the C4bC2 proconvertase obtained by electron microscopy together rationalize how hC4Nb8 prevents proconvertase assembly through recognition of a neoepitope exposed in C4b and reveals a unique C2 conformation compared with the alternative pathway proconvertase. On human induced pluripotent stem cell–derived neurons, the nanobody prevents C3 deposition through the classical pathway. Furthermore, hC4Nb8 inhibits the classical pathway-mediated immune complex delivery to follicular dendritic cells in vivo. The hC4Nb8 represents a novel ultrahigh-affinity inhibitor of the classical and lectin pathways of the complement cascade under both in vitro and in vivo conditions. The Journal of Immunology, 2020, 205: 000–000. he complement system is part of the innate immune re- emergence of multicellular organisms (1). Complement is activated sponse and a critical component for our first line of defense through three pathways: the classical pathway (CP), the lectin T against invading pathogens. Its origin dates back to the pathway (LP), and the alternative pathway (AP). The CP and the LP are initiated by a pattern recognition molecule (PRM) that binds to either pathogen-associated molecular patterns or to danger-associated *Department of Molecular Biology and Genetics, Aarhus University, DK8000 Aar- hus, Denmark; †Program in Cellular and Molecular Medicine, Boston Children’s molecular patterns (2). Pattern recognition triggers a proteolytic Hospital, Boston, MA 02115; ‡Stanley Center for Psychiatric Research, Broad Insti- cascade in which a central event is the cleavage of the protein tute of MIT and Harvard, Cambridge, MA 02142; xDepartment of Biomedicine, { complement factor C3, leading to deposition of the fragment Aarhus University, DK8000 Aarhus, Denmark; Howard Hughes Medical Institute, Boston Children’s Hospital, Boston, MA 02115; ‖Department of Neurology, Harvard C3b on the activator surface, phagocytosis of the activator, and Medical School, Boston, MA 02115; #F.M. Kirby Neurobiology Center, Boston ultimately to lysis if the activator is a susceptible cell (3, 4). Children’s Hospital, Boston, MA 02115; and **Department of Pediatrics, Harvard Medical School, Boston, MA 02115 The AP may be activated by spontaneous hydrolysis of an in- ternal thioester in C3 (5), but the AP also strongly amplifies the ORCIDs: 0000-0002-9769-2271 (A.Z.); 0000-0002-1064-7989 (E.Y.); 0000-0001- 6265-2256 (R.F.); 0000-0002-4817-155X (S.T.); 0000-0001-6909-5712 (M.B.J.); initial deposition of C3b deposited on activators through the CP 0000-0003-4226-1201 (B.S.); 0000-0002-7512-2649 (N.S.L.); 0000-0001-6292- and LP (6). The LP is initiated by binding of one of five different 3319 (G.R.A.). PRMs to specific carbohydrate patterns on the activator (7). The Received for publication May 8, 2020. Accepted for publication July 9, 2020. proteases MASP-1 and MASP-2, which are associated with the LP This work was supported by Lundbeck Foundation BRAINSTRUC Grant R155- PRMs, are activated upon clustering on the activator (8), and 2015-2666 and the Graduate School of Science and Technology, Aarhus University. MASP-2 cleaves C4 into the small fragment C4a and the large Address correspondence and reprint requests to Prof. Gregers R. Andersen, Aarhus University, Gustav Wiedsvej 10C, DK8000 Aarhus, Denmark. E-mail address: fragment 190-kDa C4b (Fig. 1A). This cleavage initiates a con- [email protected] formational change, leading to exposure of a thioester group in the The online version of this article contains supplemental material. nascent C4b that may react with a nucleophile on the activator Abbreviations used in this article: AP, alternative pathway; BLI, bio-layer interferometry; surface (9). The CP evolved from the LP, and through evolution, C4BP, C4b binding protein; C4-Dpl, C4-depleted serum; CjC4, Callithrix jacchus C4; the PRM C1q acquired the capability of recognizing Ag-bound CP, classical pathway; CR, complement receptor; CVFB, cobra venom factor-FB; 2D, two-dimensional; 3D, three-dimensional; EM, electron microscopy; FDC, follicular den- IgG and IgM, but a number of other C1q-binding and CP- dritic cell; FH, factor H; FI, factor I; FT, flow through; hC4, human C4; IC, immune activating structures have been reported (1, 10). Upon C1q acti- complex; ITC, isothermal titration; KO, knockout; LB, Luria broth; LP, lectin pathway; NHS, normal human serum; PBS-T, PBS containing 0.1% Tween 20; PDB ID, Protein vator binding, the associated C1r and C1s proteases are activated, Data Bank identification number; PRM, pattern recognition molecule; RT, room temper- and C1s, in turn, cleaves C4 to C4b, presumably in a manner very ature; RU, response unit; SCZ, schizophrenia; SEC, size-exclusion chromatography; Slp, similar to that of MASP-2 in the LP (11). sex-limited protein; SP, serine protease; SPR, surface plasmon resonance; WT, wild-type. Activator-bound C4b binds to the zymogen C2 in a Mg2+- Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 dependent manner (12, 13). The C4b2 complex is the CP proconvertase, www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000528 2 COMPLEMENT INHIBITION WITH A C4b-SPECIFIC NANOBODY the precursor of the C3 convertase. The CP C3 convertase is complex, which, together with a three-dimensional (3D) reconstruc- generated through a second cleavage conducted by C1s or MASP- tion of the C4b2 proconvertase, rationalizes its mode of action with 1/MASP-2 (Fig. 1A), releasing C2b and leaving the C3 convertase respect to inhibition of the complement CP. It is also demonstrated C4b2a attached to the surface (14). Cleavage of C3 by C4b2a that the hC4Nb8 exerts efficient CP complement inhibition in vivo in initiates a conformational change in the nascent C3 similar to that a human C4 (hC4) knock-in transgenic mouse model and in vitro in in C4b, exposing a thioester that links C3b to the surface (15). On the context of the CNS. host surfaces, C3b is degraded by the protease factor I (FI) with the help of cofactors factor H (FH), CD46/MCP, and complement Materials and Methods receptor (CR) 1 (CD35), resulting in the late opsonins iC3b and Protein purification and nanobodies generation C3dg, both ligands for CR3 (16) (Fig. 1A). In lymph nodes, iC3b/ C3dg enables uptake of immune complexes (ICs) through inter- Human C4, C4b, C2, and CR1 CCP1-3 were purified as previously de- action with CR3 on the surface of subcapsular sinus macrophages, scribed (34–36). For production of the nanobodies, after three immuni- zation boosts with a total of 400 mg of C4 and 400 mg of C4b in 3 wk from which the ICs are passed on to naive B cells and subse- intervals, blood was withdrawn from the llama and used to purify pe- quently to follicular dendritic cells (FDCs) through CR2 recog- ripheral blood leukocytes and isolate mRNA. The phage display library nition of iC3b/C3dg (17). The resulting transfer of complement was generated as described previously (37). Nanobody hC4Nb8 was se- opsonized Ags allows for long-term Ag presentation on FDCs, lected by two rounds of phage display. In the first round, 1 mg of C4 in 100 ml of PBS was coated in one well of a Nunc MaxiSorp plate. The well was leading to formation of B cell germinal centers, in which B cell then blocked by addition of 2% (w/w) BSA in PBS containing 0.1% Tween clonal expansion, class switch recombination, and somatic 20 (PBS-T). The well was washed with 3 3 300 ml of PBS-T and incu- hypermutation for the production of Abs takes place (18). C4b is bated with the phage library for 1 h. Subsequently, the well was washed 15 degraded in a similar manner by FI together with cofactors C4b times with 300 ml of PBS-T and 15 times with 300 ml of PBS. Binders binding protein (C4BP) and CR1 to C4c and C4d. However, no were eluted by addition of 100 ml of 0.2 M glycine (pH 2.2) for 10 min. The eluted phages were neutralized by addition of 15 ml of 1 M Tris (pH effector function of these C4b degradation products have been 9.1) and used for infection on ER2738 cells. The second round of selection identified, but C4b, together with C3b, functions as ligand for CR1 was done essentially as the first round but now using only 0.1 mg of C4. on RBCs and contributes to clearance of ICs (19). Whereas the After the last round of selection, 96 colonies were incubated in a 96-well function of the CP for pathogen clearance and tissue homeostasis tray, induced by addition of IPTG to a final concentration of 1 mM, and incubated overnight at 30˚C.