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. On the following day, the plate was centri- has been investigated for decades (20), more recent studies have fuged at 2000 rpm for 10 min, and the supernatant was used in an ELISA also identified an important role for this pathway during devel- to test for binding to C4. For the ELISA, 10 mg of C4 in 10 ml of PBS was opment in the brain (21, 22). Complement contributes to synaptic added to a 96-well Nunc MaxiSorp plate (100 ml/well) and incubated pruning in the CNS as a developmental mechanism for refinement overnight at 4˚C. The plate was blocked by addition of 200 ml of 2% BSA of synaptic circuits, enabling full cognitive ability in adulthood (23, in PBS-T (blocking solution) and incubated at room temperature (RT) for 2 h. The plate was then washed three times in PBS-T and 50 ml of su- 24). The pruning process is supported by the interaction between pernatant plus 50 ml of blocking solution was added to each well. After 1 h iC3b deposited on synapses with CR3 on microglia, the CNS res- of incubation at RT, the plate was washed three times in PBS-T, and 100 ml ident macrophages. Deficiencies of C1q, C3, and C4 have been of HRP-conjugated E-Tag Ab (Bethyl Laboratories) diluted 1:10,000 in demonstrated to affect synaptic pruning in mice, linking the CP blocking solution was added to each plate. The plate was incubated for 1 h at RT and washed three times in PBS-T, followed by addition of 100 ml directly to the process of neuronal connectivity refinement (25, 26). 3,39,5,59-tetramethylbenzidine substrate per well. The reaction was stop- A rapidly growing research topic concerns the molecular ped by addition of 100 ml of 1 M HCl, and absorbance was measured at mechanisms underlying disease development as a consequence of 450 nm. Phagemids of positive clones from the ELISA assays were pu- complement dysregulation. Diseases in which CP dysregulation is rified and sequenced. The sequence coding for hC4Nb8 was amplified by known to play a significant role include systemic lupus eryth- PCR using a forward primer with restriction site NdeI 59-GGGAATTC- CATATGCAGGTGCAGCTCGTGGAGACG-39 and reverse primer with ematosus, hereditary angioedema, ischemia–reperfusion injury, restriction site XhoI 59-CCCAAACTCGAGTGAGGAGACGGTGACC- sepsis, autoimmune hemolytic anemia, glomerulonephritis, Ab- TGG-39. Amplified inserts and vector pET22b+ were cut with the corre- mediated graft rejection, Alzheimer disease, schizophrenia sponding restriction enzymes. Inserts were ligated into the vector using (SCZ), and cold agglutinin disease (27–31). Numerous comple- DNA ligase (Thermo Fisher Scientific) according to supplier’s instructions. The resulting plasmids were sequenced by dideoxyribonucleotide se- ment inhibitors targeting the alternative and terminal pathways quencing with sequencing primer T7. Plasmids were transformed in have been described. However, with respect to inhibitors specific LOBSTR (38) chemically competent cells and grown on an Luria broth for the CP and LP, many fewer molecules have been described, (LB) agar plate containing ampicillin (100 mg/ml) and chloramphenicol and there is currently no well-established C4-specific complement (35 mg/ml). A single colony was picked from the plate and used to in- inhibitor (32). oculate an LB medium preculture containing antibiotics and 0.4% glucose and grown at 37˚C overnight with shaking at 150 rpm. The preculture was Nanobodies are the Ag-binding domain of H chain–only Abs diluted 50 times into 2 L LB Broth medium (Sigma-Aldrich) containing present in camelids (33). They offer a versatile platform to target antibiotics and 0.4% glucose. Cells were grown at 37˚C and 150 rpm until virtually any Ag, as they can be selected in vitro by phage display OD600 = 0.6–0.8; protein expression was induced by addition of 0.5 mM after in vivo llama immunization, isolation of lymphocytes, IPTG. Protein expression was carried out for 18 h at 20˚C and 150 rpm. The cells were pelleted by centrifugation at 6000 rpm for 20 min and mRNA extraction, and cDNA library generation. After selection, resuspended in PBS containing 400 mM NaCl and 20 mM imidazole the clones can be tested by ELISA on Ag-coated plates and pos- (resuspension buffer). For nanobody purification, the resuspended pellet itive hits sequenced and cloned into bacterial expression vectors. was sonicated, and cell debris was removed by centrifugation. The su- This workflow frequently allows production of multiple extremely pernatant was filtered through 0.45-mm cellulose filters and loaded on high-affinity binders. Nanobodies tend to have a relatively longer HisTrap FF Crude 5-ml column (GE Healthcare) equilibrated in resus- pension buffer. The column was washed with 50 ml of resuspension buffer CDR3 ranging between 15 and 20 residues, evolved to compen- and eluted with three fractions of 5-, 11-, and 11-ml volume of resus- sate for the presence of only three CDRs instead of six. This pension buffer supplemented with 400 mM imidazole. The eluate was extralong loop allows penetration of cryptic epitopes often im- dialyzed against 2 l of 20 mM NaOAc and 50 mM NaCl (pH 5.5), purified portant for protein function, a property that is also facilitated by further by cation exchange chromatography on a Source15S (GE Health- care) 1-ml column, and eluted with a 15-ml linear gradient from 50 to the small size of nanobodies (33). 500 mM NaCl. The fractions containing the nanobody were pooled after In this work, we present a potent C4b-specific–inhibiting nanobody SDS-PAGE analysis and concentrate; the sample was further purified by (hC4Nb8) and describe the crystal structure of the Ag:nanobody gel filtration on a 24-ml Superdex 75 Increase column (GE Healthcare) The Journal of Immunology 3 equilibrated in 20 mM HEPES and 150 mM NaCl (pH 7.5). For the in vivo CR1-mediated FI cleavage assay experiments, endotoxin was removed from the samples as in (39), and the endotoxin level that was quantified by the Thermo Fisher Scientific En- For the FI cleavage assay, C4b was mixed with CR1 CCP1-3, FI (Com- dotoxin Quantitation Kit was lower than 2 endotoxin units/ml for all plement Technology), and hC4Nb8 at the molar ratio of 1:10:0.5:1 in samples. For immunostaining the nanobody insert fused to the IgY H chain 20 mM HEPES-NaOH and 50 mM NaCl (pH 7.5) and incubated for 2, 4, 8, was cloned into pcDNA3.1, and the plasmid DNA was purified using the 16, and 24 h at 37˚C. A control reaction with C4b/CR1/FI = 1:10:0.5 was QIAGEN Gigaprep Purification Kit. HEK293f cells were transfected with carried out in parallel and analyzed by reducing SDS-PAGE analysis at the 1:3 DNA/PEI ratio, and protein expression was carried out for 5 d. All the 24 h time point (shown in Fig. 2). The cleavage reactions were stopped by purification steps were carried out using single-use plastic to avoid en- addition of 3 mM PMSF and reducing SDS-PAGE loading buffer. The dotoxin contamination. The cells were harvested by centrifugation at 4000 experiment was repeated twice. rpm for 15 min, and the supernatant was filtered through 0.2-mm filters, the Complement deposition assay pH was adjusted to 8.5, and the sample was loaded on a HisTrap excel 5-ml column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 8.5), For a test of the influence of nanobodies on the human CP, each well in a 96- 500 mM NaCl, and 10 mM imidazole. The protein was eluted with 30 ml well MaxiSorp plate (catalog no. 446612; Thermo Fisher Scientific) was of equilibration buffer supplemented with 500 mM imidazole. The sample coated with 100 mlof15mg/ml heat-aggregated human IgG diluted in was diluted 10-fold in 50 mM Tris-HCl (pH 8.5) and loaded on a HisTrap 50 mM sodium carbonate (pH 9.6) (Ampliqon) and incubated overnight. FF 5-ml column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH Residual binding sites in the wells were blocked by TBS (10 mM Tris [pH 8.5), 500 mM NaCl, and 10 mM imidazole. The protein was eluted with 7.4] and 140 mM NaCl) supplemented 1 mg/ml human serum albumin for 10 ml of equilibration buffer supplemented with 500 mM imidazole; 1 h at RT, then washed trice with TBS-T. Nanobodies were diluted in the elution was concentrated using 50-kDa MWCO Filters (Sartorius veronal buffer (4 mM barbital, 145 mM NaCl, 2 mM CaCl2 and 1 mM Stedim Biotech) and desalted in PBS using PD-10 Desalting Columns MgCl2 [pH 7.4]) supplemented with 0.2% normal human serum (NHS), (GE Healthcare) according to manufacturer’s instructions. The endo- and 100 ml was added to wells in duplicates. The wells were incubated for toxin level calculated using the Thermo Fisher Scientific Endotoxin 1.5 h at 37˚C in a humidity box, then washed trice with TBS-T containing Quantitation Kit was below 2 endotoxin units/ml. For site-specific bio- 5 mM CaCl2 (TBS-T/Ca). Deposited C3 was detected using 100 mlof tinylation, the AVI-tag peptide (GLNDIFEAQKIEWHE) was introduced at biotinylated rabbit anti-C3d Ab (cat no. A0063; Dako [anti-C3d]) diluted the C-terminal end of hC4Nb8 with the QuikChange Lightning Site-Directed to 0.5 mg/ml in TBS-T and incubated for 2 h at RT followed by three Mutagenesis Kit (Agilent Technologies) with forward primer 59- washes in TBS-T. Then, 100 mlof1mg/ml europium-labeled streptavidin GCACCACGGCCTGAACGATATTTTTGAAGCGCAGAAAATTGAA- (catalog no. 1244-360; PerkinElmer) diluted in TBS-T supplemented with TGGCATGAATGAGATCCGGCTGC-39 and reverse primer 59-GATCT- 25 mM EDTA were added to the wells and incubated for 1 h at RT. The CATTCATGCCATTCAATTTTCTGCGCTTCAAAAATATCGTTCAGGC- wells were washed trice in TBS-T, then 200 ml of enhancement buffer CGTGGTGGTGGTGGTG-39. The protein was purified as the wild-type (catalog no. Q99800; Ampliqon) was added to each well. The fluorescence (WT) hC4Nb8 and dialyzed against 20 mM HEPES-NaOH and 150 mM signal, read as time-resolved fluorometry, was measured using a VICTOR5 NaCl (pH 7.5). The buffer was supplemented with 0.15 mM biotin, 2 mM Multilabel Plate Reader (PerkinElmer) with excitation wavelength of 350 nm and emission 610 nm; the output was in counts per second. The test for ATP, and 5 mM MgCl2, BirA ligase was added at 1:5 w/w ratio, and the biotinylation reaction was carried out for 16 h at 25˚C. BirA ligase was the influence of the nanobodies on the LP assay was performed in a similar removed by ion exchange chromatography, and after testing the bio- manner, except that a serum concentration of 1% or 50% was used, as tinylation success by a pull-down assay of hC4Nb8-AVI-biotin with described before (37). In this assay, the surface is coated with mannan streptavidin-agarose beads (Thermo Fisher Scientific), the biotinylated instead of IgG. The effect of nanobodies on the murine LP was similarly nanobody was stored at 280˚C until use. Single point mutations were in- tested on mannan-coated surfaces. In this case, murine serum from male troduced in the hC4Nb8 WT sequence using the QuickChange Lightning C57Bl6 mice diluted to 0.33% was used, and the deposited C3 was Site-Directed Mutagenesis Kit (Agilent Technologies). quantified using a rat anti-mouse C3 Ab (catalog no. CL7503NA; CEDARLANE) at 0.25 mg/ml Tris-buffered saline containing 0.1% Tween mC4 expression, purification, and mC4b generation 20 and 5 mM Ca2+, followed by wash and incubation with biotinylated rabbit anti-mouse IgG (E0468; Dako) at 0.25 mg/ml Tris-buffered saline An expression plasmid encoding mouse C4B with the endogenous signaling containing 0.1% Tween 20 and 5 mM Ca2+. As for the assays described peptide was designed with a C-terminal His-tag. The construct was obtained above, this is followed by europium-labeled streptavidin and reading of the by PCR amplification of the full C4 insert from pC427A-K1324N, kindly signal. All experiments were carried out in duplicates. provided by D. Isenman, and subcloning into pcDNA3.1(+) using NotI and XbaI restriction sites. The recombinant mC4 was expressed in HEK293f Pull-down experiments from marmoset plasma cells maintained at 37˚C, 8% CO , and 125 rpm in serum‐free FreeStyle 2 Five micrograms of biotinylated hC4Nb8-AVI (0.87 mg/ml) were diluted in 293 Expression Medium (Invitrogen). Cells were transiently transfected 150 ml of PBS and incubated with 20 ml of Streptavidin Plus UltraLink using final concentrations of 2 mg/l polyethyleneimine (PEI 25K; Pol- resin beads (Thermo Fisher Scientific) equilibrated in PBS for 30 min at 4˚ ysciences) and 1 mg/l plasmid DNA. The conditioned medium was C with inversion. The supernatant was removed after centrifugation, and harvested 4 d posttransfection and adjusted to pH 7.8 with 25 mM the beads were washed three times for 10 min at 4˚C with 150 ml of PBS. HEPES-NaOH. The secreted mC4-His was applied to a 5 ml of HisTrap For each pull-down experiment, 15 ml of marmoset plasma (kindly pro- (GE Healthcare) in 300 mM NaCl and 20 mM HEPES (pH 7.8), washed vided by Q. Zhang and G. Feng) was thawed on ice and incubated with 20 with 25 mM and eluted with 300 mM imidazole, followed by a final ml of streptavidin-agarose beads, either conjugated to hC4Nb8 or uncon- polishing step on a 24-ml Superdex 200 Increase (GE Healthcare) jugated, and equilibrated in PBS for 30 min at 4˚C with inversion. After equilibrated in 20 mM HEPES (pH 7.5) and 150 mM NaCl. To generate incubation, the serum flow through (FT) was removed, and the beads were mC4b, the same protocol was used as for hC4b generation (35). washed six times for 10 min at 4˚C (W1–W6) with 150 ml of PBS. After Binding to C4 and C4b and disruption of the washes, the samples were analyzed by reducing SDS-PAGE. For the SDS-PAGE analysis, the input and the FT were diluted 1:100 in PBS, and proconvertase formation 10 ml were loaded onto the gel. Eight microliters of W2 and ten microliters To test binding to C4 and C4b, the nanobody was preincubated with the Ag of W4 to W6 were loaded on the gel, respectively. Residual proteins bound in a 5-fold molar excess for 5 min at 4˚C, and the sample was analyzed by to the beads were analyzed by loading 10 ml of the beads resuspended in 3 analytical gel filtration on a 24-ml Superdex 200 Increase column (GE 20 ml of PBS. A 4 concentration of Laemmli buffer containing 2-ME Healthcare) equilibrated in 20 mM HEPES-NaOH and 150 mM NaCl (pH was added, and the samples were boiled at 95˚C for 5 min. Ten microliters 7.5). Complex formation was monitored by the shift to a lower retention of Precision Plus Protein Kaleidoscope Prestained Protein Standards was volume of the peak compared with a control run in which the same amount used as molecular mass marker (kDa). of C4 or C4b was injected. To monitor proconvertase formation in the Structure determination of the C4b:hC4Nb8 complex presence of hC4Nb8, the nanobody was incubated with the proconvertase for 5 min at 4˚C at a C2/C4b/Nb ration of 1.3:1:6 M, and the sample was Deglycosylated C4b, prepared according to (35), was mixed in a 1:5 M injected onto a Superdex 200 Increase column (GE Healthcare) equili- ratio with hC4Nb8. The complex was purified by size-exclusion chroma- brated in 20 mM HEPES-NaOH, 150 mM NaCl, and 2 mM MgCl2 (pH tography (SEC) on a 24-ml Superdex 200 Increase column equilibrated in 7.5). Disruption of proconvertase formation was monitored by an increased 20 mM HEPES-NaOH and 150 mM NaCl (pH 7.5). The fractions containing intensity of the C4b (11.5 ml) and C2 (13 ml) peaks on the chromatograms the complex were concentrated to 8 mg/ml and used for crystallization and a decrease in the C4b2 peak (10.5 ml). screening with ProPlex commercial screens (Molecular Dimensions), 4 COMPLEMENT INHIBITION WITH A C4b-SPECIFIC NANOBODY

Structure Screens (Molecular Dimensions), or PEGRx screens (Hampton were recorded at concentrations of 56.25, 112.5, 225, 450, and 900 nM for Research). The screens were dispensed using an Oryx robot (Douglas In- C4, and at 1.5625, 3.125, 6.25, 12.5, and 25 nM for C4b. Fitting of the data struments), in which the drops contained a 0.15-ml sample and 0.15-ml were performed for all the measured concentrations simultaneously, using reservoir. After 5 d, plate-shaped crystals appeared in 100 mM HEPES- BIAevaluation software. The apparent KD were calculated from the ratio NaOH (pH 7), 10% w/w PEG4000, and 10% v/v 2-propanol. Crystals between the association and dissociation rate constants. The presented data were cryoprotected in the reservoir buffer containing 20% ethylene glycol or are the mean 6 SE from three replicate experiments. For the competition 25% glycerol, and x-ray diffraction data from cryocooled crystals were experiments, the coupling procedure was the same, but the RU of immo- collected at PETRA III beamlines P13 and P14 (European Molecular Biology bilized nanobody were 50. The complexes were preincubated for 1 h on ice Laboratory, Hamburg, Germany). The diffraction data were processed using prior to injection on the sensor chip at the following molar ratios: C4b/C2 XDS (40). C4b coordinates (Protein Data Bank identification number [PDB = 1:1.1; C4b/CR1 = 1:100; C4b/hC4Nb8 WT and mutants = 1:1; the ID] 5JPN) without the C345c domain were used for initial structure determi- running buffer was 20 mM HEPES, 150 mM NaCl, 3 mM MgCl2, and nation by molecular replacement in phenix.phaser (41). The nanobody in PDB 0.05% Tween 20 (pH 7.5); and all experiments were done in triplicates. ID 5IMM with 80% sequence identity without CDRs was used as starting The calorimetric titration experiments were carried out using a MicroCal model for the nanobody and placed manually with Coot (42) after density VP-ITC instrument in 20 mM HEPES-NaOH and 150 mM NaCl (pH 7.5). modification with phenix.density_modification (43), and the CDRs were built The proteins were either dialyzed or repurified by gel filtration in running manually. Refinement was carried out in phenix.refine (44) with options rigid- buffer right before the experiments. All experiments were carried out at 30˚ body, translation-liberation-screw-rotation, individual sites, individual B fac- C, and the ligands were titrated with 30 injections of a 10-ml volume. C4 tors, and use of torsion angle noncrystallographic symmetry restraints. Data was used at a 2.5-mM concentration, C4b was used at 1.25 and 2.5 mM, collection and refinement statistics are presented in Table I. Structure factors whereas hC4Nb8 and the W53A plus R107A mutant were used at 12.5 and and coordinates are deposited under PDB ID 6YSQ (www.rcsb.org). 25 mM. All experiments were conducted in duplicates. For subtraction of heats of dilution, 12.5 and 25 mM solutions of the ligands were titrated in Negative stain electron microscopy grid preparation and running buffer. Before each run, the samples were centrifuged at 24,000 rcf data processing for 5 min at 4˚C, degassed for 5 min, and centrifuged again prior to concentration measurement on a NanoDrop ND 1000 Spectrophotometer The samples were applied to glow-discharged carbon-coated copper (Saveen Werner). GC400 grids for 5 s at 20 mg/ml. After application, the sample was blotted and stained with 3 ml of 2% w/v uranyl formate by two sequential rounds Immunostaining and passive immunization experiments of staining immediately followed with blotting, followed then by a 1 min stain and blot before the grid was air-dried. The micrographs were All animal experiments were carried out in agreement with the institutional recorded automatically using Leginon on a Tecnai T12 G2 transmission guidelines at Harvard Medical School, following approval of ethical pro- electron microscope operating at 120 kV and equipped with a TemCam- tocols by the local Institutional Animal Care and Use Committee (protocol F416 detector (Tietz Video Image Processing Systems). The defocus range numbers IS00000748, IS00000111, and IS00002660) and per applicable was 20.7 to 21.7 mm, exposure time was 750 ms, and original magnifi- laws and regulations. Five- to-ten- week-old mixed sex C57BL6/C4 cation was 67,000, yielding a pixel size of 3.15 A.˚ The data were processed knockout (KO) mice backcrossed with hC4A and hC4B as previously without performing contrast transfer function correction, and particles described (M. Yilmaz, E. Yalcin, J. Presumey, E. Aw, C. W. Whelan, B. were automatically picked by CisTEM (45) and extracted with a box size Stevens, S. A. McCarroll, and M. C. Carroll, manuscript in preparation) 2 of 96 3 96 pixels. The particles used for 3D classification were selected were used in all experiments (hC4 AB/ ). For immunostaining experi- 2 after reference-free two-dimensional (2D) classification in RELION 3.0.7 ments, 14-mm spleen sections were cryosectioned and fresh frozen at 80˚ (46), using a mask of 250 A.˚ The remaining particles were used for 3D C and re-equilibrated at RT for 20 min prior to fixation with acetone. classification based on automated generation of the initial model in Afterward, acetone evaporation blocking buffer containing PBS, 2% w/v RELION for the C4b2 complexes or with C4b low pass filtered to 25 A˚ for BSA, 5% v/v FCS, and 0.1% Tween 20 was added to each slide and in- the hC4Nb8:C4b complex. Fitting of the of the crystal structure of C4b cubated for 1 h at RT, after which the primary Ab consisting of rabbit anti- (PDB ID 5JPN) (35) into the 3D electron microscopy (EM) envelope of the hC4c (Dako) or IgY-hC4Nb8 was added. The Abs were diluted 1:300 in hC4Nb8:C4b complex or of the hC4Nb8:C4b crystal structure in the 3D PBS-T, and the slides were incubated for 1 h at RT before washing three EM envelope of the C4b2 complex was performed manually in PyMOL times for 3 min in PBS, 0.1% Tween 20, and addition of secondary anti- (version 2.3.0). A total of 29,274 and 6,631 particles were used for the rabbit–Alexa 568 or anti-chicken–Alexa 488 at 1:300 dilution in PBS and hC4Nb8:C4b or C4b2 3D reconstruction, respectively. For fitting of C4b, 0.1% Tween 20. After 1 h of incubation at RT, the slides were washed three C2a, and C2b in the NSEM envelope, C3bB (PDB ID 2XWJ) was first times for 5 min in PBS and 0.1% Tween 20 and mounting medium fitted in UCSF Chimera version 1.14 using the built in “Fit in Map” (electron microscopy) without DAPI was added, and the slides were sealed 3 function (47). C3bB models without the C3b TE domain, the FB serine and dried at 4˚C overnight. The slides were imaged at magnification 20 protease (SP) domain, or missing both of these domains were also used for on a confocal microscope (Olympus FluoView FV1000 confocal system). fitting the EM envelope; however no difference was obtained in the output For the hemolytic assay, the experiments were repeated in triplicates (n = compared with the fitting of the full complex. A coordinate file was written 3 mice). Approximately 100 ml of mouse blood was collected into a tube for the fitted C3bB, and all the following steps were carried out in PyMOL containing 5 ml of 0.1 M EDTA and kept on ice for the whole experimental (version 2.3.0). C4b in the C4b:hC4Nb8 complex was aligned on the MG6 procedure. The serum (supernatant) was collected after centrifugation at domain of C3b, the FB CCP domains were used to model the C2 CCP 10,000 rpm for 8 min, and samples were diluted 1:60, 1:240, 1:1000, or domains, whereas the structure of C2a (PDB ID 2I6S) was superimposed 1:50,000 for treated and nontreated conditions. Each experimental condi- 6 on the VWA domain in the fitted C3bB. The structure of the cobra venom tion contained a 25-ml sample (mouse serum 10 mg/ml hC4Nb8), 25 ml 3 8 factor-FB (CVFB) complex (PDB ID 3HRZ) was superimposed on the C4-deficient guinea pig sera and 15 ml of EA cells (1 10 cells/ml; 65 ml fitted C3bB. Although an improved fit to the 3D reconstruction could be working volume). The absorbance value obtained from mixing 50 mlof obtained by further manual adjustment of the C4b C345c and the C2 water and 15 ml EA cells represented complete hemolysis (100%), whereas VWA-SP domains, this was avoided in the model presented in this study 50 ml gelatin veronal buffer and 15 ml of EA cells represented the back- for simplicity and because of the limited resolution. ground (negative control). The plate was incubated at 37˚C for 30 min, centrifuged at 1600 rpm for 5 min, the supernatant was collected, and Surface plasmon resonance and isothermal titration absorbance was measured at 415 nm. calorimetry experiments For passive immunization experiments, mice were injected with 1 mg of rabbit anti-PE Ab (Rockland Immunochemicals) 24 h before subcutaneous The surface plasmon resonance (SPR) measurements were conducted on a injection of 5 mg of hC4Nb8 or Lag16 and 1 mgofPEin10ml of HBSS in Biacore T200 instrument (GE Healthcare). Streptavidin was diluted to 10 each leg. The mice were sacrificed 24 h after administration of the treat- mg/ml in 10 mM sodium acetate (pH 4.5) and immobilized to 100 response ment and popliteal lymph nodes were collected in 4% paraformaldehyde; units (RU) on the carboxymethylated dextran surface of a Sensor Chip after 2 h they were transferred to 30% w/v sucrose and incubated at 4˚C CM5 (XanTec bioanalytics GmbH) using an amine coupling kit. The overnight. No distinction was made between lymph nodes coming from the biotinylated nanobody was injected on the immobilized streptavidin at 30 left or from the right leg. The lymph nodes were cryosectioned in 14 mm mg/ml, giving 24 RU of captured nanobody. The binding measurements sections and the slides were fresh frozen at 280˚C, prior to staining with were performed in 20 mM HEPES, 150 mM NaCl, 3 mM MgCl2, and the different markers with the same protocol used for spleen staining. In 0.05% Tween 20 (pH 7.5) at 30 ml/min flow rate. At the end of each the first round of experiments, the primary Abs used were 8C12-biotin for concentration, measurement of the surface was regenerated by injection of FDCs and B220-FITC for the B cell follicle, whereas in the second round 100 mM glycine (pH 2.7) for three cycles of 10 s contact time. Sensorgrams of experiments the FDCs were labeled with 7E9-pacific blue. The Abs The Journal of Immunology 5

FIGURE 1. The hC4Nb8 nanobody is a potent inhibitor of the CP and LP C3 convertase. (A) The proteolytic cascade initiated upon activation of the CP and LP. The proteolytic degradation of C3b to iC3b and C3dg is enabled on host cells because of the presence of cofactors. (B and C) The hC4Nb8 has no effect on C4b deposition but potently inhibits C3b deposition upon CP activation of NHS by the IgG surface. (D and E) Same as in (B) and (C) but with LP- driven C4b and C3b deposition from NHS onto a mannan surface. (F) In murine serum, hC4Nb8 also inhibits C3 convertase activity (i.e., suppresses C3 fragment deposition via the LP onto a mannan surface). The percentages of C4b and C3b deposition are relative to the signal at the same serum dilution without nanobody addition. The average C4 concentration in the NHS dilutions used for the assays in (B)–(E) is depicted by a dashed line. The nanobody hC3Nb1 W102A is an inactive mutant of the AP inhibitor hC3Nb1 (37); (G) hC4Nb8 inhibits LP-driven C3b deposition onto a mannan (Figure legend continues) 6 COMPLEMENT INHIBITION WITH A C4b-SPECIFIC NANOBODY were diluted 1:300 in PBS-T and the slides were incubated for 1 h at RT. site-specifically biotinylated hC4Nb8 was immobilized on a streptavidin The slides were washed three times for 3 min in PBS, 0.1% Tween 20 prior biosensor (ForteBio) at 1.25 mg/ml, and mC4b was present in the 2-fold to addition of streptavidin conjugated to Alexa488 at 1–300 dilution in dilution series from 12.5 to 0.78 nM in the association phase. Durations of PBS, 0.1% Tween 20. After 1 h incubation at RT, the slides were washed 600 s for the association and 1200 s for the dissociation phase were used. three times for 5 min in PBS, 0.1% Tween 20 and mounting medium The data were processed by subtraction of a 0 nM measurement and fitted (Electron microscopy) without DAPI was added and the slides were sealed in the Octet System Data Analysis software with a 1:1 Langmuir binding and dried at 4˚C overnight. The slides were imaged on a confocal mi- model. croscope (Olympus FluoView FV1000 confocal system) at 403 magnifi- cation and one B cell follicle was imaged in each field of view. Four images were collected for each mouse without side segregation of the Results lymph nodes. In the first round of experiments one slide was imaged for hC4Nb8 generation and its inhibitory effects each mouse, whereas in the second round three slides per mouse were A llama was immunized with a total of 400 mg of hC4 and 400 mg imaged, for a total of 12 images per mouse. For data analysis, the images were analyzed in ImageJ. The images were thresholded and the colocalized of hC4b in three consecutive immunization boosts separated by area of the FDC channel with the PE channel was calculated. The area intervals of 3 wk. The llama blood was collected, and lymphocytes around the FDC network was isolated, transformed to binary area and the were isolated for mRNA extraction and cDNA synthesis. Nano- ratio with the binary PE colocalized area was calculated and plotted in bodies were cloned as fusions to protein pIII expressed on the GraphPad Prism 6.0. The statistical analysis was carried out in GraphPad Prism 6.0 with one-way ANOVA and multiple comparisons, in which the surface of M13 phages. Potential complement inhibitors were mean of each column was compared with the mean of every other column isolated by two rounds of phage display selection on plates coated (Supplemental Table II). with hC4. The positive clones were identified by ELISA, se- quenced, and cloned into a bacterial expression vector. The in- Neuronal complement deposition assays hibitory activity of multiple nanobodies was investigated in CP Neurons were differentiated from induced pluripotent stem cells by ex- and LP deposition assays on aggregated IgG and mannan-coated pression of Neurogenin2 and grown on a Matrigel matrix for 14 d after surfaces, respectively. After these preliminary experiments, differentiation. All the assay steps were carried out using an INTEGRA VIAFLO 96/384 with 96-well tips and a 300-ml head. All aspirations and hC4Nb8 appeared to be the strongest inhibitor of CP- and LP- dispenses over cells were done at the lowest speed. The cells were sen- driven C3 fragment deposition but not C4 fragment deposition in sitized to complement deposition by addition of anti-NCAM Ab (AB5032; human serum (Fig. 1B–E). The nanobody also inhibited LP-driven Millipore) at 2.5 mg/ml in 10 mM HEPES-NaOH, 140 mM NaCl, 5 mM C3 deposition in murine serum (Fig. 1F). To test whether the KCl, 1 mM MgCl2, 2 mM CaCl2 (pH 7.2) (binding buffer), and incubation at RT for 20 min to generate ICs. The cells were then washed with 100 ml nanobody would inhibit complement in conditions where C4 is of binding buffer in each well prior to addition of 10% NHS or C4- present in high concentrations and thereby competes with C4b for depleted serum (C4-Dpl NHS; Complement Technology) diluted in gela- hC4Nb8 binding, we carried out C3 deposition assays in 50% + + tin veronal buffer including Ca2 and Mg2 (Complement Technology) NHS, triggering LP activation on a mannan-coated surface. In this with the desired nanobody dilution (hC4Nb8 or Lag16). Complement study, we included the C3-specific nanobody hC3Nb1 (37), which deposition was carried out for 30 min at 37˚C. The cells were washed twice with prewarmed neurobasal medium and live stained with FITC- efficiently suppresses the activity of the AP C3 convertase while conjugated anti-C3c Ab (F0201; Dako) diluted 1:200 for 20 min at 37˚ allowing the CP C3 convertase to turn over C3. In this manner, we C. The cells were washed once in PBS and fixed for 7 min in 4% para- were able to selectively measure the LP contribution without an formaldehyde, washed again in PBS, and stained for 1 h at 4˚C with eFluor overwhelming AP-driven C3 deposition (Fig. 1G). Also, in these 660–Tuj1 (anti–b-tubulin) Ab (50-4510-82; Invitrogen) in 50 ml per well. The cells were washed once in PBS, and the plate was kept at 4˚C until near-physiological conditions, we observe that the hC4Nb8 imaging. The plates were imaged on a PerkinElmer Opera Phenix instru- nanobody inhibits C3 deposition in a dose-dependent manner. The ment; 11 fields of view per well were collected at 633 magnification in level of C3 deposition in the presence of both hC4Nb8 and z-stack mode. Data were analyzed using a pipeline created in the Perki- hC3Nb1 is comparable to the addition of 10 mM EDTA in 50% nElmer Harmony software. Briefly, images of C3 and Tuj1 were thresh- NHS. To evaluate whether hC4Nb8 may act as a C4-specific in- olded and masked: Tuj1+ neurites were identified by the FindImageRegion module using a common threshold, spots of C3 were segmented with the hibitor in nonhuman primate disease models, we also tested the FindSpots module and masked with the Tuj1+ neurite region (C3/Tuj1+ cross-reactivity of hC4Nb8 with marmoset (Callithrix jacchus)C4 objects) and area was quantified per C3/Tuj1+ object and then summed (CjC4) by pull-down experiments. We consistently observed + across all C3/Tuj1 objects. C3 deposition was then calculated per well as presence of the CjC4 bands (CjC4 a-, b-, and g-chains) on the the sum of C3/Tuj1+ object area as a percentage of total Tuj1+ neurite area. Three plates were analyzed for a total of (n = 24) wells per dose of hC4Nb8 streptavidin beads in eight experiments with plasma from hC4Nb8 or Lag16 in 10% NHS and (n = 6) wells per hC4Nb8 or Lag16 in different animals compared with the bands observed with strep- C4-Dpl NHS. Statistical analysis was carried out in R version 3.6.1 with tavidin beads without hC4Nb8 (Fig. 1H). This result demonstrates one-way or two-way ANOVA and Tukey honestly significantly different that hC4Nb8 cross-reacts with CjC4. post hoc tests, in which the mean of each condition was compared with The strong inhibition of C3 deposition in human serum sug- the mean of every other condition. ANOVA was performed both across serum conditions (10% NHS and C4u-Dpl) at matching 20 mg/ml doses of gested that hC4Nb8 prevents either proconvertase assembly or hC4Nb8 and Lag16 (two-way) and within the deposition-permissive serum interferes with C4b2a recognition of the C3 substrate. To gain (10% NHS) condition to examine dose effects of hC4Nb8 versus Lag16 functional insight into the mechanism of inhibition, we evaluated control (one-way) (Supplemental Table III). the binding of the nanobody to native C4 and C4b by SEC. For C4b, Bio-layer interferometry experiments the presence of the nanobody resulted in a clear shift in the elution volume, indicating formation of a stable C4b-hC4Nb8 complex. In Bio-layer interferometry (BLI) measurements were carried out using an Octet RED96 System (ForteBio) with the plate at 30˚C and shaking at 1000 contrast, C4 eluted identically in the presence and absence of rpm. The running buffer for all experiments was 20 mM HEPES-NaOH, hC4Nb8, suggesting that the epitope may only be accessible in C4b 150 mM NaCl, 3 mM MgCl2, and 0.05% Tween 20 (pH 7.5). The AVI-tagged or that the affinity for native C4 is too low to be detected in this

surface in 50% NHS to levels comparable to addition of 10 mM EDTA when the AP is simultaneously inhibited by the hC3Nb1 nanobody. (H) SDS-PAGE analysis of pull-down experiment from Cj plasma using biotinylated hC4Nb8 bound to streptavidin beads (+hC4Nb8, beads lane), whereas no C4 binding is observed when using streptavidin beads but no biotinylated hC4Nb8 (2hC4Nb8, beads lane). The a-, b-, and g-chains of Cj C4 are assigned based on comparison with the molecular mass of the these chains in hC4, but a minor content of a9-chain from C4b is possible. Bds, beads; Inp., input; W, wash fractions. The Journal of Immunology 7

assay (Fig. 2A, 2B). We next investigated the formation of the CP and LP proconvertase C4b2 in the presence of the nanobody (Fig. 2C, 2D). In the presence of molar excess of hC4Nb8, C2 was unable to interact with C4b, suggesting that one mechanism whereby hC4Nb8 inhibits C3 deposition is by interfering with proconvertase assembly. Based on the knowledge that the nanobody competes with C2 binding and, by comparison, with the known structures of C3b in complex with FH and FI (48), we hypothesized that hC4Nb8 inhibited CR1-mediated FI cleavage of C4b in vitro. In the ex- perimental conditions used, the cleavage of C4 to C4c and C4d was complete after 2 h of incubation at 37˚C (result not shown). When present in a 1:1 M ratio with respect to C4b, the nanobody strongly inhibits cleavage by FI, and substrate cleavage is only starting to become apparent after 24 h of incubation at 37˚C (Fig. 2E). The hC4Nb8 recognizes a C4b neoepitope To define the structural basis for the inhibition of CP proconvertase assembly exerted by hC4Nb8, we crystallized the hC4Nb8:C4b complex and collected x-ray diffraction data extending to a maximum resolution of 3.3 A˚ (Table I). The structure was deter- mined by molecular replacement, using the structure of C4b (35, 49) as search model, whereas the nanobody was modeled from PDB ID 5IMM. The final atomic model (Fig. 3A) was obtained after a few iterations of rebuilding–refinement cycles, and refined to an Rfree value of 27%. An example of the electron density for the C4b:hC4Nb8 interface is displayed in Fig. 3B. The asym- metric unit of the unit cell contains two copies of the C4b:hC4Nb8 complex that do not differ significantly with respect to the C4b:hC4Nb8 interaction. The C4b epitope for hC4Nb8 is primarily formed by residues Pro-773-Asn781 in the last half of the a9 N-terminal region (Nt-a9) of C4b and the following two b-strands in the MG6 domain. Analysis with PISA indicates a buried surface area upon complex formation of 1736 A˚ 2 and a shape complementarity of 0.63 be- tween the two proteins. Both hydrophobic and polar residues in hC4Nb8 are engaged in interactions with C4b. In hC4Nb8, Trp53 from CDR2 and Ile105 from CDR3 form the hydrophobic core of the complex, together with Phe777, Phe778, and Trp798 presented by the C4b MG6 domain (Fig. 3C–E). A hydrogen bond appears to bridge Arg31 in the hC4Nb8 CDR1 with C4b Ser776 (Fig. 3C), whereas a second hydrogen bond possibly bridges Ser54 in hC4Nb8 CDR2 and the main chain of Val774 in C4b (Fig. 3D). Electrostatic interactions are formed between hC4Nb8 CDR3 residues Glu102, Glu110, and Arg107 and residues Arg785 and Glu787 residues in the C4b Nt-a9 region MG6 domain (Fig. 3E). Of notice, at the resolution of 3.3 A,˚ hydrogen bonds can only be considered as putative. In addition, water molecules present at the intermolecular interface cannot be modeled but most likely form additional bridging contacts between hC4Nb8 and C4b. As the Nt-a9 region undergoes an extensive conformational change upon C4 cleavage, the binding site for hC4Nb8 is a C4b- specific neoepitope. In native C4, the future Nt-a9 region is po- sitioned in a channel formed by the MG2 and MG3 domains and the linker region, whereas it becomes exposed in C4b (11, 35).

FIGURE 2. hC4Nb8 binds selectively to C4b and disrupts C4b inter- actions with C2 and CR1. (A) SEC binding assay with hC4b, (B) hC4, and hC4Nb8 mixed with hC4b2) with retention volume of each sample given on (C) hC4b2. The chromatograms for elution of C4, C4b, or C4b2 in the top of the lane. (E) CR1 cofactor activity on FI-mediated cleavage assay presence of a 5-fold molar excess of hC4Nb8 are depicted with a full line, with a 1:10:0.2:1 = C4b/CR1/FI/hC4Nb8 M ratio. The C4b/FI ratio cor- whereas the chromatograms of C4b, C2, and hC4Nb8 are depicted with a responds to that present with physiological C4 and FI concentrations. In- dashed line. The C4b2 chromatogram is depicted with a dashed and dotted cubation time is given above the gel. The positions of the C4b chains (a9-, line. (D) Reducing SDS-PAGE analysis of the chromatogram in (C) (i.e., b-, and g-) and C4d and CR1 are indicated. 8 COMPLEMENT INHIBITION WITH A C4b-SPECIFIC NANOBODY

Table I. Refinement statistics for structure determination of the Alignment of the hC4b:hC4Nb8 complex with this 3D recon- hC4Nb8:C4b complex struction reveals a major spatial overlap between hC4Nb8 and a volume of the 3D reconstruction likely to contain the C2 CCP Data Collection domains (Fig. 4D). Fitting of the CCP domains of the C2 homolog Resolution range 48.2623.3 (3.41823.3) FB in the EM envelope suggests that steric clashes arise because Space group P 1 21 1 of the dramatic overlap of hC4Nb8 with the C2 CCP2 domain Unit cell 131.2 89.51 231.2 90 97.552 90 (Fig. 4D, inset), suggesting that the nanobody prevents C2 from Total reflections 538,319 (49,811) Unique reflections 79,980 (7,914) binding through direct competition rather than by allosteric Multiplicity 6.7 (6.7) effects. Completeness (%) 99.87 (99.94) The AP functional homolog of C2, FB, adopts two different Mean I/s(I) 11.73 (0.65) conformations named closed and open. These were captured in the Wilson B-factor 147.94 crystal structures of the CVFB complex (52) and the C3bB R 0.09005 (2.284) merge complex (53), respectively. The open conformation allows cleav- Rmeas 0.09786 (2.473) CC1/2 0.998 (0.341) age by the fluid phase protease FD, and it is adopted by FB only Refinement after binding to C3b (52, 53). The two FB conformations mainly Reflections used in refinement 80,456 (7,913) differ by the location of the catalytic SP domain. In the CVFB Reflections used for Rfree 1,566 (155) complex, the SP domain is close to the first two CCP domains in Rwork/Rfree 0.2212 (0.3613)/0.2698 (0.3852) Number of nonhydrogen 26,297 FB, whereas in the open conformation the SP domain has rotated atoms 80˚ and contacts the C3b CUB domain. Importantly, our 3D re- Macromolecules 26,141 construction reveals that the C2 SP domain appears to be in a Ligands 156 rather different position compared with these two FB conforma- Protein residues 3,384 tions. The C2 conformation is extended with the SP domain far Root mean square (bonds) 0.004 Root mean square (angles) 0.91 from both the C2 CCP domains and the C4b CUB domain Ramachandran favored, 92.08, 7.20, 0.72 (Fig. 5C–E). Our model of the C4b2 complex derived by rigid- allowed, outliers (%) body modeling against small-angle x-ray scattering data (34) also Clash score 3.36 suggested a C2 conformation different from the known FB con- Average B-factor 195.53 formations for both unbound and C4b bound C2, which is also in 2 2Ι ̅ Rmeas = Shkl(N/(N 1))1/2Si|Ii(hkl) (hkl)|/ShklSiIi(hkl). CC1/2 is the correla- agreement with the cleavability of C2 in absence of C4b binding tion between random half-datasets. Statistics for the highest-resolution shell are shown in parentheses. (54). In summary, our structural studies show that hC4Nb8 binds to a neoepitope in C4b formed by residues in the Nt-a9 and the MG6 domain presented upon C4 cleavage by MASP-2 or C1s. Moreover, if hC4Nb8 were to bind the C4 MG6 domain in the This epitope overlaps strongly with the binding site for C2 CCP same manner as observed in the C4b complex, the C4 MG7 do- domains, rationalizing the observed hC4Nb8 inhibition of CP C3 main would apparently sterically clash with the nanobody convertase formation and activity. (Fig. 4A, 4B), suggesting that only weak binding to C4 may occur, hC4Nb8 binds with picomolar affinity to C4b in line with our SEC experiment (Fig. 2A). The observed hC4Nb8 epitope (Fig. 4C, left) also rationalizes the complete lack of in- The C3 deposition assays, our SEC analysis, and the crystal hibition with respect to C4 deposition (Fig. 1B, 1D). A projection structure of the hC4b:hC4Nb8 complex collectively suggested that of the hC4Nb8 binding site in C4b onto C4 reveals that the the nanobody bound with high affinity to C4b and with much nanobody epitope is located far from the three regions in C4 weaker affinity to C4, but accurate values for binding constants and contacted by MASP-2 and by homology C1s: 1) the scissile bond rate constants are needed to predict the potential of hC4Nb8 for region, 2) the sulfotyrosine region in the C-terminal part of the C4 inhibition of the CP C3 convertase in vivo. We therefore conducted a-chain, and 3) a basic patch centered around residue 1720 in the SPR experiments in which the nanobody was C-terminally AVI- C345c domain of C4 (11). The hC4b:hC4Nb8 atomic model is tagged, site-specifically biotinylated, and immobilized on a sen- also consistent with the observed inhibition of CR1-mediated FI sor chip coated with amine-coupled streptavidin. Binding kinetics degradation of C4b. A structural alignment of C4b with C3b in the were studied by flowing 2-fold dilution series of the analytes C4 structure of the C3b:FH:FI complex (48) suggests that hC4Nb8 and C4b over the immobilized ligand hC4Nb8 surface. For C4b, the prevents binding of the CCP1-2 domains of the C4b regulators sensorgrams could be fitted well with a 1:1 binding model, giving a CR1, C4BP, and MCP and the subsequent interaction with FI fitted value of KD = 15.5 6 1.3 pM (Fig. 6A). In contrast, sen- required for degradation of C4b to C4c and C4d. sorgrams obtained with C4 could not be fitted with this model, and The interaction of C2 with C4b is mediated through two contact 40-times-higher concentrations of C4 were required to obtain a points (50), with one of the binding sites in the C2b CCP domains response in the same range as acquired for C4b, resulting in very and the second provided by the MIDAS site in the C2a VWA high analyte concentrations, possibly giving rise to unspecific domain interacting with the C4b C terminus presented by the binding effects (Fig. 6B). C345c domain. Located next to the hC4Nb8 epitope are multiple We also used the SPR approach to confirm the competition of the acidic residues (E763, E764, D768, E769, E770, E771) in the C4b nanobody with C2 suggested by both our SEC analysis and our Nt-a9 previously identified as important for the interaction with structural studies (Figs. 2C, 4D) and competition with CR1 in the C2 (51) (Fig. 4C, left). Our SEC assays showed that the nanobody assisted FI cleavage assay (Fig. 2C, 2D). The extent of competi- interferes with the C2 binding to C4b but cannot distinguish tion was quantified as a percentage of signal for C4b binding to mechanistically between direct competition with C2 or an allo- immobilized hC4Nb8 in the presence of C2 or CR1 compared steric effect of hC4Nb8. To obtain the structural basis for hC4Nb8 with the signal for an experiment in which only C4b was injected. inhibition of C2 binding, we reconstituted the CP C3 pro- The signal was almost fully recovered for the C4b:CR1 complex, convertase and determined a 3D reconstruction at a resolution of whereas for C4b2 only 17% of the signal was recovered (Fig. 6C, 21 A˚ of the C4b2 complex with negative stain EM (Fig. 5). dashed and dotted line), suggesting that C2 is a much stronger The Journal of Immunology 9

FIGURE 3. The crystal structure of the hC4Nb8:hC4b complex at 3.3 A˚ resolution. (A) The nanobody (sand) binds to the Nt-a9 region (spheres) of C4b

(blue) and two b-strands in the MG6 domains. (B) Omit 2mFo-DFc map for the epitope region and the hC4Nb8 CDR3 contoured at 1 s.(C–E) Detailed presentations of interactions formed between C4b and the hC4Nb8 CDR1, CDR2, and CDR3 regions, respectively. The majority of the electrostatic in- teractions are formed by CDR3. Putative hydrogen bonds and electrostatic interactions are indicated by dashed lines. competitor than the CR1 CCP1-3 fragment used in this study. could contribute to the less efficient inhibition of LP-driven C3b These results are in qualitative agreement with previously deter- deposition observed in murine serum (Fig. 1F) compared with mined dissociation constants for complexes of C4b with C2 and human serum. We therefore measured the binding affinity of the the CR1 ectodomain, 72 nM (50) and 0.9 mM, respectively (55). hC4Nb8 nanobody to murine C4b by BLI (Fig. 6D). Our data Sequence alignment of human, marmoset, and mouse C4 reveals show that the nanobody binds to mouse C4b with a dissociation that residues 791–794 in the epitope are the least conserved in constant KD = 0.176 nM, which is comparable to the dissociation mouse C4 (Fig. 4C, right). Possibly, the presence of a lysine in constant of 16 pM observed for hC4b by SPR. Overall, our SPR mouse C4 at the position corresponding to Gln793 that is near to experiments documented that hC4Nb8 binds with picomolar af- Arg107 in the hC4Nb8 CDR3 (red in Fig. 4C) introduces elec- finity to hC4b and provided further evidence of hC4Nb8 compe- trostatic repulsion weakening the binding to mouse C4b. This tition with the physiological C4b binding partners C2 and CR1. 10 COMPLEMENT INHIBITION WITH A C4b-SPECIFIC NANOBODY

FIGURE 4. The hC4Nb8 epitope is a neoepitope in C4b and overlaps with the C2 binding site. (A) structure of hC4Nb8 (sand) in complex with C4b (gray) with MG6 and Nt-a9 in blue; (B) model of hC4Nb8 in complex with C4 (gray) with MG6 and Nt-a9 in blue. hC4Nb8 is shown as transparent surface in sand. Notice the predicted clash with the MG7 domain. (C) Left, Epitope of hC4Nb8 on C4b with residues interacting with hC4Nb8 in sand and remaining residues in blue. Right, Sequence alignment of the epitope regions (top, MG6; bottom, the Nt-a9 region and downstream residues in the MG6 domain). Interface residues are labeled with a star. His582, Lys793 in mouse C4 and Lys793, Arg798 in Slp are colored red. (D) Comparison of the C4b:hC4Nb8 structure with the NSEM envelope (gray transparent surface) of the C4b2 complex reveals that hC4Nb8 overlaps directly with the predicted C2b (red) binding site.

The difference in the inhibitory efficacy of hC4Nb8 in human and consumes substantial amounts of material, we first conducted pull- murine serum appears not to stem from a significantly weaker down experiments with very long incubation times to establish interaction with murine C4b. which hC4Nb8 single mutations had the strongest effect on the affinity for C4b. The WT hC4Nb8 nanobody was immobilized on hC4Nb8 binds C4b 105-fold better than native C4 streptavidin beads, whereas C4b, together with different hC4Nb8 In light of the limitations of the SPR approach described above, we mutants, was added in the supernatant. After prolonged incubation decided to take advantage of isothermal titration (ITC) experiments at 4˚C, SDS-PAGE analysis of material eluted from the beads by to determine the effect of mutations in hC4Nb8 and to clarify the boiling was conducted. After normalization by hC4Nb8 band in- strength of the interaction between hC4Nb8 and native C4. As ITC tensity, the effect of the mutations on the hC4Nb8:C4b interaction The Journal of Immunology 11

FIGURE 5. In the proconvertase, C2 adopts an extended conformation distinct from the closed and open states of FB. (A) Examples of 2D classes used in the 3D classification. The 2D classes are 400 A˚ in each direction. (B) Resolution estimate based on Fourier shell correlation calculation in RELION. (C–E) Fitting of the C4b structure in the C4b:hC4Nb8 complex and of the crystal structures of C2b and C2a in the NSEM density obtained from C4b2 reveals significant differences in the location of the SP domain (C) compared with FB open conformation in C3bB (PDB ID 2XWJ) (D) and to FB closed con- formation in CVFB (PDB ID 3HRZ) (E). The noncatalytic subunit is in blue, the zymogen CCP domains are in red, whereas the VWA-SP domains are in green in all panels. The location of the SP domain is denoted by a star. could be ranked in the order W53A . R107A . E102A . E104A thermodynamic parameters determined by SPR and ITC can be . A75R = R31A = WT (Fig. 6E). Based on this evidence, the compared, the binding constant for the C4b complex decreased double-mutant W53A, R107A was selected for further analysis 3000-fold for the double-mutant W53A, R107A, going from 15.5 with ITC. The structure of the C4b:hC4Nb8 complex suggests that pM to 48 nM (Fig. 7C). For both WT hC4bNb8 and the W53, the introduction of the W53A, R107A mutation in the nanobody R107A double-mutant, binding was highly exothermic, with en- will primarily affect the interaction of hC4Nb8 with the C4/C4b thalpies of 217 and 225 kcal∙mol21, respectively. The titration of MG6 domain. hC4Nb8 into native C4 resulted in a titration curve to which a Before proceeding to the titration experiments, the efficacy of the dissociation constant for the hC4Nb8:C4 complex of 2 mM could double-mutant W53A, R107Awas examined in a CP C3 deposition be fitted (Fig. 7D), confirming the very low response for C4 assay. This demonstrated that hC4Nb8 W53A, R107A needed to be binding in SPR (Fig. 6B). The double-mutation W53A, R107A present in an ∼4-fold higher concentration to inhibit the CP C3 completely abolished binding of hC4Nb8 to native C4 (Fig. 7E). convertase to the same degree as the WT nanobody (Fig. 7A). In The five order of magnitude difference in affinity for C4 compared the ITC experiments, the dissociation constant for the with C4b provides further evidence for the relevance of our crystal C4b:hC4Nb8 complex could not be determined, as the heat- structure, as the epitope of the nanobody encompasses the Nt-a9 evolved measure jumped from the nonbound to the bound state region of C4b, which only becomes exposed after C4 activation, without intermediate points needed for the fitting and binding whereas it is tucked inside a cavity formed by the MG2 and MG3 constant determination (Fig. 7B). This observation is in agreement domains in native C4 (11) (Fig. 4B). The 2-mM measured KD for with the 16 pM KD measured by SPR, which is outside of the binding to C4 is likewise in agreement with the absence of limits that can be measured directly by ITC (56). Assuming that complex formation in our SEC experiment. 12 COMPLEMENT INHIBITION WITH A C4b-SPECIFIC NANOBODY

hC4Nb8 inhibits the CP-mediated transport of ICs to FDCs and C3 deposition on neuronal cell cultures We next addressed whether hC4Nb8 may be useful for the analysis of complement-driven immune response and pathogenesis. We genetically fused the nanobody to the Fc moiety of chicken Ig IgY, creating a bivalent C4-specific reagent for immunostaining of tissue sections from transgenic mice expressing hC4, whereas deleted for murine C4. The hC4Nb8–IgY was used for staining of a spleen section in parallel with a validated commercial anti-C4 polyclonal Ab. The staining revealed colocalization between the two imaging protocols, confirming the binding of hC4Nb8 to hC4 or hC4b in transgenic mouse tissue (Fig. 8A). The hC4Nb8 nanobody was also tested in hemolytic assays to detect its inhibitory effect in the transgenic hC4 mouse serum. Incubation of the serum with 10 mg/ ml hC4Nb8 inhibited CP-driven hemolysis of EA cells (n =3) compared with nontreated serum at all the dilutions tested (1:60, 1:240, 1:960) (Fig. 8B). After these preliminary validation experiments, we adopted a local immunization strategy to evaluate the function of hC4Nb8 in vivo. The transport of ICs to FDCs depends on activation of the CP (18). In our experiments, hC4-expressing or C4KO mice were passively immunized with anti-PE Ab injected i.p. After 24 h, PE was injected s.c. in the mice hock along with hC4Nb8 or the Lag16 control nanobody. Sections of the popliteal lymph nodes were stained with anti-B220 (CD45R) Ab in Far Red (647 nm), 7E9 Ab binding to CD21/CD35 on FDCs was stained in Pacific Blue (405 nm), and PE was stained in the red channel (568 nm). The colocalized deposition of red fluorescent PE with blue FDCs on popliteal lymph nodes was quantified by ImageJ after thresh- olding the images (57). The overall analysis of two independent experiments is presented in Fig. 8C. The hC4Nb8 nanobody in- hibits PE colocalization on the FDCs compared with the Lag16 control nanobody (p , 0.0001), and colocalization is comparable to that observed for C4KO mice with a 0.1 ratio (Fig. 8C). In contrast, the calculated average ratio of colocalized area was 0.2 when the Lag16 control nanobody was administered. The total FDC area was not dependent on the treatment received by the mouse (Supplemental Table I). To assess the ability of the nanobody to inhibit complement deposition in the context of the CNS, the nanobody was assayed in neuronal complement deposition assays [modified from (58)]. Human induced pluripotent stem cell–derived neurons sensitized to complement deposition were treated with NHS and live stained for C3c, and the proportion of neuronal processes (red in Fig. 9A) colocalized with C3 deposition (green in Fig. 9A) was quantified. In this experimental setup, there is a strong dose-dependent in- hibitory effect of the hC4Nb8 nanobody on deposition of C3 onto neurites compared with the Lag16 control nanobody (p , 0.0001, Fig. 9B). Overall, our functional experiments with hC4Nb8 in different contexts demonstrate that the nanobody can be applied broadly in experiments in which complement inhibition is needed. In vivo, we have shown that hC4Nb8 inhibits the CP-dependent transport of

injected onto the hC4Nb8-coated sensor. All the experiments were carried

FIGURE 6. hC4Nb8 binds with a picomolar dissociation constant to out in triplicates at 25˚C. (D) BLI triplicate measurement of KD, in which C4b. (A) C4b injected as analyte at concentrations 25, 12.5, 6.25, 3.13, and the biotinylated hC4Nb8 was immobilized on a streptavidin sensor and 1.56 nM over an SPR sensor with immobilized hC4Nb8. (B) Same as in mC4b in the binding phase was present in the 2-fold dilution series from (A) but with C4 as analyte injected at concentrations of 900, 450, 225, 12.5 to 0.78 nM. Signal is shown as dashed lines; fitting is shown as full 112.5, and 56.25 nM, respectively. Signal is shown as dashed lines, fitting lines. (E) Pull-down assay of C4b by mutants of hC4Nb8 to rank the single is shown in full lines. (C) SPR competition experiments, in which C4b mutants for a weaker interaction with C4b; reducing SDS-PAGE analysis preincubated with CR1 (dashed line) or C2 (dashed and dotted line) were of the pull-down eluates are shown. The Journal of Immunology 13

Except for a 56-kDa chimeric protein based on the endogenous LP regulator MAP-1 and C4BP that is an efficient regulator of C4b in vitro, there is no well-characterized man-made C4-specific in- hibitor (60). In this study, we present, to our knowledge, the first structurally characterized inhibitor of the CP and LP C3/C5 convertases acting at the level of C4b. Our 14-kDa hC4Nb8 nanobody is highly specific for C4b and potently inhibits CP- and LP-driven C3 deposition in human serum as well as LP-driven C3 deposition in mouse serum. Our structural and functional studies show that the dominating mechanism by which hC4Nb8 inhibits the CP C3 convertase is by preventing assembly of the pro- convertase. If the active C4b2a was formed, comparison with our prior structural model of the CP C3 convertase (34) suggests that the nanobody would most likely also compete with the catalytic C2a subunit and hence act as a decay acceleration factor. A pos- sible disadvantage of hC4Nb8 is that our experiments with FI degradation of C4b suggest that the nanobody will prevent the endogenous regulators C4BP, CR1, and MCP from assisting FI with C4b conversion to C4d on host cells. This may be less of an issue, as FI regulation of C3b remains intact, and the FI cleavage- resistant C4b present is effectively prevented by the hC4Nb8 from forming CP C3 and C5 convertases. As a powerful demonstration of the efficacy of the nanobody framework for the development of complement inhibitors, hC4Nb8 binds a neoepitope in C4b with a dissociation constant of 16 pM, using its three CDR regions. For comparison, the therapeutic mAb eculizumab, with six CDR regions and used for over a decade in the clinic for treatment of paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, binds its Ag comple- ment C5 with a dissociation constant of 18 pM (61). Nanobodies are 14–15-kDa proteins and therefore normally have short circu- lation times in vivo because of renal clearance (62). Nevertheless, we could show that hC4Nb8 is efficient in vivo in the passive immunization model, in which the nanobody was administrated together with the PE Ag (57). The main reason for this in vivo efficacy is likely to be the picomolar KD for the hC4Nb8 inter- action with C4b deposited at the CP and LP activation site. In addition, although 1 3 105 times weaker than the C4b:hC4Nb8 interaction, complex formation with the abundant native C4 pre- sent at 2–3 mM in plasma potentially delays clearance compared with an unbound nanobody. The uptake of complement iC3b opsonized Ags examined in the passive immunization model is FIGURE 7. Validation of the crystal structure and determination of the hijacked by HIV in the chronic stage of AIDS (63, 64), suggesting dissociation constant for the C4:hC4Nb8 complex. (A) Assay for deposi- a therapeutic potential for hC4Nb8 administration in the context tion of C3 fragments in 0.2% NHS using hC4Nb8 WT and W53A, R107A of retroviral infection. mutant as inhibitor. The C4 concentration at the tested serum dilution is The hC4Nb8 offers a small versatile protein module that may marked with a vertical dashed line. (B and C) ITC experiments to quantify help to deduce the molecular mechanism behind complement- B D the interaction of the ligands hC4Nb8 WT ( and ) and the W53A, driven pathogenesis, in which evidence suggests activation C E D E R107A mutant ( and ) with the Ags C4b and ( and ) C4. Titrations through the CP or LP. For in vivo applications, hC4Nb8 could were performed at 30˚C in a 1.8-ml sample cell and 300-ml total titrant volume. All the experiments were carried out in duplicate. simulate C4 KO conditions for a short period of time, avoiding the development of auto-reactive B cells observed in C4KO mice (65). To improve its circulation time, PEGylation or fusion of the ICs to FDCs in lymph nodes, suggesting that a functional effect could nanobody to an IgG Fc or an albumin binding molecule are all be observed for other effector functions of complement as well. Our established means to increase circulation times to days (reviewed demonstration of in vitro inhibition of complement on neuronal cell in Ref. 66). In addition, fusion of nanobodies with fragments of cultures also opens the possibility of using the nanobody in so- endogenous complement inhibitors or targeting molecules is phisticated cell-based assays modeling complement-mediated syn- easily achieved by genetic engineering and could further increase aptic pruning. tissue specificity and complement inhibition, as demonstrated for the MAP-1:C4BP fusion protein (60). For future, more general Discussion animal studies not involving the hC4 knock-in mouse used in this In contrast to the steadily growing number of inhibitors targeting study, the effect of hC4Nb8 administration should be carefully the AP and the terminal pathway developed with the aim of reg- investigated. In vivo, the protease MASP-2 can cleave C3 in a C4- ulating complement activation as a therapeutic strategy (27, 59), bypass pathway (67). Because the affinity of hC4Nb8 for mC4b is the availability of CP- and LP-specific inhibitors is more limited. still in the subnanomolar range, the weaker inhibition observed in 14 COMPLEMENT INHIBITION WITH A C4b-SPECIFIC NANOBODY

FIGURE 8. hC4Nb8 binds to transgenic mice hC4 ex vivo and blocks the CP in vivo. (A) Immunostaining of spleen sections from a hC4 transgenic mouse. Red: anti-hC4 Ab; green: IgY-hC4Nb8. Scale bar, 40 mm. White arrows indicate examples of colocalization. (B) Hemolytic assay on Ab-coated erythrocytes in hC4 AB/minus mouse serum treated with 10 mg/ml hC4Nb8 (red) or untreated (black) (n = 3). (C) Analysis of all the images in which the FDC binary area colocalized with PE divided by the total FDC binary area was plotted, summary of two independent experiments (****p , 0.0001, ***p , 0.001).

C57BL/6 serum could arise from this unconventional activation (narsoplimab) and the C1s-specific BIVV009 (sutimlimab) (59) pathway or from stronger competition by mouse C2 compared interfere with both C4 and C2 cleavage. Several C1q inhibitors with human C2. The C57BL/6 serum does not contain the C4 (ANX005, ANX007, ANX009) are also in the pipeline for the homolog sex-limited protein (Slp) which has hemolytic activity treatment of neurologic disease (Annexon Biosciences). Systemic (68–70); however, in other mouse strains in which Slp is and permanent therapeutic regulation of the CP C3 convertase expressed, the nanobody may show even less efficiency because in could be problematic because C1q and C4 deficiencies predispose Slp an arginine substitutes for a tryptophan within the hC4Nb8 for systemic lupus erythematosus, and both the CP and LP are epitope (Fig. 4C). important for clearance of pathogens. The drawbacks of C4b in- The CP is known to contribute to pathogenesis in ischemia– hibition compared with inhibition of the upstream C1 complex reperfusion injury, sepsis, autoimmune hemolytic anemia, glo- may be smaller, as C4-independent functions of C1q (80) are merulonephritis, Ab-mediated rejection, Alzheimer disease, SCZ preserved. Direct C3 cleavage by MASP-2 activated through the (27–30), multiple cancer models (71), glaucoma (72), and cold LP can also still occur (81); the AP will still be able to initiate agglutinin disease (31). The LP is known to contribute to patho- through C3 tickover (82), and the activity of the AP convertases genesis in rheumatic heart disease (73), inflammatory arthritis will not be affected. (74), viral infection (75), infection by protozoan microbes (76), Of special interest for future studies resolving the role of pneumococcal infection (77), and renal disease (78), and very complement in neurodegenerative disease, we demonstrated in this recent findings implicate uncontrolled activation of the LP and study in a cell culture model, that hC4Nb8 can prevent C3b de- propagation into the alternative and terminal pathways in patients position from human serum in a dose-dependent manner on neu- infected with COVID-19 (reviewed in Ref. 79). rites to a level close to that observed with C4-deficient serum, in There are currently three mAbs under preclinical development which residual C3b deposition is likely to occur through low level or in clinical trials that, like hC4Nb8, prevent CP and LP C3 AP activity. Recent genome-wide association studies linked SCZ convertase activity: the C2-specific mAb PRO-02 (Prothix) inhib- to a higher copy number and brain mRNA levels of the C4A isotype iting the C3 convertase, whereas the MASP-2–specific OMS721 (25), as well as to a single nucleotide polymorphism in the The Journal of Immunology 15

fluid (87). It is long known that increased levels of the components C1q, C3, and C4 associate with Alzheimer disease (88, 89). Re- cent evidence implies the CP in spinal motor circuit refinement during development and in the pathogenesis of spinal muscular atrophy (90). If the hC4Nb8 can be administrated within the CNS without breaking the blood–brain barrier, therapeutic blockade of the CP C3 convertase may be a promising strategy for treating SCZ and other neurodegenerative diseases. In addition to the perspective of developing hC4Nb8 into a therapeutic agent, it also bears sub- stantial potential for in vitro research involving complement. Because of its specificity and very tight binding, it could be ap- plied for diagnostic purposes to quantitate C4b deposition. In vitro, hC4Nb8 would be a high-affinity C4b and C4c binder in ELISAs and flow cytometry experiments, and introduction of, for example, a hemagglutinin tag could allow direct detection with secondary Abs in immunostaining (91). For in vivo localization, a radioactive tag could be added and followed by positron emission tomography imaging (92). The presence of divalent cations in cell culture assays is necessary for long-term cell viability, replacing EGTA and EDTA with the hC4Nb8 nanobody to specifically block the CP and LP C3 convertase would therefore eliminate cytotoxic effects. In addition, in synaptic pruning models and other cellular models involving Mg2+ and Ca2+-dependent binding of iC3b to CR3, phagocytosis can be evaluated whereas inhibiting the CP C3 convertase with hC4Nb8. In conclusion, the hC4Nb8 nanobody is a C4-specific complement- blocking reagent with potential for very broad applications. The linkage of C4 with several neurologic diseases leads to a rise in interest toward CP therapeutic inhibition targeting the CNS. Our study provides evidence that hC4Nb8 holds promise as a research tool for the study of the molecular mechanisms of disease patho- genesis and as a potential therapeutic inhibitor. Further investigations are needed to understand whether administration of hC4Nb8 could ameliorate CP- and LP-mediated disease pathogenesis in animal models. The cross-reactivity of hC4Nb8 with marmoset C4 opens the possibility of applying the reagent in the study of CNS disease with nonhuman primate models. FIGURE 9. Incubation of hC4Nb8 with 10% NHS inhibits C3 deposi- tion on neurites in a dose-dependent manner. (A) Example image of C3c deposition (green) colocalizing with Tuj1+ neurites (red) in 10% NHS. (B) Acknowledgments Analysis of all wells in the presence of hC4Nb8 or control (Lag16, Ctrl We acknowledge Christine Schar for excellent assistance with SPR and ITC Nb) nanobody in 10% NHS (n = 24 wells per dose) or in C4-Dpl NHS (n = experiments, the beamline staff at the European Molecular Biology Labo- 6 wells per condition) shown with 95% confidence intervals. hC4Nb8 at 5, ratory (Hamburg, Germany) and Karen Margrethe Nielsen for technical 10, and 20 mg/ml doses led to significantly less C3c deposition compared support, and Neal Lojek for the differentiation of neuronal cell cultures. with control (Lag16) nanobody (p , 0.0005, p , 0.0001, p , 0.001). The authors are grateful for the pC427A-K1324N mC4 expression plas- Under C4-Dpl conditions; both hC4Nb8 and control nanobody had sig- mid, a kind gift from David Isenman, Toronto University. nificantly decreased deposition relative to the control condition in 10% NHS (p , 0.0001), further demonstrating that nanobody inhibition is Disclosures specific to the CP and LP. A.Z., S.T., N.S.L., and G.R.A. are listed as inventors on a patent describing the hC4Nb8 nanobody (patent number WO2019238674A1). The other au- CNS-specific complement regulator CSMD1 (83), which possibly thors have no financial conflicts of interest. performs a function analogous to that of CR1 in the periphery (58, 84). In the proposed disease mechanism, either C4A over- expression and/or reduced levels of CSMD1 would lead to in- References 1. Dodds, A. W., and M. Matsushita. 2007. 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