Development by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor This information is current as of September 26, 2021. Xavier Peyrassol, Toon Laeremans, Mieke Gouwy, Vannessa Lahura, Maja Debulpaep, Jo Van Damme, Jan Steyaert, Marc Parmentier and Ingrid Langer J Immunol published online 10 February 2016 http://www.jimmunol.org/content/early/2016/02/09/jimmun Downloaded from ol.1500888 http://www.jimmunol.org/ Why The JI? Submit online.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 10, 2016, doi:10.4049/jimmunol.1500888 The Journal of Immunology

Development by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor

Xavier Peyrassol,* Toon Laeremans,†,‡ Mieke Gouwy,x Vannessa Lahura,* Maja Debulpaep,†,‡ Jo Van Damme,x Jan Steyaert,†,‡ Marc Parmentier,*,{ and Ingrid Langer*

The generation of Abs that recognize the native conformation of G –coupled receptors can be a challenging task because, like most multimembrane-spanning , they are extremely difficult to purify as native protein. By combining genetic immunization, phage display, and biopanning, we identified two functional monovalent Abs (nanobodies) targeting ChemR23. The two nanobodies (CA4910 and CA5183) were highly specific for the human receptor and bind ChemR23 with moderate Downloaded from affinity. Binding studies also showed that they share a common binding site that overlaps with that of , the natural ligand of ChemR23. Consistent with these results, we found that the nanobodies were able to antagonize chemerin-induced intracellular calcium increase. The inhibition was partial when chemerin was used as agonist and complete when the chemerin (149-157) nonapeptide was used as agonist. Engineering of a bivalent CA4910 nanobody resulted in a relatively modest increase in affinity but a marked enhancement of efficacy as an antagonist of chemerin induced intracellular calcium mobilization and a much higher potency against the chemerin(149–157) nonapeptide-induced response. We also demonstrated that the fluorescently labeled http://www.jimmunol.org/ nanobodies detect ChemR23 on the surface of human primary cell populations as efficiently as a reference mouse mAb and that the bivalent CA4910 nanobody behaves as an efficient antagonist of chemerin-induced of human primary cells. Thus, these nanobodies constitute new tools to study the role of the chemerin/ChemR23 system in physiological and pathological conditions. The Journal of Immunology, 2016, 196: 000–000.

he G protein–coupled receptor (GPCR) ChemR23 is Chemerin, a 18-kDa protein, was first identified as the natural structurally related to receptors for chemoattractant mol- ligand of ChemR23 (11). The structure of chemerin is unrelated to

T ecules, such as formyl peptides (FPR1, FPR2, and FPR3), that of or other chemoattractant factors for leukocytes by guest on September 26, 2021 complement fragments (C5a and C3a), and PG D2 receptors, as (12). It is predicted to share a “cystatin fold” with a set of ex- well as to GPR1 and the orphan receptors GPR32 and GPR33 (1). tracellular proteins, which includes type 2 cystatins, cathelicidin ChemR23 is expressed by various leukocyte populations, includ- precursors, and kininogen. Chemerin is secreted by most tissues ing monocytes, , myeloid and plasmacytoid dendritic (with the notable exception of leukocytes) as an inactive precur- cells, NK cells, and microglial cells (2–4). ChemR23 expression sor, prochemerin, which is 143 aa long in humans. This precursor was also described in nonleukocyte cell populations, including is processed by proteolytic enzymes within its C terminus, gen- preadipocytes and (5, 6), osteoclasts (7), chondrocytes erating bioactive chemerin in inflammatory conditions (13, 14). (8), skeletal muscle cells (9), and endothelial cells (10). As expected from the ChemR23 expression profile, chemerin promotes chemotaxis of monocytes, macrophages, dendritic cells, and NK cells (3, 4). As a chemotactic factor generated in in- *Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Uni- flammatory conditions and acting on cells involved in innate im- versite´ Libre de Bruxelles, B-1070 Brussels, Belgium; †Structural Biology Brussels, Vrije Universiteit Brussels, B-1050 Brussels, Belgium; ‡Structural Biology Research mune responses, chemerin was initially expected to behave as a Center, Vlaams Instituut voor Biotechnologie, B-1050 Brussels, Belgium; xLabora- proinflammatory agent. However, recent data point toward more tory of Molecular Immunology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven - University of Leuven, B-3000 Leuven, complex activities that are pro- or anti-inflammatory, according to Belgium; and {Welbio, Universite´ Libre de Bruxelles, B-1070 Brussels, Belgium the disease model investigated (8, 15, 16). In addition to its role as Received for publication April 15, 2015. Accepted for publication January 5, 2016. a chemoattractant agent, chemerin was reported to be an adipo- This work was supported by the 3D4Health Program (Brussels region), Welbio, the kine regulating adipogenesis and metabolism (17). Interuniversity Attraction Poles Programme (P7/40) - Belgian State - Belgian Science Besides chemerin, E1 (RvE1) was proposed as a ligand Policy, the Actions de Recherche Concerte´es of the Communaute´ Franc¸aise de Bel- gique, the Fonds National de la Recherche Scientifique of Belgium, and the Fe´de´r- for ChemR23 (18). RvE1 is an oxygenated product of eicosa- ation Belge contre le Cancer. pentanoic acid and is believed to mediate the beneficial effects of Address correspondence and reprint requests to Prof. Ingrid Langer, Institut de Re- this essential fatty acid in a number of inflammation-associated cherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Universite´ Libre de human disorders. However, the RvE1/ChemR23 connection is Bruxelles, 808 Route de Lennik CP602, B-1070 Brussels, Belgium. E-mail address: [email protected] highly controversial, and our laboratory has been unable to con- Abbreviations used in this article: ChemR23-EGFP, ChemR23 fused at its C terminus firm it (15, 19). end to EGFP; EGFP, enhanced GFP; GPCR, G protein–coupled receptor; MCF, Although ChemR23 is the only receptor unambiguously medi- median cell fluorescence; MDDC, monocyte-derived ; RvE1, resolvin ating the chemotactic activities of chemerin, two other GPCRs E1; V H, N-terminal variable domain; VLP, virus-like particle; WT, wild-type. H were described to bind chemerin with high affinity: GPR1 and Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 CCRL2 (20, 21). GPR1 is not expressed in leukocyte populations

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500888 2 ANTAGONIST NANOBODIES TARGETING ChemR23 but is found in the CNS, skeletal muscle, skin, and culture medium supplemented with 500 mg/ml G418 (Invitrogen), indi- (20). In response to chemerin exposure, GPR1 is a poor activator vidual colonies were expanded as described above, and clones expressing of classical G protein–mediated pathways but is internalized ef- human ChemR23 were selected by FACS using an anti-human ChemR23 mouse mAb (eBioscience; 1/50). ficiently. Therefore, GPR1 might behave as a decoy receptor for chemerin. CCRL2 belongs to the receptor family and Human leukocyte populations is closely related to CCR1, CCR2, CCR3, and CCR5. In humans, Human CD14+ monocytes were isolated from 1-d-old buffy coats derived CCRL2 is expressed in monocytes, macrophages, dendritic cells, from healthy donors (Blood Transfusion Center, Mechelen, Belgium), neutrophils, T cells, NK cells, mast cells, and CD34+ bone marrow as described by Gouwy et al. (29). Human immature monocyte-derived precursors (22). Chemerin binding to this receptor does not seem dendritic cells (MDDCs) were generated by incubating purified peripheral blood CD14+ monocytes (1 3 106 cells) in plastic culture dishes (55 cm2; to promote any signaling or internalization response. Therefore, it International Medical Products) in 10 ml RPMI 1640 medium containing was suggested that CCRL2 might increase local concentrations 10% FCS (Life Technologies Europe, Gent, Belgium), 50 ng/ml GM-CSF of chemerin and present the protein to other surrounding cells (Miltenyi Biotec, Bergisch Gladbach, Germany), and 20 ng/ml IL-4 expressing ChemR23 (21, 23). (PeproTech, London, U.K.) at 37˚C in humidified air with CO2 (5%) for In addition to conventional Abs, Camelidae produce a special 6 d. Macrophages were differentiated from monocytes in the presence of recombinant human M-CSF (50 ng/ml; PeproTech) for 6–11 d. The purity type of Abs devoid of L chains that are called “H chain–only Abs” of the cell preparations was evaluated by flow cytometry, and .85% of (24). Ags are bound through the 15-kDa N-terminal variable do- CD11b+/CD206+ macrophages were consistently obtained. main (V H) of the H chain, which is frequently referred to as a H Genetic vaccination and immune repertoire cloning nanobody. For several years, nanobodies have generated a growing interest as diagnostic and therapeutic tools because of their po- Camelid single-domain Ab fragments (VHHs, nanobodies) specific for Downloaded from tential advantages over conventional Abs (25, 26). Nanobodies human ChemR23 were identified from Ig repertoires of genetically immunized llamas following a prime-boost regimen (30). The human were reported to display low immunogenicity, high solubility, ChemR23 coding sequence (GenBank accession number: Y14838.1 http:// superior stability, and greater tissue penetration. Their smaller www.ncbi.nlm.nih.gov/genbank) was cloned into the mammalian expres- paratope gives them access to cavities on the surface of proteins, sion vector pVAX1 (Invitrogen), and endotoxin-free plasmid DNA was epitopes not usually recognized by conventional Abs or engi- prepared using the EndoFree Giga (QIAGEN), according to the man- ufacturer’s instructions. Four llamas (Llama glama) were immunized by neered Fab and scFv fragments. Finally, they are easier and http://www.jimmunol.org/ intradermal administration of plasmid DNA (1–2 mg) at days 0, 17, 28, 42, cheaper to produce in microbial-expressing hosts. and 74 using a DERMOJET device (AKRA DERMOJET, Pau, France). In the current study, we used genetic vaccination and phage Twenty-three days after the last DNA immunization, 107 Dubca cells display to generate nanobodies recognizing human ChemR23. overexpressing ChemR23 were injected s.c. ChemR23-specific serum These nanobodies were characterized as antagonists of this re- conversion was monitored via flow cytometry during immunization (31). One week after the fourth DNA vaccination and 4 and 8 d after the cell ceptor and did not recognize murine ChemR23 or related human boost, 100-ml blood samples were collected from each llama, and PBLs GPCRs. They bind ChemR23 with an affinity in the range of were prepared by Ficoll-Paque Plus density gradient centrifugation, 100 nM, and their binding site overlaps with that of chemerin-9, a according to the manufacturer’s instructions (LeucoSep tubes; Greiner peptide corresponding to the C terminus of bioactive chemerin. As Bio-One). Total RNA was extracted from the PBLs, as described by

2 by guest on September 26, 2021 a bivalent construct, one of the nanobodies inhibited the activity of Chomczynski and Sacchi (32), and stored as ethanol precipitate at 80˚C. First-strand cDNA synthesis was performed using hexanucleotide random such peptide, with an IC50 as low as 1.7 nM. Following fluorescent priming and the SuperScript III First-Strand Synthesis System (Invitrogen), labeling, they detected human ChemR23 in FACS and immuno- according to the manufacturer’s instructions. The nanobody repertoire was fluorescence staining. Thus, these nanobodies are innovative tools amplified and cloned in pXAP100, with minor modifications of the method to study the distribution and role of ChemR23. described (33). pXAP100 is a pMESy4 derivative allowing expression of SfiI/BstEII cloned C-terminally His6-cMyc–tagged nanobody. For the nested PCR, the 59 primer was adapted to contain the SfiI restriction site. Materials and Methods Identification of nanobodies recognizing native extracellular Cell lines ChemR23 epitopes CHO-WTA11 cells, coexpressing apoaequorin and Ga16, were cultured in Ham’s F12 medium supplemented with 10% FBS, 100 U/ml penicillin, Nanobody-displaying bacteriophage particles were produced according to 100 mg/ml streptomycin, and 250 mg/ml Zeocin (Invitrogen, Groningen, standard protocols following helper phage superinfection (33). ChemR23- specific phages were enriched by a first round of biopanning using solid- The Netherlands) at 37˚C with a constant supply of 5% CO2 and split every 3 d. The cell lines expressing wild-type (WT) human or mouse ChemR23 phase immobilized virus-like particles (VLPs) harboring human ChemR23 were described previously (11, 27). To obtain the cell line expressing (custom made by Icosagen, Ossu, Estonia). After thorough washing to human ChemR23 fused at its C terminus with enhanced GFP (ChemR23- remove nonspecific binders, phages were eluted via trypsin treatment and Escherichia coli EGFP), the EGFP coding sequence was inserted in frame at the 39 end amplified by infection of logarithmically growing TG1. of ChemR23 cDNA in a homemade bicistronic vector (pCDneo). The In a second selection round, phage particles were incubated with CHO resulting plasmid (2 mg) was transfected into CHO-WTA11 cells using cells expressing ChemR23 or VLPs. Subsequently, individual clones were FuGENE transfection reagent (Roche), according to the manufacturer’s picked, and periplasmic extracts were prepared as described (33). The specificity of individual nanobodies was assessed by flow cytometry, instructions. Selection of stably transfected cells was carried out in culture medium supplemented with 250 mg/ml G418 (Invitrogen). After 10– staining ChemR23-overexpressing CHO cells and WT CHO cells. Unfixed, 15 d of selection, individual colonies were transferred to 24-well plates, unpermeabilized cells were incubated with 5-fold diluted periplasmic ex- grown to confluence, trypsinized, and expanded further in 6-well plates. tracts containing the nanobody in FACS buffer (PBS, 10% FBS, 0.1% The identification of cell clones expressing the ChemR23-EGFP fusion sodium azide) for 60 min at 4˚C. After washing in FACS buffer, the bound was performed by measuring EGFP emission using FACS. A similar nanobodies were detected with a FITC-conjugated anti-His Tag Ab (AbD protocol was used to obtain the cell lines expressing human GPR1 or Serotec; 1/50), and the median cell fluorescence (MCF) intensity was human CCRL2, cloned in pEFin3 (Euroscreen). The identification of cell determined. The data were processed using FACSDiva software (Becton clones expressing GPR1 or CCRL2 was performed by FACS using mouse Dickinson). mAb anti-GPR1 (kindly provided by Euroscreen; 1/20) or anti-CCRL2 Production and purification of anti-ChemR23 nanobodies (R&D Systems; 1/50). Dubca cells (28) were cultured in RPMI 1640 medium supplemented The two nanobodies, CA4910 and CA5183, were expressed in E. coli with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37˚C WK6Su2 and purified by metal-affinity chromatography. Briefly, bacteria with a constant supply of 5% CO2 and split every 3 d. Dubca cells were grown in Terrific Broth supplemented with 100 mg/ml ampicillin, expressing human ChemR23 were obtained following electroporation 0.1% glucose, and 2 mM MgCl2 to an OD600 of 0.6–0.9. The expres- of 20 mg the pCDneo-ChemR23 plasmid. After 10–15 d of selection in sion of nanobodies was induced by adding 1 mM isopropyl b-D-1- The Journal of Immunology 3 thiogalactopyranoside, and bacteria were grown overnight at 30˚C. The 400,000 cells in a final volume of 100 ml and 0.2 nM radioiodinated bacteria were centrifuged at 7000 3 g for 10 min, and the pellets were [Y145,F149]-chemerin(145–157) (Phoenix Pharmaceuticals) as tracer. Total resuspended in 15 ml TES buffer (0.2 M Tris-HCl [pH 8], 0.5 M sucrose, binding of the tracer was measured in the absence of competitor, and 0.5 M EDTA) and incubated for 1 h at 4˚C under mild agitation. A total of nonspecific radioligand binding was measured in the presence of 300 nM 30 ml 4-fold diluted TES buffer was added, and the samples were incu- unlabeled peptide. For competition-binding assays, samples were incu- bated for 45 min at 4˚C under mild agitation. The samples were centri- bated for 50 min at 27˚C with increasing amounts of nanobodies. Bound fuged at 8000 3 g for 30 min at 4˚C, and the supernatants containing and free radioligand were separated by filtration through GF/B filters nanobodies were collected for purification on Ni-NTA resin (QIAGEN) presoaked for 16 h at 4˚C in 1% BSA. Filters were washed three times with (33). Binding of nanobodies on Ni-NTA resin was performed at room ice-cold binding buffer supplemented with 500 mM NaCl. IC50 values temperature for 1 h, the columns were washed with 50 mM phosphate were calculated using nonlinear regression (Prism software; GraphPad). buffer (pH 6), 1 M NaCl, and nanobodies were eluted with 1 M NaCl in 50 mM sodium acetate buffer (pH 4.5). The protein solution was neutralized Aequorin-based calcium-mobilization assay by adding 1 M Tris-HCl buffer (pH 7.5). Nanobody purity was verified by Calcium mobilization was measured in CHO cells expressing ChemR23 by SDS-PAGE. an assay based on the luminescence of mitochondrial aequorin, as previ- ously described (27). Briefly, cells were collected from plates with 5 mM Assessing nanobody interaction with extracellular epitopes of EDTA in PBS, pelleted, resuspended at a density of 5.106 cells/ml in cell surface–expressed ChemR23 DMEM/Ham’s F12 (Invitrogen) supplemented with 0.1% BSA, and in- m CHO cells were detached from culture dishes using PBS containing 5 mM cubated with 5 M coelenterazine H (Promega) for 4 h at room temper- ature under gentle agitation in the dark. Cells were then diluted to a density EDTA, harvested by centrifugation (560 3 g, 4˚C, 3 min), and resuspended 6 6 of 10 cells/ml and incubated for an additional hour. For the agonist assay, at a density of 10 cells/ml in cold FACS buffer (PBS, 0.1% BSA, 0.1% 50 ml cell suspension was added to microplate wells containing agonists sodium azide). One hundred microliters of the cell suspension was incu- diluted in a volume of 50 ml DMEM-F12. Calcium increase was evaluated bated with anti-receptor Ab. The Abs used were mouse mAbs anti-human Downloaded from by measuring, for 30 s, the luminescent signal (integration of area under ChemR23 (eBioscience; 1/50), anti-CCRL2 (R&D Systems; 1/50), anti- the curve) resulting from the activation of the aequorin-coelenterazine GPR1 (Euroscreen; 1/20), or anti-mouse ChemR23 (eBioscience; 1/50) or complex using a Centro LB 960 luminometer (Berthold Technologies). nanobodies (300 nM). After 1 h of incubation at 4˚C, cells were washed The data were normalized for basal (0%, background removal) and max- with 2 ml cold FACS buffer and incubated for 30 min at 4˚C in the dark imal (100%) luminescence, corresponding to the signal measured fol- with an FITC-conjugated anti-mouse IgG (Sigma; 1/100) for mAbs or with lowing exposure to 20 mM ATP. For the antagonist assay, 25 ml cell an FITC-conjugated anti-His Tag Ab (AbD Serotec; 1/50) for nanobodies. suspension was added to 25 ml increasing concentrations of nanobodies The cells were washed and resuspended in FACS buffer, and the fluores- and incubated for 45 min at room temperature and then 50 ml chemerin http://www.jimmunol.org/ cence level was analyzed using an LSRFortessa flow cytometer (Becton (1 nM) or chemerin(149–157) (5 nM) solution was added, and the lumi- Dickinson). The data were processed using FACSDiva software (Becton , nescent signal (integration of area under the curve) was recorded for 30 s Dickinson). Dead cells (forward scatter 25) were excluded from the in a Centro LB 960 luminometer (Berthold Technologies). The data were analysis, and nonspecific fluorescence was determined using untransfected normalized for basal (0%, background removal) and maximal (100%) lu- CHO cells. minescence, corresponding to the signal measured following exposure to Immunofluorescence staining 1 nM chemerin or 5 nM chemerin(149–157). Dose-response curves and IC50 values were calculated using nonlinear regression (GraphPad Prism CHO cells were cultured on 22-mm glass coverslips for 48 h and washed in software). PBS. Nonspecific binding was prevented by a 30-min incubation at 4˚C in PBS containing 10% FBS (FBS/PBS). The cells were then incubated for Chemotaxis assays m by guest on September 26, 2021 1 h at 4˚C with 1 M nanobodies diluted in FBS/PBS. After two washes The chemotactic potency of chemerin and CCL3 on immature MDDCs in with FBS/PBS, the cells were incubated for 30 min at 4˚C with a DyLight the presence or absence of nanobodies was determined in the Boyden 549–conjugated anti-His Tag Ab (AbD Serotec) diluted (1/500) in FBS/ microchamber assay (Neuro Probe, Cabin John, MD). Suspended immature PBS. After three rinses in FBS/PBS, cells were fixed for 15 min with 4% MDDCs were collected from the dishes by pipetting up and down. Then the paraformaldehyde solution at room temperature and washed three times dishes were incubated with PBS for 10 min at 4˚C, and the adhered im- with PBS and once with water. Glass slides were mounted with ProLong mature MDDCs were detached by scraping. The immature MDDCs were DAPI mounting medium (Invitrogen) before viewing under a fluorescent pelleted by gentle centrifugation and resuspended in chemotaxis buffer microscope (Axioplan 2; Zeiss). (i.e., HBSS [Invitrogen] supplemented with 1 mg/ml human serum albu- min [Belgian Red Cross, Leuven, Belgium]). Chemerin or CCL3 (posi- Nanobody-binding assays tive control) was added to the lower compartment of the microchamber Purified CA4910 and CA5183 nanobodies were fluorescently labeled with and separated from the upper compartment by a 5-mmporesize DyLight 650 according to manufacturer’s instructions (Microscale Ab polyvinylpyrrolidone-treated polycarbonate membrane (GE Water & Pro- Labeling Kit; Pierce). Binding experiments were performed using DyLight cess Technologies, Manchester, U.K.). The wells in the upper compartment 6 650-labeled nanobody as tracer. For saturation experiments, 100,000 WT of the Boyden chamber were filled with 50 ml cell suspension (1 3 10 or ChemR23-overexpressing CHO cells were incubated for 1 h at 4˚C with cells/ml) in the presence or absence of nanobodies (500 nM). After in- increasing concentrations of labeled nanobodies (0.1–3000 nM) in 100 ml cubation at 37˚C for 1.5 h in humidified air with 5% CO2, the filters were cold FACS buffer. After addition of 2 ml cold FACS buffer, the cells were removed and stained, and the cells migrated across the filter were counted 3 harvested by centrifugation (560 3 g, 4˚C, 4 min) and resuspended in cold using a microscope ( 500 magnification). The chemotactic response displayed in the figures was calculated as the number of migrated cells to FACS buffer, and fluorescence was evaluated by FACS. Kd and Bmax values were calculated by nonlinear regression (Prism software; GraphPad) us- chemerin (minus cells that migrated spontaneously to the chemotaxis ing as total and nonspecific binding the MCF obtained with ChemR23- buffer)/number of migrated cells to CCL3 (minus cells that migrated 6 expressing and WT CHO cells, respectively. For competition-binding ex- spontaneously to the chemotaxis buffer). Data are shown as mean SEM. periments, 100,000 ChemR23-expressing CHO cells were incubated for 1 h at 4˚C in 100 ml cold FACS buffer containing 10 nM DyLight 650– Results labeled CA4910 or CA5183 and various concentrations of competitors Generation of nanobodies that interact with extracellular [unlabeled nanobodies, chemerin (R&D Systems) or chemerin(149–157) nonapeptide]. Following a washing step with 2 ml cold FACS buffer, the epitopes of native ChemR23 fluorescence of cells was evaluated by FACS. IC50 values were calculated To generate nanobodies directed against the extracellular domain by nonlinear regression using as nonspecific binding the MCF obtained in of human ChemR23, four llamas were immunized by genetic vac- the presence of 3 mM unlabeled nanobody. cination following a prime-boost strategy. Immune responses were Chemerin competition-binding assays using [125I]-[Y145,F149]- primed with five shots of 1–2 mg endotoxin-free pVAX1 plas- chemerin(145–157) mid DNA encoding human ChemR23. For the single-cell boost, ChemR23-expressing CHO cells were detached from the plates by 5 mM ChemR23-overexpressing camelid cells (Dubca) were chosen as EDTA in PBS and resuspended in binding buffer (50 mM HEPES [pH 7.4], cell background to bias the response toward ChemR23 and to 0.5% BSA, 5 mM MgCl2, 1 mM CaCl2). Binding was performed using avoid immunization against other cell surface markers. Mounting 4 ANTAGONIST NANOBODIES TARGETING ChemR23 of the ChemR23-specific immune response was monitored by bind to CHO cells expressing human ChemR23, but they do not testing the sera of animals, using FACS, on CHO cells expressing recognize mouse ChemR23 or human GPR1 or CCRL2. the receptor. Only one of the llamas (named 2T) showed a robust The ability of purified nanobodies to label ChemR23 by im- ChemR23-specific seroconversion. After DNA vaccination and munofluorescence was evaluated on nonpermeabilized CHO cells after the cell boost, PBLs were collected from this animal, RNA expressing an engineered ChemR23 receptor fused to EGFP at was prepared, and a phage library displaying the VHH repertoire its C terminus. Cells expressing the ChemR23-EGFP fusion and was constructed (33). A first round of panning was performed on emitting green fluorescence were also labeled with the two nano- ChemR23-expressing VLPs, and second rounds of panning were bodies detected by DyLight 549–conjugated anti-His mAb (Fig. 2). made on ChemR23-expressing CHO cells or VLPs, using WT Although EGFP fluorescence allowed detection of the receptors CHO cells or irrelevant VLPs as negative controls. After two on the plasma membrane and in intracellular compartments, rounds of panning, clear enrichment of ChemR23-specific phages nanobodies only labeled cell surface receptors. Control experi- was detected. Twenty-four clones resulting from two rounds of ments performed with cells expressing unrelated receptors (Fig. 2) panning on ChemR23-expressing CHO cells and 48 clones se- or WT CHO cells, as well as incubation with irrelevant nano- lected by one round of panning on ChemR23-expressing CHO bodies or with a secondary Ab only, did not show any signal above cells followed by one round on VLPs were analyzed further. background fluorescence (data not shown). Accordingly, from each clone, periplasmic extract was prepared to partially purify the encoded nanobody. Binding of these nano- Binding of CA4910 and CA5183 to ChemR23 bodiestoChemR23wasassessedbyflowcytometryon To estimate the affinity of CA4910 and CA5183 for ChemR23, ChemR23-overexpressing CHO cells, using WT CHO cells as the Downloaded from the two nanobodies were labeled with DyLight 650 fluoro- negative control. Nanobodies resulting in MCF $ 5overWT chrome. Saturation-binding experiments were performed via CHO cells were sequenced. Two nanobodies were identified flow cytometry using DyLight 650–labeled CA4910 (DL650- (CA4910 and CA5183) to bind ChemR23, sharing a highly similar CA4910) or CA5183 (DL650-CA5183) as tracers. K values CDR3 sequence with few amino acid differences. Following the d were calculated from saturation curves from MCF of CHO cells, expression and purification, by affinity chromatography, on Ni- expressing (total binding) or not expressing (nonspecific binding)

NTA, the specificity of the two nanobodies was reconfirmed by http://www.jimmunol.org/ ChemR23, following incubation with increasing concentrations of FACS. As shown in Fig. 1, CA4910 and CA5183 nanobodies the tracer. DL650-CA4910 and DL650-CA5183 bound ChemR23 with Kd values of 130 6 30 and 160 6 10 nM, respectively (Fig. 3). We next evaluated whether the two nanobodies recognize similar epitopes by performing competition-binding experiments using DL650-CA4910 and DL650-CA5183 as tracers. We found that CA4910 and CA5183 displaced DL650-CA4910 binding with IC50 values of 127 6 4 and 187 6 6 nM, respectively.

Similarly, CA4910 and CA5183 displaced DL650-CA5183 by guest on September 26, 2021 binding with IC50 values of 150 6 10 and 190 6 9 nM, re- spectively (Fig. 4). These data show that CA4910 and CA5183 share a common epitope, in agreement with the sequence simi- larity of these two nanobodies. The IC50 values obtained from competition experiments were similar to the Kd values derived from saturation-binding curves, suggesting that labeling of nanobodies with DL650 did not affect their interaction with ChemR23.

CA4910 and CA5183 inhibit chemerin binding We further tested whether the two nanobodies were able to affect the binding of chemerin, the natural ligand of ChemR23, on CHO cells expressing the human receptor. In a first set of experiments, we used the same competition-binding assays as above, with DL650-CA4910 and DL650-CA5183 as tracers. We found that chemerin displaced DL650-CA4910 binding with an IC50 value of 100 6 20 nM, and it displaced DL650-CA5183 binding with an IC50 value of 66 6 6 nM (Fig. 4). These results suggest that CA4910 and CA5183 share a common or overlapping binding site with chemerin. Therefore, we performed additional competition-binding experiments using the iodinated peptide [125I]-[Y145,F149]-chemerin(145–157) as tracer, which is the sole high-affinity radioiodinated tracer commercially available FIGURE 1. Assessment of nanobody specificity. The specificity of for ChemR23. It is derived from a bioactive C-terminal peptide nanobodies was investigated by FACS using WT CHO cells (dashed lines) of chemerin (13). In agreement with previous experiments, we and CHO cells expressing human and mouse ChemR23 or the related found that CA4910, CA5183, and chemerin(149–157) com- human receptors GPR1 and CCRL2 (filled graphs). Cells were incubated 125 145 149 with a reference mouse mAb for each receptor (left panels) and the pletely displaced [[ I]]-[Y ,F ]-chemerin(145–157) bind- 6 6 6 nanobodies CA4910 (middle panels) and CA5183 (right panels). Bound ing with IC50 values of 40 6, 21 4, and 23 6nM, Abs were detected with FITC-conjugated secondary Abs. The data are respectively (Fig. 5). An irrelevant nanobody was also tested, representative of three independent experiments. but it was unable to compete for tracer binding (data not shown). The Journal of Immunology 5

FIGURE 2. Detection of ChemR23 by im- munofluorescence. CHO cells expressing a ChemR23-EGFP fusion were incubated with CA4910 (top panels) or CA5183 (middle pan- els). Nuclei of the cells were labeled with DAPI (blue), and receptors were visualized with DyLight 549–conjugated anti-His Tag (red) or emission of EGFP (green) on an Axioplan 2 microscope (original magnification 340). In- cubation of CHO cells expressing an unrelated GPCR (VPAC1) fused to EGFP with CA4910 (bottom panels) gave no signal. Downloaded from http://www.jimmunol.org/ CA4910 and CA5183 behave as antagonists of ChemR23 20 nM). Similarly, we found that the bivalent nanobody com- 125 145 149 Cell-based signaling assays were performed to evaluate whether the pletely displaced [[ I]]-[Y ,F ]-chemerin(145–157) bind- two nanobodies were able to modulate the activity of ChemR23. ing in a competition-binding assay, with increased potency 6 Using a calcium-mobilization assay based on aequorin lumines- (IC50 =18 4 nM) compared with the monovalent nanobody. cence performed on CHO cells expressing ChemR23, the nano- Interestingly, we found that bivalent CA4910 almost completely bodies were unable to activate the receptor (data not shown). inhibited chemerin-induced intracellular calcium release with 6 However, both nanobodies inhibited chemerin- or chemerin(149– an IC50 of 17 2 nM, whereas monovalent CA4910 be- 157)–induced intracellular calcium release (Fig. 6). However, in- haved as a partial antagonist (Fig. 6). Moreover, the bivalent hibition of signaling was partial when full-size chemerin was used nanobody was 30-fold more potent at inhibiting intracellu- by guest on September 26, 2021 lar calcium release promoted by the chemerin(149–157) non- as the agonist, with IC50 values of 60 6 10 nM for CA4910 and 35 6 3 nM for CA5183. The nanobodies behaved as full antag- apeptide, displaying an IC50 value in the low nanomolar range 6 onists when the nonapeptide chemerin(149–157) was used as (1.7 0.3 nM, Fig. 6). 6 6 agonist, with IC50 values of 17 2 nM for CA4910 and 11 4nM Fluorescently labeled nanobodies recognize ChemR23 on for CA5183. An irrelevant nanobody did not modulate the activity human primary cells as efficiently as a reference mouse mAb of ChemR23 (data not shown). The ability of nanobodies to detect ChemR23 on human primary Bivalent CA4910 displays enhanced antagonist activity cells was tested. Monocytes were collected from buffy coats of Because previous studies showed that generation of bi- or healthy donors and derived into immature dendritic cells or mac- multivalent nanobodies resulted in increased affinity, we engi- rophages using standard culture conditions. Expression of neered a bivalent CA4910 construct. For that purpose, a second ChemR23 was monitored by FACS, using a commercial mouse CA4910 coding sequence, preceded by a GGGGSGGGG linker, mAb as reference, as well as DyLight 650–conjugated CA4910 was inserted in-frame at the 39 end of CA4910 cDNA in a (monovalent or bivalent) and CA5183 nanobodies. As shown derivative pXAP100 vector in which the sequence encoding in Fig. 7A, labeling of ChemR23 on the surface of human III was deleted. In saturation-binding experiments, this monocyte-derived macrophages or dendritic cells was as efficient bivalent nanobody displayed a moderate increase in affinity (Kd = with the fluorescently labeled nanobodies as with the reference 95 6 4 nM) compared with monovalent CA4910 (Kd =1506 mouse mAb.

FIGURE 3. Saturation-binding experiments. CHO cells expressing (total binding) or not expressing (nonspecific binding) ChemR23 were incubated with increasing concen- trations of DyLight 650–conjugated CA4910 (left panel)or CA5183 (right panel). MCF was evaluated by FACS, and binding parameters were calculated from saturation curves. Results are the mean 6 SEM of three independent experi- ments performed in duplicate. 6 ANTAGONIST NANOBODIES TARGETING ChemR23

FIGURE 4. Competitive binding of nanobodies. Binding of DyLight 650– conjugated CA4910 (upper panels) and CA5183 (lower panels, 10 nM) on CHO cells expressing ChemR23 was tested in the presence of increasing concentra- tions of CA4910 (left panels), CA5183 (middle panels), or chemerin (right panels). Results are the mean 6 SEM of three independent experiments per- formed in duplicate. Downloaded from

Monovalent and bivalent CA4910 inhibit chemerin-induced phage display, and biopanning on cells and VLPs, we identified chemotaxis of human immature dendritic cells nanobodies targeting ChemR23 and modulating its function. In- The chemotactic potency of chemerin on human immature MDDCs deed, we developed two nanobodies that specifically recognize http://www.jimmunol.org/ was evaluated in the presence or absence of nanobodies. In the human ChemR23, but not human GPR1 and CCRL2, the two absence of nanobodies, chemerin tested at 30, 100, and 300 ng/ml other chemerin receptors. They bind ChemR23 with moderate concentrations promoted a chemotactic response. The maximal affinity and share a common binding site, because they were both chemotactic response was obtained at different chemerin con- able to interfere with the binding of the other. This is consistent centrations, depending on the donor. Therefore, the responses to the with the fact that the sequences of the two nanobodies are closely three chemerin concentrations were averaged and normalized related, because they vary at only a few amino acid positions. We according to the migration obtained for the reference chemokine, also found that chemerin, the natural ligand of ChemR23, com- CCL3, used at 3 ng/ml (100%), after deduction of background pletely inhibited the binding of the nanobodies. However, the migration toward control medium. The overall relative migration measured IC50 was relatively high compared with the established by guest on September 26, 2021 response of chemerin was 12 6 2% (Fig. 7B). In the presence of Kd of chemerin for ChemR23, suggesting that the binding sites of 500 nM of monovalent or bivalent CA4910 nanobodies, the rel- chemerin and the nanobodies overlap but are not identical. Sim- ative migration response toward chemerin was reduced to 6 6 1% ilarly, the nanobodies completely inhibited the binding of a pep- (p = 0.004) and 2 6 1% (p , 0.001), respectively. Addition of 500 tide tracer derived from the chemerin C terminus. Consistent with nM of an irrelevant nanobody had no significant effect. Similarly, these binding data results, we found that the nanobodies were none of the nanobodies tested significantly modified the CCL3- able to antagonize chemerin- and chemerin(149–157)-induced induced chemotaxis of immature MDDCs (mean, 87 6 9%). intracellular calcium release. However, the inhibition was partial when chemerin was used as agonist and complete when the Discussion chemerin(149–157) nonapeptide was used as agonist. Altogether, Over the past years, nanobodies have generated a growing interest these results show that the two nanobodies share a common as new diagnostic and therapeutic tools because of their potential binding site that overlaps with that of chemerin and strongly advantages over conventional Abs (25, 26). Multimembrane- suggest that the common epitope recognized by the nanobodies spanning proteins, such as GPCRs and ion channels, are extremely is close to the binding pocket recognized by the C terminus of difficult to purify as native proteins. Consequently, the generation chemerin, which likely involves the extracellular part of the of good Abs that recognize the native conformation can be chal- transmembrane helix bundle of ChemR23. Indeed, although lenging. Following a procedure combining genetic vaccination, chemerin is structurally different from chemokines, it was sug-

FIGURE 5. Chemerin-9 competition-binding experiments. The ability of nanobodies to compete with the binding of a radioiodinated peptide tracer was assessed. Competition binding was performed on CHO cells expressing ChemR23, using [125I]-[Y145,F149]-chemerin(145–157) as tracer and increasing concentrations of CA4910 (left panel), CA5183 (middle panel), or chemerin(149–157) (right panel) as competitors. Results are the mean 6 SEM of three independent experiments performed in duplicate. The Journal of Immunology 7

nanobody displayed only a slight increase in affinity in a saturation- binding assay, but its efficacy as an antagonist of chemerin-induced intracellular calcium mobilization was enhanced significantly, probably resulting from increased nanobody size and steric hin- drance. The bivalent nanobody also displayed a much higher po- tency (30-fold) as antagonist when the chemerin(149–157) nonapeptide was used as agonist, reaching low nanomolar IC50 values. Similarly, the bivalent CA4910 nanobody inhibited chemerin-induced chemotaxis of human immature MDDCs more efficiently. The known ability of nanobodies to bind to cavities, in contrast to classical Abs, also fits with the expected interaction of the C-terminal nonapeptide with the transmembrane helix bundle of ChemR23. Therefore, the nanobodies generated in this study might constitute interesting tools to further investigate the two- step binding model of chemerin to its receptor. Although the C-terminal peptides of human and mouse chemerin cross-react fully with human and mouse ChemR23, the nanobodies do not

recognize the mouse receptor efficiently. Therefore, the binding Downloaded from site of nanobodies and of the chemerin C terminus is overlapping but different. Additional experiments involving site-directed mu- tagenesis will be necessary to investigate more precisely the re- FIGURE 6. Antagonist activity of nanobodies. The antagonist activity of nanobodies was assessed in an aequorin-based calcium-mobilization assay. spective residues involved in chemerin and nanobody binding. The response of CHO cells expressing ChemR23 to 1 nM chemerin (upper The precise role of chemerin in physiological and pathological panels) or 5 nM chemerin(149–157) nonapeptide (lower panels) was tested processes is incompletely understood. Chemerin was described http://www.jimmunol.org/ in the presence of increasing concentrations of CA4910 (left panels) and initially as a chemoattractant factor for leukocyte populations CA5183 (right panels). Results are the mean 6 SEM of three independent expressing ChemR23. However, prochemerin is expressed con- experiments performed in duplicate. stitutively by different tissues in noninflammatory conditions, and its bioactivity depends primarily on the complex proteolytic pro- gested that these proteins could bind to their respective receptors cessing of its C terminus. Numerous proteases belonging to dif- according to a two-step binding model (34). Accordingly, the ferent classes and involved in various pathways were described to cystatin-like domain of chemerin would interact with the N termi- regulate chemerin bioactivity (14, 35–39), rendering the regulation nus and extracellular loops of ChemR23, whereas the C-terminal of this system particularly complex. In addition, ChemR23 ex- peptide of chemerin, known to trigger activation of the receptor, pression was described in many cell populations other than leu- by guest on September 26, 2021 would engage the helix bundle of the receptor (13, 19). Although kocytes, including preadipocytes and adipocytes (5, 6), skeletal this hypothesis regarding ChemR23 has not been confirmed ex- muscle cells (9), and endothelial cells (10), and additional roles perimentally, our present results are consistent with such a hy- for the chemerin/ChemR23 system were proposed in the control of pothesis. This was further supported by the results obtained with the lipid and glucose metabolism (9, 17, 40), blood pressure (41), and bivalent CA4910 nanobody that we generated. Indeed, this bivalent angiogenesis (10). Finally, chemerin was demonstrated to bind

FIGURE 7. Immunodetection of ChemR23 and inhibition of chemerin-induced chemotaxis on human primary cells. (A) ChemR23 labeling was in- vestigated on human monocyte-derived macrophages (upper panels) and immature MDDCs (lower panels), using a reference mouse mAb and the DyLight 650–conjugated CA4910 and CA5183 nanobodies (or the control isotype/nanobody, dashed lines). The data are representative of three independent ex- periments. (B) The antagonist activity of nanobodies was assessed in a chemotaxis assay using human immature MDDCs. The response of the cells to chemerin at 30, 100, and 300 ng/ml was tested in the absence (control) or the presence of 500 nM monovalent CA4910, bivalent CA4910, or irrelevant nanobodies. Results are the mean 6 SEM of five independent experiments performed with cells derived from different donors in triplicates. The data corresponding to the three concentrations of chemerin were averaged, because migration was achieved in all cases but the concentration providing maximal response was different according to donor. **p , 0.005, ***p , 0.001. 8 ANTAGONIST NANOBODIES TARGETING ChemR23 with high affinity to two additional receptors: GPR1 and CCRL2. chemerin; ChemR23 promotes inflammatory signalling upon binding the ligand chemerin(21-157). Arthritis Res. Ther. 12: R228. However, ChemR23 is the only receptor through which biologi- 9. Sell, H., J. Laurencikiene, A. Taube, K. Eckardt, A. Cramer, A. Horrighs, cal activities of chemerin have unambiguously been ascribed, P. Arner, and J. Eckel. 2009. Chemerin is a novel adipocyte-derived factor in- including in vivo. Although chemerin is a chemotactic factor ducing in primary human skeletal muscle cells. Diabetes 58: 2731–2740. generated in inflammatory conditions, it behaves as a pro- or anti- 10.Kaur,J.,R.Adya,B.K.Tan,J.Chen,andH.S.Randeva.2010.Identification inflammatory agent, according to the disease model studied (15, of chemerin receptor (ChemR23) in human endothelial cells: chemerin- 16, 42–45). GPR1 and CCRL2 are receptors with limited and no induced endothelial angiogenesis. Biochem. Biophys. Res. Commun. 391: 1762–1768. signaling properties, respectively, and their roles are poorly un- 11. Wittamer, V., J. D. Franssen, M. Vulcano, J. F. Mirjolet, E. Le Poul, I. Migeotte, derstood. Only one antagonist of ChemR23 has been developed: S. Bre´zillon, R. Tyldesley, C. Blanpain, M. Detheux, et al. 2003. Specific re- the small molecule CCX832. It was described as an inhibitor of cruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J. Exp. Med. 198: 977–985. chemerin signaling through ChemR23 across multiple species 12. Allen, S. J., B. A. Zabel, J. Kirkpatrick, E. C. Butcher, D. Nietlispach, and (human, mouse, and rat) using radioligand-binding and calcium- T. M. Handel. 2007. NMR assignment of human chemerin, a novel chemo- attractant. Biomol. NMR Assign. 1: 171–173. mobilization assays (41). No antagonist of GPR1 or CCRL2 has 13. Wittamer, V., F. Gre´goire, P. Robberecht, G. Vassart, D. Communi, and been described. The nanobodies described in this article might M. Parmentier. 2004. The C-terminal nonapeptide of mature chemerin activates constitute useful tools to delineate the complex activities of the chemerin receptor with low nanomolar potency. J. Biol. Chem. 279: 9956–9962. 14. Zabel, B. A., S. J. Allen, P. Kulig, J. A. Allen, J. Cichy, T. M. Handel, and chemerin as antagonists able to discriminate the effects of E. C. Butcher. 2005. Chemerin activation by serine proteases of the coagulation, chemerin mediated by ChemR23, GPR1, and CCRL2. We showed fibrinolytic, and inflammatory cascades. J. Biol. Chem. 280: 34661–34666. that our nanobodies detect ChemR23 on the surface of human 15. Luangsay, S., V. Wittamer, B. Bondue, O. De Henau, L. Rouger, M. Brait, J. D. Franssen, P. de Nadai, F. Huaux, and M. Parmentier. 2009. Mouse leukocyte populations as efficiently as a mouse mAb widely used ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates Downloaded from as reference and that a bivalent form was able to inhibit the mi- an anti-inflammatory activity of chemerin in a lung disease model. J. Immunol. gration of MDDCs toward chemerin. Because nanobodies can 183: 6489–6499. 16. Bondue, B., O. Vosters, P. de Nadai, S. Glineur, O. De Henau, S. Luangsay, easily be engineered and modified, they also will constitute the F. Van Gool, D. Communi, P. De Vuyst, D. Desmecht, and M. Parmentier. 2011. basis of additional tools useful for detecting ChemR23 on cells ChemR23 dampens lung inflammation and enhances anti-viral immunity in a and in tissue sections. mouse model of acute viral pneumonia. PLoS Pathog. 7: e1002358. 17. Ernst, M. C., and C. J. Sinal. 2010. Chemerin: at the crossroads of inflammation

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